Biological Control and Growth-Promoting Activities of Burkholderia vietnamiensis FBCC-B8049 Isolated from Freshwater
Article information
Abstract
Colletotrichum and Fusarium are globally important plant-pathogenic fungi that cause serious diseases in chili pepper and other crops, leading to substantial yield losses due to their broad host range, environmental persistence, and the limited effectiveness of chemical control, thereby highlighting the need for sustainable alternatives such as biological control. We investigate the biological control and plant growth-promoting potential of Burkholderia vietnamiensis FBCC-B8049, isolated from freshwater environments. The strain exhibited significant antifungal activity against Colletotrichum gloeosporioides, Colletotrichum acutatum, and Fusarium oxysporum in dual-culture assays, with stronger effects against Colletotrichum species. The inhibition was likely due to direct antagonism and volatile organic compounds (VOCs) produced by FBCC-B8049, which were particularly effective against Colletotrichum species. Additionally, FBCC-B8049 demonstrated plant growth-promoting activities including siderophore production for iron acqusition, phosphate solubilization for enhanced nutrient availability, and indole-3-acetic acid synthesis to promote root development. These combined activities enhance nutrient availability and promote seed germination and seedling growth in chili pepper. Ex vivo assays further revealed the effectiveness of FBCC-B8049 in suppressing anthracnose disease on pepper fruits through both direct application and bacterial VOCs emission. Phylogenetic analysis of 16S rRNA and recA gene sequences positioned FBCC-B8049 closely to Burkholderia vietnamiensis, a known plant growth-promoting rhizobacterium. These findings highlight FBCC-B8049 as a promising candidate for sustainable agricultural applications.
Plant diseases caused by pathogenic fungi, including species from the Colletotrichum and Fusarium genera, pose significant threats to crop yields, particularly in high-value crops like green peppers (Sun et al., 2020). Specifically, Colletotrichum is one of the most widespread and economically important genera of plant-pathogenic fungi, causing anthracnose, foliar blights, and postharvest rots in nearly all major crops worldwide. Severe anthracnose in chili pepper has resulted in significant yield losses in multiple countries, including up to 80% in Vietnam and around 10% in Korea, primarily due to both pre- and postharvest infections, resulting in annual economic losses of several million dollars in major producing countries. (Byung, 2007; Don et al., 2007; Saxena et al., 2016). Also, Fusarium oxysporum Schlecht. is a soil-borne pathogen that causes vascular wilt in various crops, with symptoms such as vascular browning and wilting. As a species complex comprising multiple formae speciales (f. sp.), it infects over 100 plant species and leads to significant yield losses worldwide. Due to its long-lived chlamydospores and host specificity, chemical control is challenging, highlighting the need for alternative strategies (Dean et al., 2012).
Traditional chemical control, while effective, raise concerns about environmental damage, resistance development in pathogens, and human health risks (Aktar et al., 2009). Moreover, chemical fungicides are often non-specific and may fail to provide long-term disease suppression, especially against latent and soil-borne fungal pathogens. These issues have led to growing interest in safer and more sustainable disease management strategies. As a result, research has increasingly focused on biological control methods that offer more sustainable and environmentally friendly alternatives, including the use of beneficial bacteria. In response to growing global demand for sustainable agricultural practices, biological control agents (BCAs) have gained considerable attention as alternatives to chemical pesticides. In particular, antagonistic microbes offer several advantages over chemical agents, including target specificity, reduced ecological impact, and potential for long-term suppression through colonization and competition (Lugtenberg and Kamilova, 2009). Among the diverse microorganisms being explored for their biological control potential, species of the genus Burkholderia have attracted attention due to their dual ability to suppress plant pathogens and promote plant growth (Eberl and Vandamme, 2016). Burkholderia species are commonly isolated from various environments, including soil, water, and plant rhizospheres, where they interact with plants through various mechanisms, such as nutrient solubilization, production of growth-promoting substances, and secretion of antimicrobial compounds (Eberl and Vandamme, 2016). Moreover, some species occur freely in freshwater environments as planktonic bacteria, where they may play ecological roles in nutrient cycling such as phosphorous solubilization and nitrogen fixation (Caballero-Mellado et al., 2007; Estrada-De Los Santos et al., 2001; Martínez-Aguilar et al. 2008; Peix et al., 2001).
Among these, the Burkholderia cepacia complex (Bcc) represents a closely related group of species that present both challenges and opportunities. While Bcc bacteria are recognized as opportunistic pathogens, particularly in cystic fibrosis patients, they have also been widely studied for their biotechnological applications, including biological control, plant growth-promoting rhizobacteria (PGPR), and bioremediation (Depoorter et al., 2016). One such species, Burkholderia vietnamiensis, is known for its antifungal and plant growth-promoting (PGP) activities (Liu et al, 2022). Recent studies have demonstrated its potential to enhance plant growth and suppress fungal pathogens through mechanisms such as phosphate solubilization, siderophore production, indole-3-acetic acid (IAA), and the emission of volatile organic compounds (VOCs) which are small, volatile molecules capable of diffusing through the atmosphere to inhibit pathogens (Bitas et al., 2013; Effmert et al., 2012; Mpinda et al., 2024; Schmidt et al., 2015). Despite these promising activities, whether Burkholderia strains derived from freshwater environments can exhibit biological control and PGPR functions remains largely underexplored. Given the ecological uniqueness of freshwater niches and their underutilized microbial diversity, further investigation is warranted.
