Genomic insights into biosurfactant-mediated antifungal activity of native Bacillus amyloliquefaciens EG6.1 against Fusarium oxysporum

Insights genômicos sobre a atividade antifúngica mediada por biossurfactante de Bacillus amyloliquefaciens EG6.1 nativo contra Fusarium oxysporum

Abstract

The soil-borne fungal pathogen Fusarium oxysporum is a major contributor to reduced agricultural productivity and degradation of agroecosystem health. Meanwhile, a molecular approach using microbial biocontrol agents offers a sustainable alternative to the use of chemical fungicides. This study aims to evaluate the potential of biosurfactant produced by Bacillus spp. in controlling Fusarium which integrated with Whole Genome Sequencing (WGS) analysis. Biosurfactant activity was screened using a hemolytic assay (qualitative), an emulsification assay and surface tension measurements (quantitative). Species identification was performed using 16S rRNA gene detection via the CTAB method. Antifungal activity was tested using swab and block methods with a completely randomized factorial design (3x3x3). The presence of biosurfactant biosynthesis gene clusters was detected using Whole Genome Sequencing (WGS) analysis. The results showed that isolates ES7.1, ESD1.6, and EG6.1 were identified as B. velezensis, B. cabrialesii, and B. amyloliquefaciens, with positive potential in producing biosurfactant compounds through indications of b-hemolysis in emulsification activity, a decrease in surface tension value > 20 mN/m, and the presence of an emulsion layer. Among the isolates, B. amyloliquefaciens EG6.1 showed the strongest antifungal activity against F. oxysporum, achieving 78% inhibition in both sonicated pellet and culture fractions. A complete genome analysis of B. amyloliquefaciens EG6.1 was performed the presence of non-ribosomal peptide synthetase (NRPS) gene clusters that encode the biosynthesis of surfactin, bacillomycin D, and fengisin, with significant potential to support antifungal activity. The presence of multiple biosynthesis clusters indicates the genetic capacity of strain EG6.1 to produce bioactive compounds with synergistic fungal inhibition mechanisms. Overall, these results confirm that B. amyloliquefaciens EG6.1, in its new formulation consisting of pellet fractions and sonicated culture, shows great potential as a green biocide and contributes to the development of disease management strategies, while supporting SDG’s 2 and 12.

Keywords:
Bacillus spp.; surfactin; plant-protection; whole genome sequencing; SDG’s 2; SDG’s 12

Resumo

O fungo patogênico do solo Fusarium oxysporum é uma das principais causas da redução da produtividade agrícola e da degradação da saúde dos agroecossistemas. Por outro lado, a abordagem que utiliza agentes de controle biológico microbianos oferece uma alternativa sustentável ao uso de fungicidas químicos. Esta pesquisa tem como objetivo avaliar o potencial dos biossurfactantes produzidos por Bacillus spp. no controle do Fusarium, integrado à análise de Sequenciamento Completo do Genoma (WGS). O método de triagem da atividade dos biossurfactantes foi realizado por meio de teste hemolítico (qualitativo), teste de atividade emulsificante e medição da tensão superficial (quantitativo). A identificação das espécies foi realizada por meio da detecção do gene 16S rRNA pelo método CTAB. O teste de atividade antifúngica utilizou os métodos de swab e bloco, com um delineamento fatorial inteiramente causalizado (3x3x3). A presença de clusters gênicos de biossíntese de biossurfactantes foi detectada por meio da análise de sequenciamento do genoma completo (WGS). Os resultados mostraram que os isolados ES7.1, ESD1.6 e EG6.1 foram identificados como B. velezensis, B. cabrialesii e B. amyloliquefaciens, com potencial positivo na produção de compostos biosurfactantes, indicado pela atividade de emulsificação na forma de β-hemólise, redução do valor da tensão superficial > 20 mN/m e presença de camada de emulsão. Entre esses isolados, B. amyloliquefaciens EG6.1 apresentou a atividade antifúngica mais forte contra F. oxysporum, atingindo 78% de inibição na fração de pellets e na cultura sonicada. A análise do genoma completo da B. amyloliquefaciens EG6.1 revelou a presença de clusters gênicos da sintetase de peptídeos não ribossômicoss (NRPS) que codificam a biossíntese da surfactina, da bacilomicina D e da fengicina, com potencial significativo para apoiar a atividade antifúngica. A presença de vários clusters de biossíntese demonstra a capacidade genética da cepa EG6.1 de produzir compostos bioativos com mecanismos sinérgicos de inibição fúngica. De modo geral, esses resultados confirmam que a B. amyloliquefaciens EG6.1, com sua nova formulação na forma de fração de pellets e cultura sonicada, apresenta alto potencial como biocida verde e contribui para o desenvolvimento de estratégias de manejo de doenças, alinhando-se aos Objetivos de Desenvolvimento Sustentável (ODS) 2 e 12.

Palavras-chave:
Bacillus spp.; surfactina; proteção de plantas; sequenciamento completo do genoma; ODS 2; ODS 12

1. Introduction

Pathogen attacks, such as pests and microbes on crops, are a major challenge in modern agricultural production and have a significant impact on global food security. Plant pathogens such as bacteria and fungi infect various organs through soil, air, and water, causing damage during production, post-harvest, and storage. The activity of pathogenic microbes potentially inhibits the growth of crops, reduces the quality and quantity of crops, and even causes plant death. Various pathogenic fungi that cause diseases in crops have been reported in Indonesia. For example, Fusarium sp., Bipolaris sorokiniana, and Helminthosporium sp. attack wheat crops in East Lombok (Rinjani et al., 2019), while Colletotrichum magnum damages papaya crops in Bali (Sudirga et al., 2022). Colletotrichum sp., Fusarium sp., and Aspergillus sp. have been reported as pathogens against shallots in Boyolali (Haryuni et al., 2024). Aspergillus flavus, Aspergillus niger, Fusarium sp., Curvularia sp., Rhizoctonia solani, and Trichoderma sp. were reported as the dominant pathogens in rice plants in West Sumatra (Rahma et al., 2025). In addition, Alternaria soalni, Colletotrichum spp., Fusarium solani, and Phytophthora infestans attacked local potatoes in South Central Timor (Simamora et al., 2025). Among these various pathogenic fungi, the genus Fusarium is one of the most harmful groups because it can survive in the soil for long periods (chlamydospores) (Mehdinia et al., 2024), producing mycotoxins that can contaminate soil and plant residues (Almeida et al., 2025), disrupting microbial communities and soil ecology (He et al., 2024), and causing vascular wilt disease in various crop plants. F. oxysporum is known as a soil-borne pathogen that attacks plant vascular tissues, thereby inhibiting the transport of water and nutrients (Erbay and Hayit, 2025). Infection by this pathogen often causes symptoms of permanent wilting (Erbay and Hayit, 2025), root necrosis (Punja et al., 2026), reduced productivity and economic losses in agriculture (Dong and Fang, 2021), and even plant death. Its high ecological adaptability and wide host range make F. oxysporum difficult to control using conventional methods.

