Durian (Durio zibethinus Murray) is a high-value tropical fruit crop widely cultivated in Southeast Asia, particularly in Thailand, Malaysia, Indonesia and Vietnam. Owing to its unique flavor, aroma and rich nutritional composition, including carbohydrates, fatty acids, vitamins, minerals and antioxidants, durian has become an important commodity in both domestic and export markets Le et al., (2024). In Vietnam, durian cultivation has expanded rapidly in recent years, reaching nearly 180,000 ha with an annual production of approximately 1.5 million tons in 2024 (MAE, 2025).
       
Despite its economic importance, durian production is constrained by several fungal diseases, among which anthracnose caused by Colletotrichum spp. is one of the most serious. The pathogen causes leaf anthracnose, leaf blight, fruit rot and postharvest decay, leading to significant yield and quality losses (Xu et al., 2025).
       
Chemical fungicides remain the main approach for controlling durian anthracnose; however, their excessive use has raised concerns regarding environmental contamination, pathogen resistance, chemical residues and risks to human health (Le et al., 2024). Consequently, sustainable and eco-friendly disease management strategies are increasingly needed.
       
Biological control using antagonistic microorganisms, particularly Bacillus spp., has emerged as a promising alternative due to their broad-spectrum antifungal activity, environmental adaptability and production of diverse bioactive compounds, including lipopeptides, hydrolytic enzymes, siderophores and volatile organic compounds (Chakraborty et al., 2022). Several studies have demonstrated the effectiveness of Bacillus spp. against Colletotrichum spp. in different crops (Heo et al., 2024; Li et al., 2022). In addition to disease suppression, many Bacillus strains exhibit plant growth-promoting traits such as IAA production, siderophore production, nitrogen fixation and mineral solubilization (Miljaković et al., 2022), making them promising candidates for sustainable agricultural applications.
       
In the present study, antagonistic bacterial strains were evaluated for their ability to suppress the growth of Colletotrichum sp. associated with durian anthracnose disease. The selected isolate was identified using morphological, biochemical and molecular approaches. Furthermore, its antifungal mechanisms, including the production of diffusible metabolites and VOCs, were investigated and its plant growth-promoting traits were evaluated. The findings generated in this study contribute scientific evidence supporting the development of eco-friendly biological control strategies for sustainable durian cultivation.

Microorganisms and culture conditions
 
The antagonistic bacterial isolate DM16 used in this study was isolated from rhizosphere soil collected from durian orchards located in Dak Lak province, Vietnam (12°44' 04.0"N, 108°15'30.0"E). The isolate was routinely maintained on LB agar medium and incubated at 30°C for 24 h prior to experimental use.
       
The phytopathogenic fungus Colletotrichum sp. was obtained from the Department of Microbial Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture. The fungal strain was maintained on PDA plates and incubated at 25°C in darkness for seven days and used as the target pathogen in all antifungal assays. All experiments were carried out at the Department of Microbial Biotechnology during 2024-2025.
 
In vitro antagonistic activity against Colletotrichum sp.
 
The antagonistic activity of isolate DM16 against Colletotrichum sp. was evaluated using the dual-culture assay according to Yoganathan and Tiwari (2021). A 5 mm agar disc was excised from the actively expanding edge of a seven day old fungal culture and placed at the center of a 90 mm PDA plate. A 24-h-old bacterial culture of isolate DM16 was streaked on both sides of the fungal plug in two parallel lines approximately 4 cm in length, maintaining a distance of 2 cm from the fungal inoculum.
       
Control plates consisted of PDA medium inoculated only with the fungal plug. All treatments were conducted with three independent replicates and maintained at 25°C for seven days. The radial growth of the fungal colony was measured and the percentage inhibition was determined according to the equation below:
 

 
Whereas,
R₁= Fungal radial growth in the control treatment,
R₂= Fungal growth measured in the presence of the bacterial isolate.
 