This study aims to evaluate the biological control and PGP potential of the freshwater-derived strain Burkholderia vietnamiensis FBCC-B8049 against major plant pathogenic fungi such as Colletotrichum gloeosporioides, C. acutatum, and Fusarium oxysporum. FBCC-B8049 was previously isolated from a freshwater environment and showed promising antifungal and PGP activities. Notably, it demonstrated strong VOC-mediated inhibition against Colletotrichum spp., suggesting the possibility of a distinct mechanism compared to previously reported strains.
In this study, we assessed both direct antifungal activity and indirect inhibition, as well as PGP activities including siderophore, phosphate solubilization and IAA production. Furthermore, its effects on seed germination, seedling growth, and fruit disease suppression in chili pepper were tested to explore its potential for agricultural application.
Materials and Methods
Bacterial strains and culture conditions
The bacterial strains used in the study were obtained from the Freshwater Bioresources Culture Collection (FBCC) in the Nakdonggang National Institute of Biological Resources (NNIBR). A total of 30 strains of Burkholderia species, all isolated from freshwater environments, were employed in the antagonistic assay. The bacteria were cultured in Reasoner’s 2A (R2A) and tryptic soy bean broth (TSB) at 28°C for 3 days. The bacterial suspension was adjusted to 100-fold dilution (1.0 × 106 colony-forming units [CFU]/mL) and 250-fold dilution (4.0 × 105 CFU/mL) by serial dilution with 1 mM magnesium sulfate (MgSO4) in sterile distilled water.
To monitor bacterial growth, optical density at 600 nm (OD600) was measured over time using a microplate reader (EPOCH2, BioTek Instruments, Inc., Winooski, VT, USA), and CFUs were determined by serial dilution and plating on tryptic soy agar plates at designated time points (0–120 h). A cell-free culture filtrate (CF) was prepared by centrifuging the bacterial culture at 5,000 rpm for 10 min, followed by filtration of the supernatant through a 0.22 μm hydrophilic filter (Minisart NML Syringe Filters, Cellulose Acetate, Cat. no. S6534-FMOSK, Goettingen, Germany).
Plant pathogenic fungi strains and culture conditions
The plant pathogenic fungi Colletotrichum gloeosporioides FBCC-F56, C. acutatum KACC42403, and Fusarium oxysporum KACC42795 were used as the test pathogen. The fungi were cultured on potato dextrose agar (PDA) plates at 25°C for 7 days until sporulation. To harvest spores, 5 mL of sterile distilled water was added to each PDA plate, and the surface of cultured fungi was gently scraped. Spores were harvested by flooding the PDA plates with 5 mL of sterile distilled water and gently scraping the surface. The spore suspension was filtered through a double layer of Miracloth (475855, Millipore, Darmstadt, Germany) to remove mycelial debris and was then adjusted to a concentration of 1 × 106 spores/mL using a hemocytometer.
In vitro dual-culture antagonistic assay
The in vitro antagonistic activity of the bacterial strains against C. gloeosporioides, C. acutatum, and F. oxysporum was assessed using a dual-culture assay. PDA plates were divided into two equal halves. A 3 μL suspension of fungal spores was spotted 2.25 cm from the center of the plate. A single colony of the Burkholderia strain was streaked in a straight line on the center of the plate. The plates were incubated at 25°C for 7 days, and the mycelial growth was evaluated by measuring the radial growth of the fungal colony towards the bacterial streak. The inhibition of mycelial growth was calculated using the following formula:
The control area of mycelial growth is the mycelial growth of plant pathogens in the control plate without bacterial inoculation, whereas the treatment area is the mycelial growth in the presence of the bacterial strains. All experiments were performed in triplicate, and results were expressed as the mean ± standard deviation.
In vitro volatile antagonistic assay
The volatile activity of FBCC-B8049 against plant pathogens was evaluated using the double Petri dish method (Li et al., 2015). A ‘top’ plate containing the bacterial culture was replaced by a ‘bottom’ plate containing PDA inoculated with 10 μL of spore suspension of plant pathogens. The Petri dishes were positioned face-to-face with FBCC-B8049 cultured on one side and plant pathogens inoculated on the other. The plates were then inoculated at 25°C for 7 days. The total area of mycelial growth in Petri dish (6 cm diameter) was 21.5 cm2. All experiments were performed in triplicate, and results were expressed as the mean ± standard deviation.
Siderophore production and phosphate solubilization activity
FBCC-B8049 was assayed for siderophore production using the blue agar Chrome Azurol S (CAS) assay (Ahmad et al., 2008; Louden et al., 2011). Siderophore detection was performed on CAS based medium. CAS agar plates were prepared and inoculated with the culture of FBCC-B8049 and incubated at 28°C for 48 to 72 h. The formation of a yellow-orange halo around the bacterial growth was considered as positive for siderophore production.