Many solutions have been implemented to control plant pathogenic fungi using chemical products, such as pesticides or synthetic fungicides. One example is the use of synthetic surfactants, which are the main components of these products; their continuous use has negative impacts on the environment, such as a decline in agricultural land fertility and contamination of water sources (Rajmohan et al., 2020), affecting human health, and increasing the resistance of pathogenic fungi (Fan et al., 2017; Panth et al., 2020) necessitate alternative strategies, particularly biosurfactant. In addition, the demand for pesticide-free food has also increased, requiring control strategies to find new sustainable and environmentally friendly alternatives based on the green economy, such as the development of green biocide products through the use of biological agents as biosurfactant producer.

One promising strategy is the use of microbial biocontrol agents from the Bacillus genus of bacteria, which can be employed to formulate green biocides as biosurfactant-producing. Bacillus is a genus of bacteria that produces volatile organic compounds, hydrolytic enzymes, and lipopeptides, playing an important role in controlling plant pathogens and enhancing plant growth. One of the potential compounds produced by Bacillus is lipopeptides, which are a group of biosurfactant consisting of surfactin, iturin, fengycin, bacillomycinD, bacilysin, and bacillibactin, with antifungal properties (Dunlap et al., 2016; Ngalimat et al., 2021). Biosurfactants offer several advantages, including effective antifungal activity against various pathogenic fungi, such as Fusarium oxysporum (Olmedo et al., 2022); high biodegradability and environmental friendliness (Aslam et al., 2023); stability under various extreme conditions (Jumpathong et al., 2022); multifunctionality, such as improving soil quality and fertilizer efficiency (Peng et al., 2025; Ezhkin et al., 2023), and low toxicity to non-target organisms (Machado et al., 2023).

Several similar studies have been conducted on rhizosphere bacteria in the production of biosurfactants. The methods used to test antifungal activity have been limited to the use of culture fractions in the form of supernatants via the spot and block methods. The presence of biosurfactant compounds from bacteria has also been detected through chemical extraction and purification methods. However, a molecular-level understanding of the relationship between gene expression and biosurfactant compounds has not yet been established. In contrast to these studies, this research tested antifungal activity using a modified method combining swab and block assays with three types of bacterial culture fractions: sonicated culture, sonicated pellet, and supernatant. This study is also supported by a comprehensive understanding of the genetic basis that plays a crucial role in bacterial biosurfactant production through Whole Genome Sequencing (WGS) analysis. The development of WGS as an analytical method is essential for identifying gene clusters involved in the biosynthesis of secondary metabolites, as well as the molecular mechanisms underlying effective biological control activity. WGS enables the identification of specific genes associated with the production of biosurfactants and fungal cell wall-degrading enzymes (Wang et al., 2023). In this context, the Bacillus spp. isolates obtained from a unique indigenous breeding site for Aedes aegypti larvae in the study by Salamun et al. (2020) represent promising candidates for innovative biocontrol applications based on ecological engineering. Therefore, this study aims to evaluate the biosurfactant and antifungal activity of Bacillus spp. against the plant pathogen F. oxysporum, as well as to conduct whole-genome sequencing (WGS) to investigate the presence of biosurfactant biosynthesis genes. It is hoped that this study will provide new insights into the development of efficient and environmentally friendly biological control agents to support sustainable agriculture.

2. Materials and Methods

2.1. Preparation of tools and materials

This study used six types of media, including Nutrient Agar (NA), Nutrient Broth (NB), Nutrient Yeast Salt Medium (NYSM), Blood Agar, Synthetic Mineral Water (AMS) with 2% molasses as a C source substrate, and Potato Dextrose Agar (PDA). NA and NB served as base media for NYSM preparation, which was used as the growth medium for the three bacteria isolates. Solid NYSM medium was prepared by mixing 28.0 g/L NA, 0.5 g/L yeast extract, 0.2 g/L MgCl2, 0.01 g/L MnCl2, and 0.1 g/L CaCl2, while liquid NYSM was prepared with a composition of 8.0 g/L NB, 0.5 g/L yeast extract, 0.2 g/L MgCl₂, 0.01 g/L MnCl₂, and 0.1 g/L CaCl₂ (Myers and Yousten, 1978). Blood Agar medium was used as a test medium for hemolytic activity, and PDA medium served as a test for antifungal activity. AMS supplemented with 2% molasses was used to evaluate biosurfactant production, following the method described by Nafidiastri et al. (2024). All types of media and other equipment needed, such as blue tips, yellow tips, cork borers, and falcon tubes, were sterilized with an autoclave at 121ºC, 1 atm for 15 minutes.

2.2. Confirmation of microscopic characteristics

The bacteria isolates included ES7.1, ESD1.6, and EG6.1, which were successfully isolated from the breeding site sediment of A. aegypti larvae in the study by Salamun et al. (2020). The morphological characteristics of the ES7.1 bacterium are irregular in shape, with a flat elevation and entire edges, and it is white in color. The ESD1.6 bacterium is circular in shape, with a raised elevation and entire edges, and it is white in color. The EG6.1 bacterium is irregular in shape, raised, and lobate, and it is whitish-cream in color. The three bacteria isolates were cultured on NYSM solid medium and incubated for 18-20 hours. Gram and endospore staining with malachite green were performed to confirm the cell shape, Gram type, and presence of endospores.

