Biopriming of soybean seeds with Bacillus spp.

Biocondicionamento de sementes de soja com Bacillus spp.

Abstract

Seed biopriming with Bacillus spp. has emerged as a sustainable alternative to chemical fungicides for soybean seed treatment. This study evaluated the effects of commercial biological formulations based on Bacillus amyloliquefaciens, B. subtilis, B. velezensis, and B. pumilus on seed health and physiological performance, comparing them with the standard chemical fungicide Captan^®. Seeds were bioprimed by immersion in treatment solutions and assessed for seed-borne fungi, germination, emergence, and early seedling growth. After surface disinfection, all biological treatments resulted in the complete suppression of detectable seed-borne fungi, including Alternaria, Aspergillus, Cladosporium, Curvularia, Diaporthe, Fusarium, and Penicillium, showing efficacy comparable to Captan^®. In particular, treatments based on these Bacillus species significantly improved the germination speed index, percentage of normal seedlings, root length, and seedling emergence index. These findings demonstrate that Bacillus-based biopriming ensures effective pathogen management while enhancing seed vigor, representing a promising strategy to reduce dependence on chemical fungicides in sustainable soybean production systems.

Keywords:
biological control; Bacillus; germination; seed health; emergence

Resumo

O biocondicionamento de sementes com Bacillus spp. tem se destacado como uma alternativa sustentável ao uso de fungicidas químicos no tratamento de sementes de soja. Este estudo avaliou os efeitos de formulações biológicas comerciais à base de Bacillus amyloliquefaciens, B. subtilis, B. velezensis e B. pumilus sobre a sanidade e o desempenho fisiológico das sementes, em comparação com o fungicida químico padrão Captan^®. As sementes foram biocondicionadas por imersão nas soluções dos tratamentos e avaliadas quanto à presença de fungos associados às sementes, germinação, emergência e crescimento inicial de plântulas. Após a desinfestação superficial, todos os tratamentos biológicos resultaram na ausência de fungos detectáveis nas sementes, incluindo Alternaria, Aspergillus, Cladosporium, Curvularia, Diaporthe, Fusarium e Penicillium, apresentando eficácia semelhante ao Captan^®. Em particular, tratamentos à base de Bacillus amyloliquefaciens, B. subtilis, B. velezensis e B. pumilus promoveram melhorias significativas no índice de velocidade de germinação, percentual de plântulas normais, comprimento radicular e índice de velocidade de emergência. Esses resultados demonstram que o biocondicionamento com Bacillus spp. assegura o manejo eficiente de patógenos e aumenta o vigor das sementes, configurando-se como uma estratégia promissora para reduzir a dependência de fungicidas químicos em sistemas sustentáveis de produção de soja.

Palavras-chave:
controle biológico; Bacillus; germinação; sanidade de sementes; emergência

1. Introduction

Seed health plays a crucial role in sustainable agricultural production. Pathogen-free and vigorous seeds ensure healthy crop establishment, high productive potential, and reduced need for subsequent interventions (Sharma et al., 2020; Farooqi et al., 2022). Seed-borne pathogens can reduce germination, weaken seedlings, increase disease incidence, and cause significant yield losses, directly affecting food security and production systems (Gebeyaw, 2020; Moumni et al., 2023).

Chemical fungicides have long been the primary strategy for seed disease control; however, concerns regarding their environmental impacts, pathogen resistance, and risks to human health are increasing (Lamichhane et al., 2020; FRAC, 2023; Manguana et al., 2025). This scenario has accelerated interest in sustainable alternatives, such as biological control agents and microbial-based bioinputs (Lahlali et al., 2022; Rahman et al., 2018). Biological control, utilizing beneficial microorganisms or their metabolites, is a promising strategy that acts through competition, antibiosis, and the induction of plant resistance (Coelho et al., 2023). Among these, species of the genus Bacillus have gained significant attention due to their broad-spectrum antagonistic activity against plant pathogens (Kenfaoui et al., 2024). Several studies have highlighted the potential of Bacillus-based bioinputs as sustainable tools for improving plant performance and reducing dependence on synthetic chemical inputs (Rahman et al., 2018; Lahlali et al., 2022; Pedrozo et al., 2025).