FBCC-B8049 was screened for phosphate solubilization activity using the Pikovskaya’s (PVK) agar medium (Gupta et al., 1994). The PVK medium was modified by adding 0.1% bromophenol blue to enhance the visibility and clarity of the solubilization halo (Nautiyal, 1999).
Glycosidase, amylase, cellulase, protease assay, and nitrogenase assay
Enzymatic activities of FBCC-B8049 were evaluated as described below. All media were prepared according to the manufacturer’s instructions. Glycosidase activity was assessed using Esculin Iron Agar, which contained esculin (1 g/L), ferric ammonium citrate (0.5 g/L), and agar (15 g/L), with the final pH adjusted to 7.1 ± 0.2 at room temperature (RT). Esculin Iron Agar powder (82981, Sigma-Aldrich, St. Louis, MO, USA) was used for this assay. Protease, amylase, and cellulase activities were assayed using AZCL-casein, AZCL-amylose, and AZCL-HE-cellulose as substrates (Megazyme, Bray, Ireland) and xanthan gum (0.3 g/mL) in R2A medium agar, respectively. The culture of FBCC-B8049 was dropped into an 8 mm filter paper disc on each specific substrate agar.
Jensen’s medium was used to evaluate the nitrogen-fixing ability of FBCC-B8049 and was modified as needed (M710, HiMedia Laboratories, Mumbai, India) (Sherpa et al., 2021). The medium contained the following key components: dipotassium phosphate (1.0 g/L), magnesium sulphate (0.5 g/L), sodium chloride (0.5 g/L), ferrous sulphate (0.1 g/L), Sodium molybdate (0.005 g/L), calcium carbonate (2.0 g/L), and sucrose (20.0 g/L). No nitrogen source was included. The formation of a halo zone around the colony was considered as a positive for nitrogenase activity.
IAA production
The ability of FBCC-B8049 to utilize L-tryptophan for IAA production, was investigated using a colorimetric quantification method (Gang et al., 2019). The culture of FBCC-B8049 was grown for 72 h in TSB and M9 minimal medium broth supplemented with or without tryptophan (0.15 g/100 mL) at 28°C. Uninoculated medium was included as negative controls to account for background absorbance.
A 1.5 mL aliquot of the fully-grown cultures was transferred to an Eppendorf tube and centrifuged at 13,500 rpm for 5 min. The supernatant (1 mL) was then mixed with Salkowski’s reagent (49 mL of 35% perchloric acid, 1 mL of 0.5 M ferric chloride solution) and inoculated at RT for 30 min in dark condition. The development of a pink color indicated IAA production. Quantitative analysis of IAA was performed using a 96-well plate, and the optical density was measured at 536 nm using the EPOCH2 microplate reader (BioTek Instruments, Inc.). The standard curve for IAA concentration was established within the range of 0 to 100 μg/mL (Supplementary Fig. 1). The results represent the mean ± standard deviation of three independent experiments.
Seed germination
Seed germination and root growth were evaluated using the between-paper method (Gupta et al., 2019). A total of 8 seeds were placed on moistened sterile paper towel. And then bacterial dilutions at 100-fold and 250-fold dilution and cell-free CF were inoculated with 10 μL volumes. After drying under sterile airflow conditions on clean bench, the papers were rolled and inoculated at 25°C for 10 days. Seedling root lengths were measured after the 10-day incubation period. The experiment was conducted in duplicate.
In vivo PGP effect of FBCC-B8049 on chili pepper seedling
Chili pepper seeds were germinated in Petri dish for one week. Germinated seeds were transplanted into individual pots. The pepper seedlings were subject to bacterial dilutions (100-f and 250-f) and cell-free CF every week, and the seedlings were subsequently grown for 45 days. The greenhouse temperature ranged from 10°C to 15°C at night and from 25°C to 35°C during the daytime throughout the period of the experiments.
Ex vivo assay to evaluate BCA of FBCC-B8049 against chili pepper anthracnose
Chili pepper fruits (Capsicum annuum L., Nokwang) were sterilized by dipping them into 1% sodium hypochlorite for 30 s, 70% ethanol for 30 s, and followed by rinsing with sterile distilled water. The anthracnose pathogen, C. acutatum, was cultured on PDA plate at 25°C for 7 days. A spore suspension of C. acutatum was prepared as described above. Each pepper fruit was wounded with a sterile needle and inoculated with the spore suspension. The fruits were treated with 10 μL of bacterial suspension at 100-fold and 250-fold dilutions and CF at each inoculation site. After drying under sterile aseptic conditions, 5 μL of the spore suspension at each site was inoculated. Additionally, a bacterial culture was spread on a 6-cm Petri dish. This dish and inoculated fruits were placed in the same chamber for VOCs treatment. The VOCs treatment was conducted in a humidified chamber at 25°C with >70% relative humidity for 7 days. Disease symptoms were assessed at 7 days post-inoculation (dpi). The lesion area was measured using a digital caliper. Disease severity was assessed by calculating the disease severity index (DSI) based on a 0–5 rating scale. The rating criteria were defined as follows: 0 = no lesion; 1 = lesion area ≤ 1.0 cm2; 2 = 1.0–2.0 cm2; 3 = 2.0–3.0 cm2; 4 = 3.0–4.0 cm2; and 5 = 4.0–5.0 cm2. The maximum score is 5. DSI was calculated using the following formula:
Each treatment group included three pepper fruits per replicate, and the experiment was independently performed in triplicate (n = 9).