2.3. Screening for biosurfactant activity

Screening for biosurfactant activity in bacteria isolates was performed qualitatively and quantitatively to determine the ability to produce biosurfactant compounds.

2.3.1. Testing the hemolytic activity of Bacillus spp. bacteria isolates

Qualitative screening of biosurfactant activity was performed using hemolytic activity. Bacteria isolates ES7.1, ESD1.6, and EG6.1 were inoculated on Blood Agar medium and incubated for 72 hours. Positive results showed a clear zone around the growing bacteria colonies.

2.3.2. Measurement of the growth curve of Bacillus spp. bacteria isolates

The growth curve of the three bacteria isolates on AMS+2% molasses medium was measured to determine the ability to use molasses as a carbon source and produce biosurfactant compounds. This measurement was carried out by measuring the bacteria density with a spectrophotometer at a wavelength of 600 nm.

2.3.3. Emulsification activity of Bacillus spp. bacteria isolates

The emulsification activity of bacteria isolates was tested by placing 2 mL of bacteria and 2 mL of kerosene and oil in a test tube. This was followed by homogenization using a vortex mixer for 2 minutes, and left for 1 hour and 24 hours at room temperature. Observations and measurements were carried out at both incubation times. Measurements were based on the height of the emulsion layer formed. The emulsification index (E1 or E24) value was calculated as the percentage of the emulsion layer height (cm) divided by the total solution height (Datta et al., 2018) (Equation 1).

$$E(\text{\%})=\frac{TE}{TL}\times 100\text{\%}$$ (1)

Where: TE: Emulsion height; TL: Total solution height.

2.3.4 Surface tension of Bacillus spp. bacteria isolates

Surface tension analysis was conducted to determine the production of biosurfactant in bacteria isolates. It is one of the preliminary methods for quantitatively testing biosurfactant activity and was conducted in this study using a Du-Nouy tensiometer. The Du-Nouy ring method is based on measuring the force required to detach a ring or wire loop from an interface or surface. The detachment force is proportional to the interfacial tension (Chauhan et al., 2013) (Equation 2).

$$\gamma =\gamma 0\frac{\theta }{\theta 0}$$ (2)

Where: γ : surface tension of the sample; γ_o: standard surface tension value of distilled water at t^oC (72 dyne/cm); θ : sample value indicated on the scale of the device; θ_o: distilled water value indicated on the scale of the device,

2.4. Detection of 16S rRNA genes (species name identification)

Bacteria species identification was performed by detecting the 16S rRNA gene using primers 27F and 1492R. DNA extraction was performed using the CTAB method (Ausubel et al., 2003) with the Wizard Genomic DNA Purification Kit (Promega). Amplification of the 16S rRNA gene was performed using a PCR (Polymerase Chain Reaction) machine. The PCR reaction was conducted under conditions of initial denaturation at 94°C for 2 minutes, denaturation at 92°C for 30 seconds, annealing at 55°C for 30 seconds, elongation at 72°C for 1 minute, final elongation at 72°C for 5 minutes, 35 cycles. The PCR products were visualized through electrophoresis using 1% agarose gel, followed by Ethidium Bromide (EtBr) staining and observed under ultraviolet light. The samples were sent to 1st Base DNA Sequencing Service Malaysia. The sequences obtained were analyzed using BioEdit and aligned with GenBank data through the Basic Local Alignment Search Tools nucleotide (BLASTn) NCBI (Altschul et al., 1997).

2.5. Antifungal activity test of bacteria isolates

The study employed a completely randomized factorial design with three replications. The positive control used was the fungicide ZPT Score 250EC, and the negative control was sterile medium. The antifungal activity test was conducted in vitro using the swab method to determine the ability of the three bacteria isolates to suppress the growth of the plant pathogen Fusarium oxysporum. The three bacteria isolates that had been grown for 72 hours were then centrifuged and sonicated, producing three culture fractions, namely cell-free supernatant, sonication pellet, and sonicated pure culture. The fractions were applied to PDA medium in Petri dishes using the swab method, with holes made in the center using a cork borer to inoculate F. oxysporum.

F. oxysporum isolates that had been rejuvenated on PDA medium for five days were cut into blocks using a cork borer and placed in the center. Fungal growth was observed daily until 168 hours of incubation. The growth zone of F. oxysporum was measured using the growth inhibition index formula adapted from Jasim et al. (2016) (Equation 3):

$$\text{\% Index of Growth Inhibition}=\frac{D1}{D1-D2}\times 100\text{\%}$$ (3)

Where: D1: diameter of control growth; D2: diameter of treatment.

2.6. Identification of crude biosurfactant compounds through Whole Genome Sequencing (WGS)

Identification of crude biosurfactant products was carried out through Whole Genome Sequencing (WGS) analysis. Bacteria genomic DNA (gDNA) was isolated and extracted using the Wizard kit. The gDNA was further analyzed using WGS with PromethlON 24 A Series (Oxford Nanopore Technologies, UK). The sequencing data were then processed bioinformatically through the Galaxy platform to obtain the complete genome sequence, with quality control (NanoPlot), filtering (Filtlong), assembly (Flye), and refinement (Medaka). Subsequently, genome annotation was performed using Bakta. The annotation results were stored in GenBank format files, which were visualized using the Proksee platform. Prediction of clusters gene in bacteria also carried out with antiSMASH.

2.7. Data analysis

All of data obtained in all stages of this study were analyzed descriptively and statistically. Microscopy characters in the form of Gram type and endospores, hemolytic activity, emulsification activity and surface tension measurement, vizualitation of 16S rRNA gene and WGS result, including prediction of clusters gene of microbe were analyzed descriptively. In addition, data obtained from antifungal activity test were analyzed statistically using R studio. Data on the growth of the pathogenic fungus F. oxysporum were analyzed using repeated measures ANOVA. Data on the inhibitory activity of Bacillus spp. biosurfactants against F. oxysporum were analyzed using two-way ANOVA..