The biocontrol potential of Bacillus spp. is associated with multiple mechanisms, including the production of antimicrobial secondary metabolites and the activation of induced systemic resistance (ISR) pathways, which enhance plant tolerance to biotic stresses (Nihorimbere et al., 2024). In addition, Bacillus spp. may act as plant growth-promoting bacteria, improving seedling vigor and early establishment (Miljaković et al., 2022). Recent evidence indicates that seed biopriming with Bacillus subtilis can enhance soybean germination and physiological performance, supporting its potential as a sustainable alternative to chemical fungicides (Silva et al., 2024).

Therefore, this study investigated the effect of biopriming soybean seeds with commercial biological products based on different Bacillus spp. strains compared with a standard chemical treatment (Captan^®). We hypothesized that Bacillus-based biopriming can effectively control seed-borne pathogens while simultaneously improving seed vigor, thereby offering a sustainable alternative to conventional chemical treatments and contributing to the development of robust biological strategies for soybean seed management.

2. Materials and Methods

2.1. Seed material

The experiment was conducted at the Plant Pathology Laboratory of the Federal University of Paraíba (CCA/UFPB), Brazil. Soybean (Glycine max (L.) Merrill) seeds of the cultivar DM 82i78 RSF IPRO (2024/2025 harvest) were used. The seeds presented an initial moisture content of 11.7% and were stored at 10 °C until the beginning of the assays. Seeds were acquired from the Large Crops Laboratory of the Federal University of Paraíba (UFPB).

2.2. Seed surface disinfection

Prior to treatment, seeds were surface-disinfected in 1% sodium hypochlorite for 3 min, followed by three rinses in sterilized distilled water. This procedure was adopted to minimize external contaminants and to standardize the evaluation of fungi associated with the seed lot. The study did not aim to quantify the epiphytic fungal diversity of non-disinfected seeds.

2.3. Seed treatments and dosage

Seeds were treated with commercial biological products based on different Bacillus spp. strains and compared with a standard chemical fungicide. Although some of these products are labeled for nematode or foliar disease management, they were selected due to the documented activity of Bacillus spp. against multiple plant pathogens, including seed-borne fungi, and their common application as seed treatments or bioinoculants in soybean and other crops (Fernandes et al., 2021; Etesami et al., 2023a; Mian et al., 2024). Doses were defined according to manufacturer recommendations for soybean seed treatment, remaining within the range previously evaluated for Bacillus-based seed inoculants (Tavanti et al., 2020; Carvalho et al., 2023; Ibanhes Neto et al., 2023).

All products were selected based on their commercial recommendation for pathogen suppression. Treatments are detailed in Table 1. Biological treatments were applied via immersion in 100 mL of an aqueous solution, whereas the chemical fungicide Captan^® was applied at the recommended rate of 250 g per 100 kg of seeds.

Table 1
Description of commercial products, microbial composition, and application rates used for soybean seed treatment.

Treatment	Product	Microorganism(s)	Rate used (manufacturer recommendation)
T1	Control	Sterilized distilled water	–
T2	Fx Protection®	Bacillus amyloliquefaciens	0.33 mL
T3	Inlayon®	B. amyloliquefaciens	0.20 mL
T4	NemaControl®	B. amyloliquefaciens	0.20 mL
T5	Twixx-A®	B. amyloliquefaciens	0.50 mL
T6	Bombardeio®	B. subtilis + B. velezensis + B. pumilus	0.26 mL
T7	Captan®	Chemical fungicide	250 g/100 kg seeds

• Note: Biological products were diluted in 100 mL of aqueous solution for seed immersion. Captan was applied as a dry/slurry chemical treatment at the manufacturer's recommended rate.

2.4. Seed health test

Seed sanitary quality was assessed using the blotter test. A total of 200 seeds per treatment were evaluated, distributed into 10 replicates of 20 seeds each. Each replicate consisted of a Petri dish containing 20 seeds placed on sterilized filter paper moistened with sterilized distilled water. Plates were incubated at 25 ± 2 °C under a 12 h photoperiod for seven days.