Molecular identification and phylogenetic analysis
For phylogenetic analysis, DNA was extracted using the Wizard Genomic DNA Purification Kit (A1120, Promega, Madison, WI, USA) according to the manufacturer’s instructions. To determine the taxonomic position of strain FBCC-B8049 within the Burkholderia genus, 16S rRNA and recA gene sequences were analyzed. The 16S rRNA gene was amplified using the universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The PCR amplification was performed by Macrogen (Seoul, Korea). To identify the strain, the obtained sequences were analyzed using BLAST searches against the National Center for Biotechnology Information database (USA). Based on 16S rRNA sequencing, FBCC-B8049 was initially identified as a member of the Burkholderia genus. To further resolve its species-level identification, the housekeeping gene recA was sequenced using the primers BUR1 (5′-GATCGARAAGCAGTTCGGCAA-3′) and BUR2 (5′-TTGTCCTTGCCCTGRCCGAT-3′) (Mahenthiralingam et al., 2000). For phylogenetic analysis, reference sequences of type strains from the Burkholderia group were collected to construct a phylogenetic tree and assess the taxonomic status of FBCC-B8049. Sequence alignment was performed using the ClustalW algorithm and tree editing were performed using the graphical tools within the Mega-X software (version 12.0.08). Phylogenetic trees for both 16S rRNA and recA genes were constructed using the maximum likelihood method. The robustness of the trees was evaluated with 1,000 bootstrap replicates to assess confidence in the branching patterns. Bordetella pertussis was used as an outgroup to root both phylogenetic trees.
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA). Student’s t-test was used for two-group comparisons, and one-way analysis of variance (ANOVA) followed by Tukey’s honest significant difference test was used for multiple-group comparisons. For ex vivo evaluation of biological control efficacy, non-parametric tests were applied due to the non-normal distribution of data. A Kruskal-Wallis test was used to determine significant differences among treatments, and Dunn’s test was performed for post-hoc multiple comparisons. Statistical significance was set up at P < 0.05.
Results
Screening of freshwater-derived Burkholderia strains against plant pathogens
The antifungal activity of 30 strains of freshwater-derived Burkholderia sp. was evaluated to plant pathogenic fungi such as C. gloeosporioides, C. acutatum, and F. oxysporum using a dual-culture assay (Fig. 1A–C). Among these, FBCC-B8049 showed the highest inhibitory effect on mycelial growth (Fig. 1D). Specifically, the mycelial growth area of C. gloeosporioides, C. acutatum, and F. oxysporum were significantly reduced from 25.19 ± 0.89 cm2 to 6.46 ± 0.62 cm2, 13.97 ± 0.17 cm2 to 4.99 ± 0.23 cm2, and 23.45 ± 0.41 to 11.31 ± 0.67 cm2, respectively (P < 0.05). Statistical analysis was performed only between the fungal control and FBCC-B8049 to assess the significance of its inhibitory effects.
Evaluation of antifungal activity of freshwater-derived Burkholderia strains against plant pathogenic fungi. Antifungal activity of 30 Burkholderia strains against (A) Colletotrichum gloeosporioides (Cg), (B) C. acutatum (Ca), and (C) Fusarium oxysporum (Fo) was assessed through in vitro dual-culture assays. The black bar represents the mycelial growth of the untreated fungal control. The dark gray, yellow, and white bars show the mycelial growth of Cg, Ca, and Fo, respectively, when exposed to the Burkholderia strains. The red bar indicates FBCC-B8049 with the strongest antifungal activity. (D) The inhibitory effects of FBCC-B8049 against Cg, Ca, and Fo. The left side shows fungal mycelial growth in the absence of FBCC-B8049, while the right side displays fungal growth in its presence. Statistical analysis was performed only between the fungal control and FBCC-B8049 using a Student’s t-test (**P < 0.01, ***P < 0.001).
Assessment of antifungal activity by bacterial VOCs
The antifungal activity of VOCs produced by FBCC-B8049 was evaluated. In the absence of FBCC-B8049, all pathogens exhibited normal mycelial growth. In the presence of FBCC-B8049, the mycelial growth of C. gloeosporioides and C. acutatum was notably inhibited (Fig. 2A). Specifically, the mycelial growth of C. gloeosporioides and C. acutatum decreased from 21.5 ± 0.00 cm2 to 3.87 ± 3.37 cm2 and from 11.3 ± 0.28 cm2 to 3.86 ± 1.74 cm2, respectively (P < 0.05) (Fig. 2B). In contrast, the mycelial growth of F. oxysprorum was not affected by VOC exposure.