3. Results and Discussion

Three bacteria isolates, ES7.1, ESD1.6, and EG6.1, were found to cause mortality in Aedes aegypti larvae at the initial screening stage, with 70% mortality for isolates ES7.1 and EG6.1, as well as 100% mortality for isolate ESD1.6 (Maswantari, 2018). The microscopic characteristics of the bacteria isolates were observed through Gram staining, as shown in Figure 1, and endospore staining in Figure 2.

Figure 1
Gram staining of bacterial isolates A) ES7.1, B) ESD1.6, and C) EG6.1, showing rod-shaped Gram-positive cells (purple color) observed under a light microscope. Scale bar = 10 µm

Figure 2
Microscopic visualization through spore staining. The red arrow indicates vegetative cells, while the orange arrow indicates endospores. A) ES7.1, B) ESD1.6, C) EG6.1.

Figure 1 shows that the three bacteria isolates found have rod-shaped cells, belong to the Gram-positive bacteria group, and appear purple when observed under a microscope. In the Gram staining process, crystal violet (Gram A) and iodine (Gram B) that enter Gram-positive bacteria cells cannot be washed away by alcohol due to the presence of a strong and thick layer of peptidoglycan in the cell wall, causing the appearance of a purple color. The cell wall structure of Gram-positive bacteria is 15-80 nm thick, consisting mostly of peptidoglycan with a lipid content of 1-4% and teichoic acid (Bishop and McCue, 2021). According to Suleyman et al. (2018), Gram-positive bacteria are more susceptible to extreme environmental conditions, such as temperature, pH, and salinity, and are more resistant to penicillin.

Figure 2 shows that the three bacteria isolates successfully isolated formed endospores. All bacteria isolates produced oval-shaped endospores located at the terminal. Bacteria endospores have a high level of resistance to environmental stress and can be widely distributed in nature (Shrestha et al., 2025). A study (Guillén-Navarro et al., 2023) showed that the sporulation process of Bacillus can influence the production of biosurfactant. The high presence of endospores found is comparable to the increase in antifungal activity of biosurfactant produced by B. subtilis MC6B-22 from 24 to 84 hours of incubation (death phase). It has been established that the condition is related to the production of biosurfactant (surfactin) which is dependent on the ComX and PhrC genes, along with several transcription factors such as CodY, DegU and PerR, which control sporulation (Hamoen et al., 2003). This can be used to combat fungal pathogens, and it can be employed in the creation of storage formulations and environmentally friendly biocides for agricultural land (Maral-Gül and Eltem, 2025).

Hemolytic activity was used to determine the ability of bacteria isolates to produce biosurfactant. Figure 3 shows that all three bacteria isolates hemolyzed blood cells, as indicated by the clear zone formed around the bacteria colonies on Blood Agar observed after 24 hours with B-hemolysis or total hemolysis. The B-hemolysis type indicated the lysis of all red blood cells and the release of hemoglobin from the cells.

Figure 3
Hemolytic activity of bacterial isolates A) ES7.1, B) ESD1.6, and C) EG6.1 on Blood Agar after 24–72 h incubation, showing β-hemolysis indicated by clear zones surrounding the colonies. Black arrow: colony bacteria, Blue arrow: clear zone.

Bacteria capable of producing B-hemolysis have the potential to generate biosurfactant. In general, biosurfactant are hemolytic substances that can function as antibodies against erythrocyte membrane antigens (Ibrahim et al., 2013). The hemolytic activity of biosurfactant can occur through two different mechanisms. The first is through increased membrane permeability to small solutes, which occurs when the biosurfactant concentration is low, causing osmotic lysis. The second is through membrane solubilization, which usually occurs at high biosurfactant concentrations (Zaragoza et al., 2010; Bamba et al., 2024).

The ability of the three bacteria isolates to use and metabolize molasses as a substrate is shown in Figure 4. All isolates showed a good growth curve from the adaptation, exponential, stationary, and death phase up to 144 hours of observation. The highest bacteria isolate density growing on molasses substrate was produced by ES7.1, with optimal growth at 48 hours of incubation. In general, the production of lipopeptide biosurfactant is induced when bacteria cells are under nutritional stress. For example, surfactin production is induced in actively growing bacteria cells during the transition from the exponential to the stationary phase, while fengycin synthesis is associated with the early stationary phase, and iturin only accumulates in the later stationary phase (Ongena and Jacques, 2008).

Figure 4
Time Course Profiles of Bacteria Growth of Bacillus spp. in AMS + 2% Molasses.

The emulsification activity of bacteria isolates on molasses substrates was identified to determine the ability to produce an emulsion layer (Figure 5).

Figure 5
Emulsification activity of bacterial isolates ES7.1, ESD1.6, and EG6.1 with kerosene/oil, showing emulsion layer formation after 1 h and 24 h incubation. The emulsification index (E24) was calculated based on emulsion height.

Figure 5 shows that bacterial isolate EG6.1 is the most effective at producing emulsions compared to bacterial isolates ES7.1 and EG6.1. From 48 to 168 hours of incubation, the emulsifying activity produced by bacterial isolate EG6.1 reached >50%; in fact, at 96–120 hours of incubation, the resulting emulsion exceeded 60%.

This mechanism of emulsification activity occurs through direct contact (adhesion) with the surface of hydrocarbon (oil) droplets that are larger than the microbial cells. In this case, the microbial cells adhere to the surface of hydrocarbon droplets that are larger than the cells, and substrate uptake is carried out by diffusion or active transport. The availability of substrate for cell attachment is a limiting factor in substrate uptake. Direct contact between hydrocarbons and cells indicates the existence of an important mechanism in substrate uptake. Biosurfactant can be classified into low and high-molecular-weight types. Low molecular weight biosurfactant include glycolipids, phospholipids, and lipopeptides, which are effective in reducing surface tension. Meanwhile, high molecular weight biosurfactant, such as proteins, lipopolysaccharides, lipoproteins, or complex mixtures of these biopolymers, are more effective in stabilizing oil-in-water emulsions (acting as emulsifiers) (Calvo et al., 2009).