The main fungal genera evaluated were Alternaria, Aspergillus, Cladosporium, Curvularia, Diaporthe, Fusarium, and Penicillium, commonly reported as important seed-associated fungi in different crops (Mertoğlu and Karaca, 2023; Pikovskyi et al., 2025). Fungal incidence was calculated as the percentage of infected seeds relative to the total number of seeds evaluated (Kumkum et al., 2022; Demissie and Sako, 2023), using the following formula (Equation 1):

$Incidence\left(\%\right)=$ $\frac{Numberofinfectedseeds}{Totalnumberofseedsevaluated}x100$ (1)

Pathogen suppression was estimated based on the percentage reduction in fungal incidence in treated seeds compared with the untreated control, an approach commonly used in studies evaluating treatments for the control of seed-borne fungi (Addrah et al., 2019), according to the following formula (Equation 2):

$Pathogensuppression\left(\%\right)=$ $\frac{Incidenceincontrol-Incidenceintreatment}{Incidenceincontrol}x100$ (2)

2.5. Germination and physiological quality

The germination test was conducted with four replicates of 50 seeds per treatment (200 seeds total per treatment) using the rolled paper towel method. Seeds were placed on Germites t^® paper moistened with sterilized distilled water (2.5 times the dry paper weight), following the rules described by Brasil (2009), and incubated in a BOD chamber at 25 ± 2 °C. Counts were performed from the 5th to the 8th day after sowing. Germination percentage, germination speed index (GSI), as well as the percentages of normal seedlings, abnormal seedlings, dead seeds, and hard seeds, were evaluated according to the official Rules for Seed Testing (Brasil, 2009).

2.6. Seedling development on paper (laboratory conditions)

Early seedling growth was assessed under controlled laboratory conditions. Five normal seedlings per replicate were randomly selected for root and shoot length measurements. Seedlings were considered normal when they presented well-developed essential structures and absence of visible damage, following the criteria established by Brasil (2009).

2.7. Dry matter determination

Following length measurements, shoots and roots were separated and dried in a forced-air oven at 65 °C for 72 h until constant weight. Dry matter was determined using an analytical balance and expressed in grams per seedling.

2.8. Experimental design and statistical analysis

The experiment was conducted in a completely randomized design (CRD). Seed health was evaluated using 10 replicates per treatment, while germination tests were performed with four replicates. Prior to the analysis of variance (ANOVA), data were tested for normality and homogeneity of variances using the Shapiro–Wilk and Levene tests, respectively. Subsequently, data were subjected to ANOVA; when significant treatment effects were detected (p ≤ 0.05), means were compared using the Scott–Knott test at the 5% probability level. All statistical analyses were performed using the R software (RStudio, version 2024.04.0).

3. Results

3.1. Pathogen control

Seed health evaluation revealed fungal incidence only in the untreated control, with the presence of Alternaria, Aspergillus, Cladosporium, Curvularia, Diaporthe, Fusarium, and Penicillium. Fusarium spp. showed the highest incidence (14%), while the other genera ranged from 2% to 10%.

All biological treatments based on Bacillus spp. (Fx Protection^®, Inlayon^®,NemaControl^®, Twixx-A^®, and Bombardeio^®), as well as the chemical fungicide Captan^®, completely suppressed fungal incidence, providing 100% control after seven days of incubation.

3.2. Germination and vigor

Physiological quality assessments of soybean seeds revealed significant differences among treatments (Tables 2 and 3). Inlayo n^®, Twixx-A^®, Bombardeio^®, and Captan^® exhibited the highest germination percentages at the 5th day (G5), ranging from 62% to 69%, while the control (SDW) and Fx Protection^® presented lower values (Table 2).

Table 2
Germination and vigor parameters of soybean seeds treated with biological products based on Bacillus spp. and a chemical fungicide.