In vitro volatile assay of FBCC-B8049 through volatile organic compounds (VOCs) against pathogenic fungi using a sandwich indirect dual culture. (A) Mycelial growth in the absence or presence of VOC exposure with FBCC-B8049. (B) Quantification of mycelial growth under exposure to bacterial VOCs. The Black bars represent mycelial growth in the control group, whereas gray bars indicate mycelial growth exposed to bacterial VOCs. Cg, Colletotrichum gloeosporioides; Ca, C. acutatum; Fo, Fusarium oxysporum. Statistical analysis was performed using a Student’s t-test (**P < 0.01; ***P < 0.001; ns, not significant).
Plant growth promotion and extracellular enzyme activities
The plant growth promotion and extracellular enzyme activities of FBCC-B8049 were evaluated (Fig. 3). FBCC-B8049 showed siderophore production on CAS agar plates, phosphate solubilization on PVK agar plates, and nitrogenase activity on Jensen’s agar plates. Glycosidase activity assay was also detected, indicating its ability to degrade sugar substrates. In contrast, protease, amylase, and cellulase activities were not detected, suggesting limited involvement in protein degradation, starch breakdown, and cellulose decomposition, respectively.
In vitro assay of plant growth promotion and enzymatic activities of FBCC-B8049. Each picture demonstrates plant growth promotion and enzymatic activities of FBCC-B8049, including siderophore production, phosphate solubilization, nitrogenase activity, and glycosidase, protease, amylase, and cellulase activities. Control plates, without bacterial inoculation, serve as negative controls for the response of the media. Positive results are visualized by clear zones, color changes, or halo formations, reflecting the activity of FBCC-B8049.
IAA production
The production of IAA by FBCC-B8049 was evaluated under nutrient-rich and nutrient-depleted conditions, with and without the addition of L-tryptophan (Fig. 4). In nutrient-rich conditions, the addition of L-tryptophan significantly enhanced IAA production from 11.53 ± 1.81 μg/mL to 20.05 ± 3.05 μg/mL. On the other hand, in nutrient-depleted conditions, it was observed that the absence of L-tryptophan resulted in 0.07 ± 0.04 μg/mL of IAA production, whereas the presence significantly increased production to 5.73 ± 0.15 μg/mL.
Indole-3-acetic acid (IAA) production by FBCC-B8049 under different culture conditions. FBCC-B8049 was grown in tryptic soy bean broth (TSB) (gray bars) and M9 minimal medium (black bars) with or without tryptophan (Trp) supplementation. Uninoculated medium under the same conditions was used as negative controls to account for background absorbance. Statistical analysis was determined using Student’s t-test between the same culture conditions (*P < 0.05, **p < 0.01). Data are presented as the mean ± standard deviation of three independent experiments.
Plant growth promotion by FBCC-B8049 in chili pepper
Root length during seed germination was measured to assess the growth-promoting activity of FBCC-B8049 (Fig. 5). In the control group, the root length was measured at 45.24 ± 6.98 mm, while in the treated groups, the measurements were 81.71 ± 6.01 mm for 100-fold dilution, 73.38 ± 10.39 mm for 250-fold dilution, and 15.97 ± 21.49 mm for cell-free CF. Root length in both the 100-fold and 250-fold dilution group were significantly increased compared to the control (P < 0.05). No significant difference was observed between the 100-fold and 250-fold dilution groups. In contrast, the CF group exhibited an inhibitory effect on seed germination.
Growth-promotion effect of FBCC-B8049 on seed germination in chili pepper. (A) Chili pepper seeds were treated with bacterial suspensions of FBCC-B8049 at 100-fold (1.0 × 106 colony-forming units [CFU]/mL) and 250-fold (4.0 × 105 CFU/mL) dilutions, and a cell-free culture filtrate (CF). Untreated seeds were used as the control (Ctrl). The growth promotion effect of FBCC-B8049 on seed germination was assessed after 10 days of incubation using the roll towel and in-between paper methods. (B) Root lengths of germinated seed were measured. The experiment was performed in three independent replicates (n = 24). Data are presented as the mean ± standard deviation (SD). Different letters above the bars indicate significant differences between groups as determined by Tukey’s honest significant difference test (P < 0.05).
Seedling height and stem thickness were measured to assess the growth-promoting activity of FBCC-B8049 (Fig. 6). In the control group, the seedling height was 290 ± 11.11 mm. In the treated groups, seedling heights were 365 ± 6.38 mm for the 100-fold dilution and 405 ± 20.46 mm for the 250-fold dilution. Seedlings treated with the 250-fold dilution showed a significantly higher compared to the control (P < 0.05). Stem thickness was evaluated to reflect the overall vigor of the seedlings. The stem thickness was 0.82 ± 0.36 cm for the control, 4.11 ± 0.06 cm for the 100-fold dilution, and 4.30 ± 0.26 cm for the 250-fold dilution. Seedling treated with the 250-fold dilution showed a significantly thicker stem compared to those in the control group (P < 0.05). In contrast, no seedlings survived in the CF-treated group.
Growth-promoting effects of FBCC-B8049 on chili pepper seedlings. (A) Representative images of chili pepper seedlings treated with FBCC-B8049 bacterial suspensions at 100-fold (1.0 × 106 colony-forming units [CFU]/mL) and 250-fold (4.0 × 105 CFU/mL) dilutions, and a cell-free culture filtrate (CF). Untreated seedlings were used as the control (control). (B) Seedling height and (C) stem diameter were measured after treatment. Bars with different letters indicate significant differences based on Tukey’s honest significant difference test (P < 0.05).