Surface tension measurements were conducted to determine the ability of the three bacteria isolates to produce biosurfactant using molasses as a carbon source (Figure 6).

Figure 6
Evaluation of Biosurfactant Production through Surface Tension Measurement of Bacillus spp. y line: Surface Tension Value, x line: Incubation Time.

The bacteria isolate that produced the lowest surface tension value was isolate EG6.1, with a value of 30.86 mN/m at 48 hours. The decrease in surface tension of the three bacteria isolates relative to the control on molasses substrate during 144 hours of incubation is shown in Table 1. The highest decrease was produced by EG6.1, with a decrease of 41.94 mN/m relative to the control at 24 hours of incubation. Bacteria isolate ES7.1 also had the best surface tension reduction ability after bacteria isolate EG6.1, which was around 41.29 mN/m compared to the control at the same incubation time.

Table 1
Measurement of surface tension and its decrease to control of ES7.1, ESD1.6, and EG6.1 bacteria isolates for 0 until 144 H incubation.

Isolate Code	Incubation Time (H)	Surface Tension (mN/m)	Decrease of Surface Tension (mN/m)
ES7.1	0	51.17	21.63
	24	31.51	41.29
	48	33.08	39.72
	72	35.52	37.28
	96	39.51	33.29
	120	41.79	31.01
	144	45.52	27.28
	Control	72.8	-
ESD1.6	0	53.24	19.56
	24	37.17	35.63
	48	47.39	25.41
	72	47.14	25.66
	96	47.30	25.50
	120	49.66	23.14
	144	46.01	26.79
	Control	72.8	-
EG6.1	0	54.49	18.31
	24	30.86	41.94
	48	31.79	41.01
	72	31.33	41.47
	96	34.06	38.74
	120	34.21	38.59
	144	35.02	37.78
	Control	72.8	-

This decrease in surface tension may be due to the activity of biosurfactant on the surface produced by isolates. Biosurfactants are amphiphilic compounds that enable interactions between hydrophilic and hydrophobic surfaces. These compounds can reduce the surface tension of a liquid by decreasing the cohesive forces between molecules at the surface. This reduction in surface tension allows for the formation of more stable and efficient interfacial films (Wardhani et al., 2022). A bacteria is considered to have the potential to produce biosurfactant when the surface tension reduction value is> 10 mN/m. Isolate EG6.1 effectively reduced surface tension to 30.86 mN/m, which is better than the study by Asadollahi et al. (2016), where B. cereus decreased surface tension to 36.4 mN/m. This difference may be due to the influence of variations in the concentration of molasses substrate used. The EG6.1 bacteria isolate used a molasses concentration of 2%, while the comparison bacteria in another study, namely B. cereus, used 10% molasses (Asadollahi et al., 2016). According to Ni'matuzahroh et al. (2003) and Nitschke et al. (2004), one obstacle that affects the efficiency of B. subtilis 3KP biosurfactant production with molasses substrate is the instability of product yield due to differences in the composition and concentration of nutrients. These differences are directly related to the sugar processing process from which the molasses is obtained. Variation in sugar content of molasses, which is the main carbon source for the growth of B. subtilis 3KP bacteria, greatly affects the productivity of the biosurfactant produced.

The bacteria 16SrRNA gene identification results were analyzed using Basic Local Alignment Search Tools nucleotide (BLASTn) and aligned with the database in GenBank (Table 2).

Table 2
Result of Basic Local Alignment Search Tools (BLAST) nucleotide analysis of ES7.1, ESD1.6, and EG6.1 bacteria isolates based on 16S rRNA gene.

Isolate Code	Description	Scientific Name	Query Cover	%ID	Number Acces
ES7.1	Bacillus velezensis strain CBMB205	Bacillus velezensis	99%	99.93%	NR_116240.1
ESD1.6	Bacillus cabrialesii strain TE3	Bacillus inaquosorum	99%	99.58%	NR_180419.1
EG6.1	Bacillus amyloliquefaciens strain MPA 1034	Bacillus amyloliquefaciens	99%	99.58%	NR_117946.1

Table 2 shows that the three bacteria isolates ES7.1, ESD1.6, and EG6.1 belong to the same genus, Bacillus. ES7.1, ESD1.6, and EG6.1 are identical to the species B. velezensis, B. cabrialesii, and B. amyloliquefaciens, respectively, with a similarity level of more than 99%. The three DNA bands appeared to be the same size as the marker when aligned, ranging from 1500 bp. The presence of DNA bands visible on the agarose gel indicates the presence of the 16S rRNA gene, which functions as a DNA barcode for bacteria species. Sequencing of the 16S rRNA gene serves as an approximate method for rapid and accurate bacteria identification. A bacteria is considered to represent the same genus when the similarity index is above 95% and represents the same species when it is above 97% (Srinivasan et al., 2015; Johnson et al., 2019). The PCR results of the three bacteria were visualized by electrophoresis using a 1% agarose gel, as shown in Figure 7.

Figure 7
Visualization Results of DNA Band Electrophoresis of B. velezensis ES7.1, B. cabrialesii ESD1.6, and B. amyloliquefaciens EG6.1 with 1500 bp.

The antifungal activity of the three isolates B. velezensis ES7.1, B. cabrialesii ESD1.6, and B. amyloliquefaciens EG6.1 was tested using three fractions, namely culture (K), pellet (P), and supernatant (S), to suppress the growth of the plant pathogenic fungus Fusarium oxysporum. The diameter growth of pathogen fungi was measured and observed for 72, 120, and 168 hours on the Figure 8. The lowest growth of F. oxysporum was observed in the presence of culture fractions and EG6.1 pellets B. amyloliquefaciens, at 11.2 and 9.0 mm, respectively.

Figure 8
F. oxysporum Growth Diameter on Antifungal Activity at 72, 120, and 168 H after Inoculated with Bacillus spp.’s Fraction.