Phenotypic and genotypic identification of isolate DM16
Morphological, physiological and biochemical characterization
 
Preliminary identification of isolate DM16 was conducted based on morphological, physiological and biochemical characteristics according to the criteria described in bergey’s manual of systematic bacteriology (Claus and Berkeley, 1986). Biochemical characterization included catalase, methyl red, voges-proskauer and citrate utilization tests, which were performed following the manufacturer’s instructions (Merck KGaA, Darmstadt, Germany).
 
16S rRNA gene sequencing and phylogenetic analysis
 
For genetic characterization, DM16 was first grown on LB agar plates at 30°C for 24 hours. Genomic DNA was extracted according to the method described by Masoomi-Aladizgeh et al., (2019). Amplification of the 16S rRNA region was carried out using universal primer pairs 27F and 1492R following Bai et al., (2019).
       
The amplified PCR products were purified and sent to 1st base (Malaysia) for bidirectional sequencing. The obtained sequences were analyzed using BLASTn against sequences available in the NCBI database. Phylogenetic analysis was conducted using the neighbor-joining (NJ) method in MEGA 11 software.
 
Antifungal activity of crude diffusible metabolites
 
The antifungal activity of extracellular diffusible metabolites produced by isolate DM16 was evaluated according to the method described by Wang et al., (2022). A single bacterial colony was inoculated into 50 mL of LB broth and incubated at 30°C with shaking at 180 rpm for 24 h. Subsequently, 500 μL of the bacterial suspension was transferred into fresh LB broth and incubated under the same conditions for an additional 24 h.
       
The culture broth was centrifuged at 6,000 rpm for 25 min at 4°C and the supernatant was collected and filtered through a sterile 0.22-ìm membrane filter to obtain a cell-free filtrate. The filtrate was then mixed with molten PDA medium (45°C) at final concentrations of 1%, 5% and 10% (v/v) before being poured into petri dishes (20 mL per plate). PDA medium supplemented with sterile LB broth (1%, v/v) served as the control treatment.
       
A 5-mm mycelial plug of Colletotrichum sp. was placed at the center of each plate and incubated at 25°C for 4 days. Fungal colony diameters were measured and the inhibition percentage was calculated using the following equation:
 

 

 
Where,
Cd= The colony diameter in the control treatment (mm).
Td= The colony diameter in the treatment containing bacterial filtrate (mm).
       
All experiments were conducted in triplicate.
 
Antifungal activity of volatile organic compounds (VOCs)
 
The antifungal activity of VOCs produced by isolate DM16 against Colletotrichum sp. was evaluated using the double-plate assay described by Li et al., (2020). Two culture media, LB agar and Tryptone Soya Agar (TSA), were used to evaluate VOCs production by the bacterial isolate.
       
A single bacterial colony was inoculated into 50 mL of LB broth and incubated at 30°C with shaking at 160 rpm for 24 h. Subsequently, 100 μL of the bacterial suspension was spread evenly onto LB agar or TSA plates. A 5-mm mycelial plug of Colletotrichum sp. was placed at the center of PDA plates and pre-incubated at 25°C for 48 h prior to the VOCs assay.
       
The bacterial and fungal plates were paired face-to-face and sealed tightly with parafilm to establish a shared headspace without physical contact between the microorganisms. Control treatments consisted of PDA plates paired with uninoculated medium plates. All treatments were incubated at 25°C and fungal growth was measured every 24 h for 72 h. Each experiment was performed in triplicate.
       
The inhibition rate caused by VOCs was calculated using the following equation:
 

 

 
Where,
Dc= The average diameter of fungal mycelial growth in the control plate (mm),
Dt = The fungal colony diameter in the treatment plate (mm),
Dd= The diameter of the initial fungal plug (mm).
 
Determination of plant growth-promoting traits
 
The plant growth-promoting characteristics of isolate DM16, including IAA production, siderophore production, nitrogen fixation and zinc solubilization, were evaluated using standard microbiological methods.
       
The production of IAA was quantified using the Salkowski colorimetric assay, following the method described Gordon and Weber (1951). Briefly, isolate DM16 was inoculated into nutrient broth supplemented with L-tryptophan and incubated at 30°C with shaking at 200 rpm for 48 h. The culture broth was centrifuged at 3,000 rpm for 15 min and 1 mL of the supernatant was mixed with 2 mL of salkowski reagent. After incubation in darkness for 30 min at room temperature, absorbance was measured at 530 nm using a spectrophotometer.