Ex vivo efficacy of BCA on anthracnose in pepper fruit
The biological control efficacy of FBCC-B8049 against anthracnose disease in chili pepper fruits was evaluated using ex vivo assays (Fig. 7). The DSI (%) of the control group was measured at 60.00 ± 0.71%. In contrast, the DSI values of the treated groups were 17.78 ± 1.67% for the 100-fold dilution, 20.00 ± 1.00% for the 250-fold dilution, and 13.33 ± 0.71% for the VOC treatment. According to Dunn’s multiple comparison test (P < 0.05), all FBCC-B8049-treated groups showed significantly lower DSI values than the control. However, no significant differences were observed among the 100-fold dilution, 250-fold dilution, and VOC-treated groups.
Ex vivo assay of the biological control effect of FBCC-B8049 on chili pepper. (A) Evaluation of chili pepper fruits after treatment with various concentrations of FBCC-B8049 and its volatile organic compounds (VOCs). The mean disease severity index (%) is shown below the corresponding images. (B) Disease symptoms on pepper fruits under different treatment conditions were measured. Gray bars represent the mean of disease severity index (%), with error bars indicating standard deviations (SD). A Kruskal-Wallis test indicated a statistically significant difference among the treatment groups (H = 17.11, P = 0.0007). Significant differences were determined using Dunn’s post-hoc test, and different letters on the bars indicate statistically significant differences (P < 0.05). The experiment was performed in three independent replicates with three fruits per treatment (n = 9). Control, untreated control; 100-fold dilution, 1.0 × 106 colony-forming units [CFU]/mL; 250-fold dilution, 4.0 × 105 CFU/mL; VOCs, VOCs emitted from bacterial spread plate.
Analysis of phylogenetic tree
Phylogenetic analyses of FBCC-B8049 were conducted based on 16S rRNA and recA gene sequences to determine its taxonomic position within the Burkholderia genus (Fig. 8). The 16S rRNA phylogenetic tree indicates that FBCC-B8049 is most closely related to B. vietnamensis TVV75 (Fig. 8). The branch connecting FBCC-B8049 to this species is supported by bootstrap values of 71. Similarly, the recA gene phylogenetic tree also placed FBCC-B8049 in close proximity to B. vietnamensis TVV75 (Fig. 8B). These results suggest that FBCC-B8049 is likely a member of the B. vietnamensis. Based on the results of these analyses, we propose renaming Burkholderia sp. FBCC-B8049 as Burkholderia vietnamiensis FBCC-B8049 to reflect its close affiliation with the B. vietnamensis species. The 16S rRNA and recA gene sequences of B. vietnamiensis FBCC-B8049 were deposited into the GenBank database under the accession number PV076870 and PV102960, respectively.
Phylogenetic trees based on 16S rRNA and recA gene sequences of FBCC-B8049. Phylogenetic trees were constructed using the maximum likelihood method with bootstrap values calculated from 1,000 replicates. Bootstrap values are indicated at the nodes, and scale bars represent the number of substitutions per site. Bordetella pertussis was used as the outgroup for both analyses. FBCC-B8049 is placed in proximity to Burkholderia vietnamiensis TVV75 in both the 16S rRNA (A) and recA (B).
Discussion
The exploration of beneficial microorganisms from freshwater environments highlights a large untapped resource for sustainable agriculture applications. Freshwater bacteria contribute significantly to biogeochemical cycles and ecosystem stability (Newton et al., 2011), yet their potential for biological control and PGP activities remains underexplored. Given that freshwater is widely utilized in agricultural irrigation systems, the discovery of microbes with biological control and PGP capabilities from these environments represents a highly practical and environmentally sustainable strategy (Lakhiar et al., 2024). In this study, we screened 30 Burkholderia strains isolated from freshwater ecosystems and identified FBCC-B8049 as a promising candidate for both biological control and plant growth-promotion activities.
The dual-culture assays revealed that FBCC-B8049 exhibited the strongest antifungal activity against plant pathogenic fungi such as C. gloeosporioides, C. acutatum, and F. oxysporum (Fig. 1). These findings suggest that the potent antifungal properties of FBCC-B8049 were associated with antagonistic effects, including nutrient competition and secretion of diffusible antifungal compounds (Compant et al., 2005). These results indicate that FBCC-B8049 has significant potential as a BCA for plant diseases caused by these pathogens.
In addition to direct antagonism, FBCC-B8049 exhibited significant antifungal activity through the emission of VOCs (Fig. 2). The VOC-mediated inhibition notably suppressed the mycelial growth of C. gloeosporioides and C. acutatum, whereas F. oxysporum showed no significant response to VOC exposure. Interestingly, FBCC-B8049 showed stronger antifungal activity against Colletotrichum spp. than Fusarium oxysporum. This difference may be explained by the structural characteristics of their cell walls. Colletotrichum spp. may exhibit relatively higher permeability to volatile compounds due to a more open or less compact wall structure. In contrast, Fusarium spp. possesses thicker, chitin-rich cell walls with compact β-glucan layers, which may act as a stronger physical barrier to volatile compounds. These differences in cell wall architecture could influence the sensitivity of each pathogen to bacterial VOCs.