The culture fraction composition consisted of growth media, bacteria cells, and secreted active compounds, while the pellet fraction consisted of bacteria cells alone that had been separated from the growth media and active compounds during the centrifugation process. Both fractions were sonicated to destroy the cell walls, ensuring that the active compounds attached to the bacteria could be released. Sonication is a method of lysing cells using ultrasonic waves with frequencies higher than human hearing (above 20,000 Hz). These ultrasonic waves cause an increase in temperature, which affects the cell walls to open and release the active compounds produced by the bacteria (Fransiska et al., 2012). The supernatant fraction consists of active compounds that have been separated from the bacteria cells.

Table 3 shows the growth inhibition values of F. oxysporum due to the antifungal activity produced by the three fractions of bacteria isolates.

Table 3
Antifungal activity of B. velezensis ES7.1, B. cabrialesii ESD1.6, and B. amyloliquefaciens EG6.1 based on inhibition level of F. oxysporum.

Isolate	Fraction Type	Inhibition Level of F. oxysporum (%)
B. velezensis ES7.1	Culture	76.7 ± 0.46
	Pellet	81.3 ± 0.39
	Supernatant	-
B. cabrialesii ESD1.6	Culture	31.7 ± 0.64
	Pellet	63.8 ± 0.35
	Supernatant	-
B. amyloliquefaciens EG6.1	Culture	78.0 ± 0.14
	Pellet	82.3 ± 0.0
	Supernatant	-
K+	Fungicide	60.9 ± 0.63
K-	Aquadest	-

Based on the results, the order of culture and pellet fractions that showed the best inhibition of F. oxysporum growth were isolates B. amyloliquefaciens EG6.1, B. velezensis ES7.1, and B. cabrialesii ESD1.6. B. amyloliquefaciens EG6.1 in the culture and pellet fraction had the best ability to suppress the growth of F. oxysporum, with inhibition rates of 78.0% and 82.3%, respectively. The inhibition of F. oxysporum growth by the bacteria isolates in three fractions is shown in Figure 9.

Figure 9
Antifungal Activity of Bacillus spp. Against F. oxysporum at 168 H incubation. ESD1.6 as B. cabrialesii, ES7.1 as B. velezensis, EG6.1 as B. amyloliquefaciens, K- as Negative Control (non-treatment), K+ as Positive Control (Chemical Fungicide). K: sonicated culture, P: sonicated pellet, S: supernatant free-cells.

According to Rochmawati and Trimulyono (2020), biosurfactant compounds that have potential as antifungal agents originate from the lipopeptide group, namely iturin, fengycin, and surfactin. Fungal growth is inhibited by iturin by damaging the cell membrane, altering permeability, and removing K+ ions. Iturin and fengisin presumably cause conidia and hyphae lysis, while also inhibiting toxin formation in fungi. A similar study has been conducted by (Guillén-Navarro et al., 2023), showing that lipopeptide biosurfactant produced by B. subtilis MC6B-22 combated plant pathogens F. nivale and F. solani in antifungal activity tests. The inhibitory activity observed can be attributed to the action of mycosubtilin, a type of lipopeptide widely known for the antifungal activity against various yeasts and pathogenic fungi. Lipopeptides, including iturin and mycosubtilin, have been identified as major factors in the antagonistic activity produced by B. subtilis (Ongena and Jacques, 2008). Moreover, B. subtilis MC6B-22 possesses various antimicrobial compounds confirmed by the lipopeptide gene cluster detected through genomic sequence analysis (mycosubtilin, surfactin, bacillaene, bacillibactin) and other secondary metabolites such as bacilysin, subtilin, terpens, rhizocticin A (Guillén-Navarro et al., 2023). The results indicate that the three bacteria isolates observed can produce biosurfactant, indicating strong potential as biological agents for use in high-performance green biocide consortia against plant pathogens in agriculture.

Based on the antifungal activity of the three bacteria isolates, B. amyloliquefaciens EG6.1 showed high potential in inhibiting plant pathogenic fungi, in line with the ability to produce biosurfactant. Therefore, further investigation was conducted on the genes implicated in biosurfactant biosynthesis to determine the mechanism of inhibiting F. oxysporum. Biosurfactant biosynthesis genes in B. amyloliquefaciens EG6.1 were identified molecularly through the Whole Genome Sequencing (WGS) method. WGS is an analytical method used to obtain comprehensive genetic information about bacteria isolates, including genome size, nucleotide composition, and the presence of functional genes implicated in primary and secondary metabolism. This high-capacity sequencing method also allows the determination of the nucleotide sequence for the entire genome, resulting in a higher resolution than conventional molecular techniques such as single PCR or 16S rRNA marker (Goodwin et al., 2016).

The WGS analysis results from the B. amyloliquefaciens EG6.1 isolate are shown in Figure 10. The assembly results show that the genome of B. amyloliquefaciens EG6.1 isolate has a total length of 4,275,032 bp with a GC content of 43.90%, in line with the characteristics of the Bacillus genus genome (GC content 35–46%) (Priest and Austin, 1993). The number of contigs produced was 1 with an N50 value of 4,275,032 bp, indicating that the assembly quality was in the good category according to de novo assembly standards. In addition, this genome had a coding density of 89.5%, suggesting high efficiency in coding capacity. Annotation results using Bakta (v1.9.4, database v5.1) identified 4,229 CDS, including 24 pseudogenes and 166 hypothetical genes, as well as 86 tRNA, 1 tmRNA, 30 rRNA, and 35 ncRNA with a total of 63 ncRNA regions. Although no CRISPR arrays were found, small elements such as 4 sORFs were successfully detected. The genome also contained two oriC regions, indicating chromosomal replication origins, while no oriV or oriT sites were detected, supporting the absence of additional contigs that might correspond to plasmids or other extrachromosomal elements.

Figure 10
Circular genome map of Bacillus amyloliquefaciens EG6.1 generated from WGS analysis, showing coding sequences (CDS), rRNA, tRNA, GC content, and predicted biosynthetic gene clusters. Key biosurfactant-related clusters are highlighted.