Siderophore production was qualitatively evaluated using Chrome Azurol S agar medium according to the method of Schwyn and Neilands (1987). The appearance of an orange halo surrounding bacterial colonies was considered positive for siderophore production.
       
Nitrogen-fixing ability was assessed based on bacterial growth on nitrogen-free medium following the method described by Döbereiner  et al. (1995). Growth on nitrogen-free medium indicated the ability of the isolate to utilize atmospheric nitrogen.
       
Zinc solubilization activity was determined using mineral salt agar supplemented with insoluble zinc compounds according to the method described by Fasim et al., (2002). The formation of a clear halo zone around bacterial colonies indicated zinc-solubilizing ability.
       
All experiments were conducted in triplicate.

Antagonistic activity of isolate DM16 against Colletotrichum sp.
 
Isolate DM16 exhibited strong inhibitory activity against fungal growth, with a mycelial growth inhibition rate of 57.04 ±1.52% (Fig 1a,b). A distinct inhibition zone was observed between the bacterial colony and fungal mycelia, indicating effective antagonism by the bacterial isolate.


       
Microscopic examination further revealed substantial morphological alterations in fungal hyphae exposed to DM16. In the control treatment, Colletotrichum sp. produced dense, uniform and highly branched hyphae with normal morphology. In contrast, fungal hyphae in the presence of DM16 became swollen, thinner and irregular in appearance. Hyphal tip enlargement and reduced branching were also observed and some hyphae appeared difficult to distinguish clearly under microscopic observation. These abnormalities suggested that isolate DM16 interfered with normal fungal cell development and hyphal elongation.
 
Identification and characterization of isolate DM16
 
Colonies of isolate DM16 cultured on LB agar at 30°C for 72 h appeared creamy white, opaque and rough, with irregular margins and a raised convex center (Fig 2a). Microscopic observation showed that the bacterial cells were gram-positive, rod-shaped with rounded ends and approximately 2.97 µm in length and 0.64 µm in width (Fig 2b). Endospore formation was also observed.

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The physiological and biochemical properties of isolate DM16 are summarized in Table 1. The isolate tested positive for Gram staining, sporulation, catalase activity, voges-proskauer reaction, starch hydrolysis, gelatin hydrolysis, chitin hydrolysis and growth in 6% NaCl. Negative reactions were recorded for methyl red, citrate utilization and motility tests.


       
Molecular identification based on partial 16S rRNA gene sequencing further supported the taxonomic placement of isolate DM16. The amplified 16S rRNA gene fragment was approximately 960 bp in length. BLASTn analysis against sequences deposited in the NCBI GenBank database showed the highest similarity (97.78%) with Bacillus amyloliquefaciens. Phylogenetic analysis using the Neighbor-Joining method clustered isolate DM16 within the Bacillus clade closely related to B. amyloliquefaciens (Fig 2c). Based on the combined morphological, physiological, biochemical and molecular characteristics, isolate DM16 was identified as Bacillus sp.
 
Effect of crude diffusible metabolites on the mycelial growth of Colletotrichum sp.
 
The antifungal activity of extracellular diffusible metabolites produced by isolate DM16 was assessed using cell-free culture filtrates incorporated into PDA medium at different concentrations. After 4 days of incubation, no significant difference in fungal colony diameter was observed between the treatment containing 1% filtrate and the control treatment (Fig 1c). However, higher filtrate concentrations markedly suppressed fungal growth (Fig 1d).
       
At concentrations of 5% and 10% (v/v), the culture filtrates inhibited the mycelial growth of Colletotrichum sp. by 21.62±2.21% and 28.83±1.27%, respectively. The inhibition effect increased with increasing filtrate concentration, indicating a concentration-dependent antifungal activity. Fungal colonies grown on filtrate-amended media exhibited reduced radial growth compared with those on the control plates.
 