Treatment	G5 (%)	G8 (%)	GSI	NS (%)	AS (%)
Control (SDW)	56 b	75 b	6.52 c	37.5 b	4.5 b
Fx Protection®	53 b	80 a	6.52 c	39.5 b	0 a
Inlayon®	65 a	85 a	6.89 b	42.5 b	0 a
NemaControl®	52 b	81 a	7.09 b	43.0 a	0 a
Twixx-A®	62 a	79 a	7.18 a	46.5 a	0 a
Bombardeio®	66 a	79 a	7.45 a	43.0 a	0 a
Captan®	69 a	72 b	7.61 a	47.0 a	0 a
CV (%)	14.36	8.19	12.93	8.19	194.41

• Note: Means followed by the same letter within a column do not differ by the Scott–Knott test at 5% probability (p ≤ 0.05). G5 = germination at the 5th day; G8 = final germination at the 8th day; GSI = germination speed index; NS = normal seedlings; AS = abnormal seedlings; SDW = sterilized distilled water; CV = coefficient of variation.

Table 3
Early growth and dry matter accumulation of soybean seedlings originated from seeds treated with Bacillus-based biological products and a chemical fungicide under laboratory conditions.

Treatment	SL (cm)	RL (cm)	ShDW (g)	RDW (g)
Control (SDW)	7.75 a	11.00 a	0.096 a	0.0645 a
Fx Protection®	5.65 b	8.35 b	0.071 b	0.0545 b
Inlayon®	5.90 b	8.25 b	0.075 b	0.0490 b
NemaControl®	5.25 b	8.50 b	0.0715 b	0.0480 b
Twixx-A®	6.80 b	14.00 a	0.0835 b	0.0675 a
Bombardeio®	4.40 c	7.30 b	0.0670 b	0.0450 b
Captan®	5.60 a	12.15 a	0.0805 b	0.0615 a
CV (%)	29.59	33.42	11.97	16.19

• Note: Means followed by the same letter within a column do not differ by the Scott–Knott test at 5% probability (p ≤ 0.05). SL = shoot length; RL = root length; ShDW = shoot dry weight; RDW = root dry weight; SDW = sterilized distilled water; CV = coefficient of variation.

Final germination at the 8th day (G8) ranged from 72% to 85%, with Inlayo n^® achieving the highest numerical value (85%) (Table 2). The control treatment presented the highest percentage of abnormal seedlings (4.5%), whereas all biological and chemical treatments resulted in 0% abnormal seedlings (Table 2).

Seed vigor, assessed by the germination speed index (GSI), was significantly higher in Twixx-A^®, Bombardeio^®, and Captan^® compared with the control (Table 2).

Regarding seedling growth (Table 3), Twixx-A^® and Captan^® showed high root length values (14.0 and 12.15 cm, respectively), similar to the control (11.0 cm), while Bombardeio^® presented the lowest shoot length (4.4 cm). Shoot dry weight was highest in the control treatment, whereas most biological treatments showed slightly lower values. Root dry weight did not differ significantly among the control, Twixx-A^®, and Captan^® treatments (Table 3).

3.3. Seedling emergence and growth in greenhouse conditions

Data regarding seedling emergence and early development under greenhouse conditions (using soil/substrate) are presented in Tables 4 and 5. On the first evaluation day (E1), no significant differences were detected among treatments for emergence percentage (Table 4). However, by the ninth day (E9), the Fx Protection^®, Inlayon^®, Twixx-A^®, and Bombardeio^® treatments exhibited the highest emergence rates (85–90%), significantly outperforming both the negative control (SDW) NemaControl^®, and the chemical fungicide Captan (Table 4).

Table 4
Seedling emergence percentage and emergence speed index of soybean seeds treated with Bacillus-based biological products and a chemical fungicide under greenhouse conditions.

Treatment	E1 (%)	E9 (%)	SEI
Control (SDW)	27 a	72 b	64.38 b
Fx Protection®	31 a	85 a	73.18 a
Inlayon®	21 a	90 a	62.63 b
NemaControl®	18 a	79 b	57.68 b
Twixx-A®	27 a	89 a	76.79 a
Bombardeio®	27 a	87 a	66.97 a
Captan®	23 a	79 b	59.90 b
CV (%)	20.25	7.70	9.36

• Note: Means followed by the same letter in the column do not differ by the Scott–Knott test at 5% probability (p ≤ 0.05). E1: emergence at the 1st day; E9: emergence at the 9th day; SEI: seedling emergence index; SDW: sterilized distilled water; CV: coefficient of variation.