The secondary metabolite gene clusters of B. amyloliquifaciens EG6.1 were predicted using antiSMASH 8.0.0 version. The results showed that there were 13 region (gene clusters) in the genome of B. amyloliquifaciens EG6.1, as shown in Table 4. There are 3 Terpene, 2 NRPS (Non-ribosomal Peptide Syntethase), 1 Sactipeptide, 1 CDPS (Cyclodipeptide Synthase), 1 RiPP (Ribosomally Synthesized and Post-translationally Modified Peptide) with azole, 1 NRP-metallophore, 1 Lanthipeptide-class-ii, 1 T3PKS (type III Polyketide Synthase), and 1 transAT-PKS (Trans-Acyltransferase Polyketide Synthase).

Table 4
Prediction of gene clusters encoding secondary metabolites in B. amyloliquifaciens EG6.1 using antiSMASH analyze.

Region	Type	From	To	Similarity Confidence	Most Similar Known Cluster
Region 1	Terpene	1	14,599
Region 2	NRPS	7237,738	789,126	High	Surfactin
Region 3	Unknown	1,452,040	1,493,458	High	Bacilysin
Region 4	Sactipeptide	1,496,431	1,518,042	High	Subtilosin A
Region 5	CDPS	1,754,052	1,774,789	High	Pulcherriminic acid
Region 6	Azole-containing-RiPP	2,077,104	2,107,207		Unknown
Region 7	NRP-metallophore, NRPS	2,121,225	2,173,006	High	Bacillibactin
Region 8	Terpene-precursor	2,832,468	2,853,358		Unknown
Region 9	Lanthipeptide-class-ii	2,918,511	2,941,588		Unknown
Region 10	T3PKS	3,052,612	3,093,709	Low	1-carbapen-2-em-3-carboxylic acid
Region 11	Terpene	3,141,147	3,163,045		Unknown
Region 12	NRPS, betalactone, transAT-PKS	3,241,281	3,372,005	High	Fengycin
Region 13	TransAT-PKS, NRPS, T3PKS, PKS-like	3,558,287	3,672,999	High	Bacillaene

The genome of B. amyloliquefaciens EG6.1 contains various gene clusters that function in the synthesis of antimicrobial compounds, such as Surfactin, Bacilysin, Subtilosin A, Pulcherriminic acid, Bacillibactin, 1-carbapen-2-em-3-carboxylic acid, Fengycin, and Bacillaene. The results of the antiSMASH analysis predicted a similarity level of 73% for Surfactin and 84% for Bacilysin (B. velezensis FZB42), 90% for Subtilosin A (B. subtilis subsp. spizizenii ATCC 6633), 89% for Pulccherriminic acid and 88% for Bacillibactin (B. subtilis subsp. subtilis str. 168), 80% for Fengycin and 77% for Bacillaene (B. velezensis FZB42). Various antimicrobial compounds are assembled by a series of enzyme machines known as NRPS (Surfactin, Fengycin/Plipastatin, Bacillomycin) and PKS (Bacillaene, Macrolactin, Difficidin) (Fazle Rabbee and Baek, 2020; Zhang and Sun, 2018). Surfactin is a strong biosurfactant molecule and is effective as an antimicrobial, anti-mycoplasma, and anti-Legionella agent (Nafidiastri et al., 2026; Loiseau et al., 2015; Vollenbroich et al., 1997). Fengycin or plipastatin has broad-spectrum antifungal activity against filamentous fungi by targeting disruption of cell membrane permeability, resulting in the death of target cells (Zhang and Sun, 2018). Bacillaene, as the latest polyene antibiotic, exhibits antibacterial and antifungal activity by targeting the inhibition of prokaryotic cell protein synthesis (Um et al., 2013; Chen et al., 2009). Bacilysin, synthesized by B. velezensis, acts as an antibacterial, anti-cyanobacterial, and antifungal agent on several groups of microbes through a mechanism of cell wall structure destruction, causing intracellular components to leak out (Wu et al., 2015; Wu et al., 2014). Bacillibactin siderophore for scavenging iron from the environment produced by Bacillus spp. is important in facilitating Fe(III) acquisition, especially when Bacillus cells experience iron limitation (Dimopoulou et al., 2021; Fukushima et al., 2013). Bacillibactin has antagonistic activity against pathogenic fungi when working together with lipopeptide compounds secreted by NRPS. Subtilisin A is a cyclic sulfur wool antibiotic protein (Algburi et al., 2017). Among the various clusters of active compound genes, the presence of two biosurfactant biosynthesis genes such as surfactin and fengicin in the WGS analysis suggests that they are responsible for the high antifungal activity of B. amyloliquefaciens EG6.1 against F. oxysporum. The inhibition exerted by these two compounds occurs through the disruption of fungal cell walls and cell membranes, which can lead to the leakage of proteins and nucleic acids, swelling, vacuolization, and ultimately cell lysis (Assena et al., 2024; Deng et al., 2024). Surfactin and fengicin are also known to inhibit the germination of F. oxysporum spores (Xue et al., 2023). The presence of these two genes implies the production of biosurfactant compounds that can inhibit plant pathogenic fungi, thereby increasing agricultural productivity.

The biosurfactant biosynthesis genes produced by B. amyloliquefaciens EG6.1 are shown in Table 5. The results show the alignment of the biosurfactant biosynthesis genes of B. amyloliquefaciens EG6.1 in the GenBank database, which indicates a high level of similarity in the surfactin, bacillomycin, and fengycin gene clusters.

Table 5
Whole genome sequencing result of biosynthesis biosurfactant genes from B. amyloliquefaciens EG6.1.