Effect of volatile organic compounds (VOCs) produced by isolate DM16
 
The volatile organic compounds (VOCs) produced by isolate DM16 significantly affected both fungal growth and colony morphology of Colletotrichum sp. The antifungal activity varied depending on the culture medium used for bacterial growth (Fig 3).

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After 24 h of incubation, VOCs produced by DM16 cultured on TSA medium inhibited fungal growth by 19.38± 2.19%, whereas VOCs produced on LB medium showed a lower inhibition rate of 5.98±1.21%. The inhibitory effects increased over time, reaching 24.48±6.55% and 14.10± 3.27% after 72 h for TSA and LB media, respectively.
       
In addition to reducing fungal growth, VOC exposure induced pronounced morphological alterations in fungal colonies. Compared with the control colonies, treated fungal colonies displayed three distinct concentric growth zones. The outermost region appeared thin and sparsely distributed, indicating reduced mycelial density. A second circular zone approximately 1 cm from the colony center exhibited elevated aerial mycelial growth, whereas the innermost region surrounding the inoculum plug appeared depressed or sunken. These morphological abnormalities indicated that VOCs produced by DM16 interfered with normal fungal colony development.
 
Plant growth-promoting traits of isolate DM16
 
In addition to its antifungal activity, isolate DM16 exhibited several plant growth-promoting characteristics. The isolate demonstrated the ability to produce indole-3-acetic acid (IAA), produce siderophores, fix atmospheric nitrogen and solubilize zinc, indicating its multifunctional potential as a beneficial rhizobacterium (Fig 4).

​
       
Qualitative assays confirmed siderophore production and zinc-solubilizing activity through the formation of clear halo zones surrounding bacterial colonies. Furthermore, the growth of DM16 on nitrogen-free medium confirmed its nitrogen-fixing capability. Quantitative analysis revealed that isolate DM16 produced 5.5 µg/mL of IAA under the tested conditions.
       
The combination of IAA production, siderophore synthesis, nitrogen fixation and zinc solubilization suggests that DM16 may enhance nutrient availability and stimulate plant growth in the rhizosphere, thereby contributing to improved plant health and productivity (Timofeeva et al., 2023).
       
The present study demonstrated that Bacillus sp. DM16 exhibits strong antagonistic activity against Colletotrichum sp. In the dual culture assay, DM16 inhibited fungal mycelial growth by 57.04±1.52% and induced distinct hyphal abnormalities. These morphological changes suggest that DM16 likely suppresses fungal growth through the production of antifungal metabolites that compromise cell wall integrity and disrupt hyphal development.
       
Comparable antifungal activities have been reported for several Bacillus species against a wide range of phytopathogenic fungi. For instance, Bacillus amyloliquefaciens FX2 inhibited the growth of Colletotrichum gloeosporioides by 53.70±2.19% (Li et al., 2022), which is comparable to the inhibitory effect observed for strain DM16 in the present study. Likewise, two Bacillus strains were reported to suppress Fusarium oxysporum f. sp. cubense Tropical Race 4, achieving inhibition rates of 79.6% and 81.3%, respectively (Fan et al., 2021). In addition, Bacillus subtilis var. amyloliquefaciens inhibited the growth of Phytophthora infestans by 35.56% (Keerthana et al., 2018), whereas Bacillus velezensis reduced the growth of Verticillium dahliae, the causal agent of Verticillium wilt, with an inhibition rate of 70.94% (Wang et al., 2022). Collectively, these studies demonstrate the broad-spectrum antifungal activity of Bacillus spp. and support the potential of strain DM16 as a promising biocontrol agent for the management of fungal plant diseases.
       
Morphological, physiological, biochemical and molecular analyses indicated that isolate DM16 belongs to the genus Bacillus. However, species-level identification remained inconclusive because the partial 16S rRNA gene sequence (~960 bp) showed only 97.78% similarity with Bacillus amyloliquefaciens. The original amplified fragment was longer, but low-quality and noisy sequences at both ends were trimmed before analysis, resulting in a shorter high-quality sequence. According to current bacterial taxonomic criteria, 16S rRNA gene similarities below 98.65-99% are generally insufficient for reliable species identification (Stackebrandt and Ebers, 2006; Kim et al., 2014). In addition, closely related species within the Bacillus subtilis complex often share highly conserved 16S rRNA sequences, making discrimination based solely on partial 16S rRNA analysis difficult (Fan et al., 2017). Therefore, DM16 was conservatively designated as Bacillus sp. and additional analyses such as multilocus sequence analysis (MLSA) or whole-genome sequencing would be required for precise species classification.
       