FBCC-B8049 effectively suppressed anthracnose disease caused by C. acutatum in chili pepper fruits (Fig. 7). Both direct treatments (100-fold and 250-fold dilutions) and VOC exposure significantly reduced DSI value compared to the control (P < 0.05). Notably, there was no significant difference between 100-fold and 250-fold dilution, suggesting that even a lower bacterial concentration effectively inhibits mycelial growth. Given its comparable efficacy and potential for reducing production costs, the 250-fold dilution could be considered a practical concentration for future field application. These results indicate the dual biological control mechanisms: direct inhibition of pathogen growth through bacterial metabolites and indirect suppression via VOCs. This study suggests a non-contact strategy that minimizes the risks associated with live bacterial application while maintaining antifungal efficacy. Previous studies have suggested that bacterial volatiles such as acetoin and 2,3-butanediol can trigger systemic resistance in plants (Rudrappa et al., 2010; Ryu et al., 2004) and inhibit fungal spore germination (Chen et al., 2022). In addition, Burkholderia and related genera are known to produce antifungal VOCs such as α-pinene and limonene, which possess strong antimicrobial activity (Chee et al., 2009; Kalemba and Kunicka, 2003; Lis-Balchin et al., 1999; Tenorio-Salgado et al., 2013). Unlike conventional BCAs, FBCC-B8049 offers a unique advantage as a freshwater-derived strain capable of suppressing fungal pathogens through both direct inhibition and the emission of antifungal VOCs. These distinctive properties support its potential as a safe, non-contact BCA and underscore its novelty within sustainable disease management strategies. Although the specific VOCs produced by FBCC-B8049 remain unidentified, these results imply that VOC production constitutes an important indirect mechanism for fungal growth suppression. Future work is needed to characterize the antifungal VOCs emitted by FBCC-B8049 using gas chromatography–mass spectrometry analysis.
The PGP activities of FBCC-B8049 were shown through multiple functional assays that are crucial for enhancing nutrient availability in the rhizosphere (Figs. 3 and 4). The production of siderophores by FBCC-B8049 can facilitate iron acquisition by plants, an essential micronutrient often limited in bioavailability due to its insoluble forms in soil environments. By chelating iron and making it more accessible to plants, siderophore-producing bacteria not only improve plant growth but may also contribute to suppressing the growth of phytopathogenic fungi through iron competition (Han et al., 2015). In addition, the ability to solubilize phosphate compounds plays a crucial role in improving plant nutrient uptake and contributing to crop production (Kucey, 1988). However, Phosphate compounds are precipitated into sparingly soluble forms such as CaHPO4, Ca3(PO4)2, FePO4, and AlPO4, which can be inaccessible to plants (Omar, 1997). FBCC-B8049 exhibits phosphate-solubilizing activity, which further enhances nutrient accessibility in the rhizosphere. Together, these PGP activities underline the potential of FBCC-B8049 as a powerful plant growth-promotion. The absence of protease, cellulase, and amylase activities in FBCC-B8049 suggests that it lacks enzymatic traits required for organic matter decomposition (Fig. 3). This limitation may restrict its application in environments where such enzymatic functions are critical for plant-microbe interactions or nutrient cycling. Although FBCC-B8049 shows strong PGP potential, its application should be carefully adjusted to environments where its strengths, such as nutrient solubilization and phytohormone production, are most advantageous.
IAA production was significantly increased in the presence of L-tryptophan, suggesting that FBCC-B8049 synthesizes IAA through a tryptophan-dependent pathway (Fig. 4). In nutrient-rich media such as TSB, which contains various amino acids, FBCC-B8049 was found to produce IAA in the absence of exogenous tryptophan (Fig. 4). FBCC-B8049 produced detectable levels of IAA even without tryptophan, suggesting utilization of endogenous amino acids as precursors. Supplementation with tryptophan resulted in a 73.77% increase in IAA production, confirming its role as a key biosynthetic precursor. Given that soil environments are typically heterogeneous and nutrient-depleted, and that plant root exudates influence microbial behavior (Ma et al., 2022), we further investigated IAA production under minimal nutrient conditions. In M9 minimal medium, FBCC-B8049 produced only trace amounts of IAA without tryptophan, suggesting the absence of a tryptophan-independent pathway. However, supplementation with tryptophan led to a notable increase in IAA levels, reinforcing the dependence on a tryptophan-mediated pathway. Such pathways are common among PGP bacteria and play crucial roles in modulating root system architecture, promoting root elongation, and enhancing lateral root formation (Duca et al., 2014; Spaepen et al., 2007). The ability of FBCC-B8049 to produce IAA under nutrient-depleted conditions implies that it could positively influence plant growth by stimulating root development, thereby improving nutrient and water uptake efficiency nature conditions. A similar mechanism has been reported in Burkholderia phytofirmans PsJN, which synthesizes IAA via tryptophan-dependent biosynthesis and improves plant resilience under various environmental stresses (Naveed et al., 2015). Although the precise biosynthetic pathway utilized by FBCC-B8049 remains to be elucidated, its responsiveness to tryptophan supplementation highlights its potential as a PGP inoculant under various agricultural conditions.