Biosurfactant	Scientific Name	Species Name	Query Cover	%ID	Access Number
Surfactin	Surfactin biosynthesis thioesterase SrfAD	Bacillus halotolerans	100%	92.98%	WP_105955226.1
Surfactin	Surfactin non-ribosomal peptide synthetase SrfAC	Bacillus subtilis	100%	94.59%	WP_187954649.1
Surfactin	Surfactin non-ribosomal peptide synthetase SrfAB	Bacillus cabrialesii	100%	97.82%	WP_407062066.1
Surfactin	Surfactin non-ribosomal peptide synthetase SrfAA	Bacillus spizizenii	100%	95.09%	WP_041520855.1
Bacillomycin D hybrid PKS/NRPS BamA	Non-ribosomal peptide synthetase/type I polyketide synthase	Bacillus amyloliquefaciens	100%	90.98%	WP_424162584.1
Bacillomycin D non-ribosomal peptide synthetase BamB	Non-ribosomal peptide synthetase	Bacillus	100%	93.98%	WP_060962666.1
Bacillomycin D non-ribosomal peptide synthetase BamC	-	-	-	-	-
FenC	-	-	-	-	-

This suggests that the lipopeptide biosynthesis pathway in the bacteria is conserved and functionally active. The WGS analysis results of the biosurfactant biosynthesis genes of B. amyloliquefaciens EG6.1 are supported by various related studies, as shown in Table 6. The integration of WGS sequence alignment data and comparisons of various biosurfactant biosynthesis genes between species show that B. amyloliquefaciens EG6.1 has a relatively superior and complementary genetic profile.

Table 6
Comparison of various biosurfactant biosynthesis genes in B. amyloliquefacines EG6.1 with other Bacillus spp.

Bacteria	Genes of Biosynthesis Biosurfactant	Biosurfactant Product	Application	References
B. velezensis ES4.3	srfAD	Surfactin	Antifungal agent	Nafidiastri et al. (2021)
B. mojavensis EG6.4	srfAD	Surfactin	Antifungal agent	Salamun et al. (2022)
B. subtilis UFAB25	sfp	4’-phosphopantetheinyl transferase	Antifungal agent	Olmedo et al. (2022)
	srfAA	surfactin non-ribosomal peptide synthetase SrfAA
	fenB	non-ribosomal peptide synthase
B. subtilis UFAB29	sfp	4’-phosphopantetheinyl transferase
	srfAA	surfactin non-ribosomal peptide synthetase SrfAA
B. licheniformis UFAB18	LichAA	lichenysin non-ribosomal peptide synthetase LicA
B. licheniformis UFAB19	LichAA	non-ribosomal peptide synthetase
B. licheniformis UFAB37	LichAA	lichenysin non-ribosomal peptide synthetase LicA
B. subtilis MB418	srfAA, srfAC, srfAD	Surfactin	Degrading oil compounds	Yasmin et al. (2022)
	fenC	Fengycin
B. subtilis MB415	srfAA, srfAB, srfAC, srfAD	Surfactin
	fenB	Fengycin
B. subtilis BK7.1	srfAA, srfAD	Surfactin	Antifungal agent	Salamun et al. (2023)
B. amyloliquefaciens 50	FenD, srfA, bac	Fengycin, Surfactin, Bacilysin	Antifungal agent	Santos et al. (2023)
B. amyloliquefaciens 2784
B. amyloliquefaciens 2785
B. amyloliquefaciens 2788
B. amyloliquefaciens 2791
B. subtilis CCMA 2031	sfp, srfAA, FenB	Surfactin, Fengycin	Enzymatic and antimicrobial activity	Prósperi de Oliveira Paula et al. (2025)
B. subtilis CCMA 0658	srfAA, FenD, ituC, ituD	Surfactin, Fengycin, Iturin
B. velezensis ES7.3	srfAD	Surfactin	Antifungal agent	Nafidiastri et al. (2026)
	ituD	Iturin
B. amyloliquefaciens EG6.1	srfAA, srfAB, srfAC, srfAD	Surfactin	Antifungal agent	This study
	BamA, BamB, BamC	Bacillomycin

The combination of the surfactin–fengycin–bacillomycin gene cluster has the potential to provide synergistic effects in the context of microbe–pathogen interactions and microecosystem stability, although further confirmation through gene expression analysis and metabolite characterization is needed. Overall, these results position B. amyloliquefaciens EG6.1 as a relevant candidate for the development of biosurfactant-based biocontrol agents, with a strong genetic basis supported by comparisons between Bacillus species. This comparative approach adds value to the interpretation of WGS analysis and enhances the ecological significance of the results.

From this results, the combination of these biosurfactant gene clusters provides a molecular basis for using EG6.1 as a biological component in nature-based solutions aimed at improving soil health and suppressing plant pathogens through environmentally friendly mechanisms. The reliance on indigenous microbial functions over synthetic products is in line with the principles of sustainable ecosystem management and supports broader goals related to SDG points 2 (Zero Hunger), 12 (Responsible Consumption and Production), and 15 (Life on Land). However, further studies integrating gene expression analysis, metabolite profiling, and ecosystem-scale assessment are needed to transform this genomic potential into validated ecological engineering applications.

4. Conclusions

This study revealed a variety of Bacillus species isolated from the indigenous breeding site of Ae. aegypti larvae, one of which, B. amyloliquefaciens EG6.1, acts as an antifungal agent capable of inhibiting 78% of the growth of the plant pathogenic fungus F. oxysporum in both culture fractions and sonicated pellets. These results are supported by the presence of biosurfactant compounds in B. amyloliquefaciens EG6.1, as evidenced by hemolytic activity, the ability to reduce surface tension, and the formation of a stable emulsion layer. These findings are supported by a WGS-based molecular approach, which shows that B. amyloliquefaciens EG6.1 contains several conservative and relatively complete biosurfactant biosynthesis gene clusters, including surfactin, fengycin, and bacillomycin, with high percent identity. It is hoped that these findings can be developed in further studies on the isolation of pure biosurfactant compounds that can later be applied and produced on an industrial scale.

Acknowledgements

The authors are grateful to the Dean of the Faculty of Science and Technology and the Chancellor of Universitas Airlangga, Surabaya, Indonesia, who have provided laboratory facilities. This study is partly funded by internal funding, which is the ARF (PDU) 2025 of Universitas Airlangga research funding with contract No. 1744/UN3.LPPM/PT.01.03/2025. Appreciation also goes to all those who participated in this study.

Data Availability Statement

Research data is available in the body of the article.

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*

e-mail: [e-mail]

Editor:

Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  21 Aug 2026
• Date of issue
  2026

History

• Received
  25 Mar 2026
• Accepted
  07 July 2026