The inhibitory effects observed in this study are likely associated with the production of extracellular antifungal metabolites. The culture filtrate assay revealed a concentration-dependent inhibition of fungal growth, suggesting that diffusible metabolites secreted by DM16 contributed significantly to antifungal activity. Numerous Bacillus strains are known to produce bioactive secondary metabolites, including cyclic lipopeptides, hydrolytic enzymes and antimicrobial peptides capable of suppressing fungal pathogens (Chakraborty et al., 2022).
       
In addition to diffusible metabolites, VOCs produced by DM16 significantly inhibited fungal growth and altered colony morphology. Interestingly, stronger inhibition was observed when DM16 was cultured on TSA medium compared with LB medium, suggesting that nutrient composition influenced VOC biosynthesis. Similar observations were reported by Huang et al., (2018), who found that VOCs produced by Bacillus mycoides cultured on TSA medium exhibited greater inhibition against Rhizoctonia solani and Pythium aphanidermatum than VOCs produced on LB medium. Likewise, Gotor-Vila  et al. (2017) demonstrated that TSA medium enhanced VOC-mediated antifungal activity of B. amyloliquefaciens CPA-8 against Monilinia laxa, Botrytis cinerea and Monilinia fructicola. The morphological abnormalities observed in fungal colonies exposed to VOCs further confirmed the inhibitory effects of volatile metabolites. Treated colonies exhibited sparse peripheral growth, abnormal aerial mycelia and depressed central regions, indicating disruption of normal fungal growth and differentiation. Previous studies have linked such abnormalities to membrane damage, oxidative stress and respiratory disruption induced by bacterial VOCs (Sunkad et al., 2023).
       
Beyond its antifungal activity, isolate DM16 exhibited several plant growth-promoting traits, including IAA production, siderophore production, nitrogen fixation and zinc solubilization. These multifunctional characteristics are important attributes of beneficial rhizobacteria for sustainable agricultural systems. Bacterial IAA production is known to stimulate root elongation, nutrient uptake and overall plant development (Miljakovic et al., 2022). The IAA concentration produced by DM16 (5.5 µg/mL) was within the range commonly reported for plant growth-promoting Bacillus strains. In addition, siderophore production may enhance iron acquisition and suppress pathogens through competition for micronutrients, whereas nitrogen fixation and zinc solubilization contribute to improved nutrient availability in the rhizosphere (Timofeeva et al., 2023).
       
Overall, the findings of the present study indicate that Bacillus sp. DM16 possesses both strong antifungal activity and multiple plant growth-promoting traits, highlighting its potential as a multifunctional microbial inoculant for sustainable disease management and crop production. Further studies are required to identify the specific antifungal metabolites and VOCs produced by DM16 and to evaluate its efficacy under greenhouse and field conditions.

In this study, the endophytic bacterial isolate DM16 was identified as Bacillus sp. based on morphological, biochemical and 16S rRNA gene sequence analyses. Strain DM16 showed strong antagonistic activity against Colletotrichum sp. via diffusible and volatile metabolites, causing marked hyphal abnormalities. Beyond its antifungal effects, DM16 also exhibited plant growth promoting traits such as IAA and siderophore production, nitrogen fixation and zinc solubilization. These multifunctional properties highlight its potential as a sustainable biocontrol and biofertilizer agent, warranting further evaluation in greenhouse and field conditions.

The authors gratefully acknowledge the family of Mr. A, residing in (tên xã A) commune, Dak Lak Province, Vietnam, for kindly granting permission to collect samples from their durian orchard for this study. The authors also sincerely thank the Department of Microbial Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture for providing access to laboratory facilities, equipment and technical resources that supported the experimental work.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.