Table 5
Shoot and root growth and dry matter accumulation of soybean seedlings originated from seeds treated with Bacillus-based biological products and a chemical fungicide under greenhouse conditions.

Treatment	SL (cm)	RL (cm)	SDW (g)	RDW (g)
Control (SDW)	9.55 d	10.00 c	0.503 a	0.2265 a
Fx Protection®	10.50 c	18.15 a	0.501 a	0.2305 a
Inlayon®	11.05 b	18.00 a	0.508 a	0.2270 a
NemaControl®	12.20 a	15.00 b	0.523 a	0.1720 b
Twixx-A®	11.90 a	11.70 b	0.505 a	0.2030 a
Bombardeio®	12.20 a	10.05 b	0.526 a	0.1895 a
Captan®	9.45 d	7.25 c	0.494 a	0.1385 c
CV (%)	10.89	36.61	7.44	12.34

• Note: Means followed by the same letter in the column do not differ by the Scott–Knott test at 5% probability (p ≤ 0.05). SL: shoot length; RL: root length; SDW: shoot dry weight; RDW: root dry weight; CV: coefficient of variation.

The Seedling Emergence Index (SEI) confirmed this trend, with Fx Protection^® (73.18) and Twixx-A^® (76.79) presenting the highest values, while other treatments remained statistically similar to the control. Regarding seedling growth (Table 5), NemaControl^®, Twixx-A^®, and Bombardeio^® yielded the greatest shoot lengths (11.9–12.2 cm). Conversely, the control (SDW) and Captan^® exhibited the most limited development, suggesting that the tested Bacillus-based bioproducts provide a superior bio-stimulatory effect compared to conventional chemical treatments.

Regarding root length, Fx Protection^® (18.15 cm) and Inlayon® (18.00 cm) exhibited the highest values. NemaControl^®, Twixx-A^®, and Bombardeio^® showed intermediate results, whereas the control (SDW) and Captan^® presented the lowest measurements (Table 5).

Shoot dry weight did not differ significantly among treatments (Table 5). However, root dry weight showed significant variation; Captan^® presented the lowest value (0.1385 g), followed by NemaControl^® (0.1720 g), which remained significantly lower than the control and the remaining biological treatments.

4. Discussion

The results demonstrate that Bacillus-based commercial products are effective for soybean seed treatment, providing both pathogen suppression and improvements in physiological performance. All biological treatments completely eliminated the incidence of seed-borne fungi, reaching control levels comparable to the chemical fungicide Captan^®. This highlights the strong potential of Bacillus spp. as sustainable alternatives for seed protection.

The effectiveness of Bacillus spp. is supported by well-established antagonistic mechanisms, including competition for space and nutrients, production of antimicrobial metabolites and lipopeptides, and secretion of enzymes capable of degrading fungal cell walls (Diniz et al., 2024; Kenfaoui et al., 2024). In addition to direct pathogen inhibition, several Bacillus strains promote plant growth through the induction of systemic resistance (ISR) and the synthesis of phytohormones (Ding et al., 2025). These dual functions reinforce their relevance as biological seed protectants, as reported in recent studies with soybean and other crops (Sun et al., 2023; Olszak-Przybyś and Korbecka-Glinka, 2024).

The performance of Bacillus-based products is driven by distinct antagonistic mechanisms that vary among species (Dimkić et al., 2021; Shahid et al., 2021). While these bacteria compete for resources, species such as Bacillus amyloliquefaciens are renowned for producing antifungal lipopeptides, including surfactin, iturin, and fengycin (Wang et al., 2022; Yi et al., 2024; Chen et al., 2025). These metabolites directly disrupt fungal lipid membranes, leading to pore formation and cytoplasmic leakage. In contrast, Bacillus subtilis is frequently associated with the secretion of hydrolytic enzymes, such as β-1,3-glucanases and chitinases, which degrade the structural integrity of fungal cell walls (Dimkić et al., 2021).