The PGP potential of FBCC-B8049 was demonstrated through seed germination and seedling growth in chili pepper (Figs. 5 and 6). Treatment with diluted bacterial suspensions (100-fold and 250-fold dilutions) significantly enhanced root elongation during seed germination compared to the untreated control. Root length is an important indicator of seedling vigor, as longer roots facilitate better water and nutrient acquisition in early developmental stages (Wang et al., 2016). Furthermore, FBCC-B8049 promoted seedling height and stem thickness under in vivo conditions compared to the control group. These observations suggest that FBCC-B8049 can effectively stimulate early plant development, likely through a combination of improved nutrient availability (e.g., siderophore and phosphate solubilization activities) and phytohormone production (e.g., IAA). In contrast, seedlings treated with a high concentration of cell-free CF exhibited reduced viability, indicating that excessive accumulation of bacterial metabolites may have a phytotoxic effect. Therefore, the application concentration of FBCC-B8049 must be carefully optimized to maximize PGP effects while minimizing potential toxicity.
Phylogenetic analysis based on 16S rRNA and recA gene sequences identified FBCC-B8049 as Burkholderia vietnamiensis. This identification provides valuable taxonomic context, situating FBCC-B8049 within a well-known species with established PGPR and antifungal properties (Liu et al., 2022; Mpinda et al., 2024). The close phylogenetic proximity of FBCC-B8049 to B. vietnamiensis suggests that it may share similar beneficial properties, including nutrient solubilization, phytohormone production, and antagonism against plant pathogens. Importantly, FBCC-B8049 was isolated from a freshwater environment, distinguishing it from most previously reported B. vietnamiensis strains, which are primarily soil-derived. This aquatic origin may confer unique ecological adaptations that enhance survival and functionality in water-based agricultural systems or humid postharvest environments, broadening its potential applications.
While FBCC-B8049 exhibited promising antifungal and PGP activities, the biosafety of Burkholderia species remains an important consideration. Members of the Bcc are known to occupy diverse ecological niches, ranging from beneficial rhizosphere colonizers and endophytes to opportunistic human pathogens, particularly in immunocompromised individuals (Coenye and Vandamme, 2003; Compant et al., 2008). FBCC-B8049, isolated from a freshwater environment, demonstrated multiple beneficial functions. Importantly, it lacked enzymatic activities such as protease and cellulase, which are often associated with host tissue degradation and pathogenicity. Whole-genome sequencing will be essential to fully assess the biosafety profile of FBCC-B8049. This step will enable the identification of virulence-related gene clusters, antibiotic resistance markers, and other mobile genetic elements that may influence its environmental or clinical risks. In addition, phytotoxicity and human pathogenicity are distinct biosafety concerns. In this study, phytotoxicity was observed only at high concentrations of the cell-free CF, likely due to the accumulation of microbial metabolites or medium-derived compounds, while no effects were detected at practical application levels. Nonetheless, further evaluation is needed to ensure the safe use of both the bacterial cells and their metabolites.
Despite the promising results, this study has several limitations. First, the experiments were conducted under controlled laboratory conditions, which may not fully reflect the complexity of agricultural environments. Factors such as temperature, soil composition, and plant-microbe interactions could influence the performance of FBCC-B8049. Second, interactions with native microbial communities may affect FBCC-B8049’s colonization and activity. Studies involving microbial community profiling before and after application will be necessary to evaluate its ecological compatibility and guide formulation strategies. Third, proper formulation not only protects bacterial viability during storage and application but also ensures compatibility with diverse field environments and agricultural practices. Formulation significantly influences the efficacy and shelf life of BCAs (Teixidó et al., 2022). Key factors such as nutrient composition and optimal growth conditions affect bacterial physiology and metabolite production (Paau, 1988). Growth curve analysis of FBCC-B8049 offers valuable insights into its physiological traits and provides a basis for future formulation development (Supplementary Fig. 2). Therefore, further studies on concentration titration and formulation optimization are necessary to ensure reproducibility and scalability of its biological control and PGPR activities under practical agricultural conditions.
These limitations emphasize the need for comprehensive field trials and ecological studies to optimize application parameters. By understanding and mitigating the effects of environmental variability and microbial interactions, we can better harness the beneficial attributes of FBCC-B8049 for sustainable agricultural practices.
Taken together, these findings highlight the potential of FBCC-B8049 as a dual-function agent for both disease suppression and plant growth promotion. Its ecological origin from freshwater, combined with its diverse bioactive properties, underscores its value as a promising microbial resource for sustainable agriculture. To enable its practical application, future research should focus on VOC profiling, genomic and transcriptomic analysis, field validation, formulation optimization, and comprehensive biosafety evaluation.
Notes
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
This study was supported by Korea Environment Industry & Technology Institute (KEITI) through the Project to make multi-ministerial national biological research resources more advanced, funded by Korea Ministry of Environment (MOE) (2021003420002) and by Nakdonggang National Institute of Biological Resources (NNIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NNIBR20253106)
Electronic Supplementary Material
Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).