Beyond direct antagonism, Bacillus spp. act as biological priming agents for the plant immune system. The interaction between microbial-associated molecular patterns (MAMPs) and soybean root receptors can trigger induced systemic resistance (ISR) (Yu et al., 2022). This response is typically characterized by the upregulation of defense-related genes (e.g., PR-1 and PDF1.2) and the accumulation of antioxidant enzymes, such as peroxidase (POD), polyphenol oxidase (PPO), and superoxide dismutase (SOD) (Rais et al., 2017; Jain et al., 2017; Zahoor et al., 2025). These enzymes help mitigate oxidative stress, creating a physiological barrier that limits colonization even in tissues not directly exposed to the biocontrol agent.

Seed surface disinfection was performed to standardize the assessment of fungi associated with the seed lot, ensuring that differences among treatments primarily reflected the control of internally associated or persistent pathogens. Beyond sanitary quality, Bacillus-based treatments positively influenced seed physiological performance. Biopriming improved germination percentage, speed, and the proportion of normal seedlings compared with the negative control. Similar benefits have been reported in soybean, where microbial inoculation enhances seed metabolic activation (Miljaković et al., 2022).

Enhanced root development, particularly in treatments such as Twixx-A^®, suggests a biostimulant effect associated with Bacillus metabolites. A more vigorous root system improves water and nutrient uptake, increasing tolerance to stressful conditions. These responses are often linked to phytohormone modulation, especially gibberellins and auxins, which regulate enzymatic activity and reserve mobilization during germination (Etesami et al., 2023b).

Although some treatments caused a temporary reduction in shoot length and dry mass compared with the control, this pattern likely reflects an initial energy cost. In this process, metabolic resources are redirected toward defense activation, microbial interaction, or root stimulation rather than immediate shoot expansion. This transient reduction did not compromise germination capacity, suggesting it is a short-term physiological adjustment.

The benefits observed under laboratory conditions were further reflected in seedling emergence assays. Several Bacillus-based products increased emergence speed compared with the control and Captan^®, corroborating that beneficial microorganisms improve performance under substrate conditions (Javed et al., 2021). Higher SEI values in Fx Protectio n^®, Twixx-A^®, and Bombardeio^® are consistent with the role of Bacillus spp. as producers of growth regulators that accelerate development (Shahzad et al., 2016; Hu et al., 2024). Faster and more uniform emergence is essential to reduce exposure to soil-borne pathogens and improve stand uniformity.

Finally, differences among products, even those based on the same species (e.g., B. amyloliquefaciens), suggest that strain-specific characteristics and formulation significantly influence performance (Martins et al., 2018). While Captan^® acts primarily as a chemical fungicide, the evaluated biological treatments combined pathogen suppression with physiological stimulation, resulting in improved vigor under conditions where plants face nutritional and abiotic challenges. These findings confirm that Bacillus-based seed treatments represent an effective strategy for soybean production.

5. Conclusion

Biopriming soybean seeds with commercial products based on Bacillus spp. provided complete suppression of seed-borne fungal incidence, demonstrating efficacy comparable to the chemical fungicide Captan^®. Beyond phytosanitary protection, several biological treatments enhanced physiological performance by increasing germination speed, eliminating abnormal seedlings, and promoting robust root development. Under greenhouse conditions, Bacillus-based products further accelerated seedling emergence and improved early growth compared with the untreated control and the chemical fungicide. These findings indicate that Bacillus spp. represent a promising, sustainable alternative for soybean seed management, effectively combining pathogen control with significant improvements in seedling establishment and vigor.

Acknowledgements

The authors thank the Federal University of Paraíba (UFPB) and the Centre of Agricultural Sciences (CCA) for providing laboratory and greenhouse facilities and institutional support.

Data Availability Statement

The datasets generated and analysed during this study are not publicly available by any means.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  27 July 2026
• Date of issue
  2026

History

• Received
  23 Nov 2025
• Accepted
  14 Apr 2026