Ralstonia solanacearum species complex (RSSC), a soilborne plant pathogen, causes bacterial wilt in a variety of crops, including potato, pepper, eggplant, and tomato, and is transmitted through soil and water (Ahmed et al., 2022; Silva et al., 2024). The RSSC is classified into four phylotypes based on geographic origin and genetic characteristics, and is composed of three distinct species: R. pseudosolanacearum (phylotypes I and III), R. solanacearum (phylotype II), and R. syzygii (phylotype IV) (Inoue et al., 2023; Sedighian et al., 2020). The pathogen initiates infection by attaching to the root surface and entering the root tissues, and rapidly colonizing the xylem (Dey et al., 2023; Inoue et al., 2023; Yadav et al., 2024). It induces disease by delivering effectors via a type III secretion system that suppress host immunity and facilitate systemic infection (Soongnern et al., 2022; Wang et al., 2023). This pathogen exhibits high genetic diversity, environmental persistence, and a broad host range, thereby constituting a major threat to global food security (Sharmila et al., 2025; Vailleau and Genin, 2023). Tomato bacterial wilt is one of the most devastating bacterial diseases worldwide, with yield losses ranging from 15% to 95% (Manda et al., 2020; Wu et al., 2023). Notably, R. pseudosolanacearum can persist in soil and plant debris for long periods, and its persistence is exacerbated under continuous cropping systems. Continuous cropping is an increasingly common practice in intensive tomato production, which further elevates the risk and severity of disease outbreaks (Sun et al., 2023). Substantial agricultural losses have been reported worldwide due to bacterial wilt, highlighting the need for sustainable control strategies (Guo et al., 2022; Seleim et al., 2023; Soliman et al., 2024).
Conventional management strategies, such as chemical pesticides, plant-derived antimicrobials, antibiotic treatments, and the development of resistant cultivars, have shown limited success in controlling bacterial wilt in the field (Wang et al., 2023). Moreover, these strategies face limitations due to environmental concerns, reduced effectiveness under field conditions, high costs, inconsistent performance, and the rapid evolution of pathogen resistance (Ababa, 2024; Wang et al., 2023; Wu et al., 2023). Therefore, more sustainable and eco-friendly approaches are needed. Biological control using beneficial microorganisms has emerged as a promising alternative (Li et al., 2024; Shah et al., 2025). Among various mechanisms, including biofilm formation and induced systemic resistance (ISR) in plant (Ramírez et al., 2022), the use of microbial volatile organic compounds (VOCs) represents a particularly attractive approach. Due to their low molecular weight, VOCs are able to diffuse through the soil or atmosphere, and various VOCs have been reported to inhibit pathogen growth, and in some cases, induce resistance in plants (Ahmed et al., 2022; Almeida et al., 2022; Poulaki and Tjamos, 2023; Wu et al., 2023).
Some microbial VOCs have been reported to exhibit antibacterial activity against Ralstonia species and to reduce bacterial wilt; Bacillus velezensis K0T24 has antagonistic activity against R. pseudosolanacearum and suppressed mulberry bacterial wilt (Jiao et al., 2024). VOCs emitted by endophytic bacteria isolated from potato, B. safensis and B. aerius, significantly reduced the incidence of bacterial wilt and exhibited morphological abnormalities of R. solanacearum (Yousefvand et al., 2023). In addition, the genus Pseudomonas also plays a significant role in the biocontrol of various pathogens. P. fluorescens VSMKU3054 exhibited inhibitory effects against bacterial wilt by inducing the plant immune systems in tomato plants (Suresh et al., 2022). An acid-tolerant strain, P. protegens CLP-6, produced VOCs that exhibited antagonistic activities against R. solanacearum in tobacco plants (Zhao et al., 2023), and the antagonistic effect was strongly enhanced under acidic conditions (pH 5.5). Strain CLP-6’s VOCs such as 2-ethylhexanol and 1,3-benzothiazole which were produced more abundantly at low pH, completely inhibited R. solanacearum. These findings underscore the potential of VOCs producing by Pseudomonas as effective antibacterial agents, due to their ability to suppress a broad range of pathogens and adapt to diverse soil environments, including acidic conditions (Win et al., 2023).
Microbial VOCs have been shown to affect virulence of plant pathogens by modulating the expression of pathogenicity-related genes as well as by inhibiting their growth (Tahir et al., 2023). For example, VOCs from Bacillus amyloliquefaciens significantly reduce the expression of the transcriptional regulator phcA in R. solanacearum, a critical gene controlling virulence factors such as extracellular polysaccharides and bacterial motility (Raza et al., 2016; Almeida et al., 2022). VOCs also influence the expression of motility-related genes, such as motA and motC, in Xanthomonas oryzae, thereby limiting the pathogen’s ability to infect host plants (Xie et al., 2018). Moreover, VOCs downregulate genes associated with antioxidant activity and central metabolism, disrupting the pathogen’s stress responses and overall growth. Importantly, VOCs have also been found to induce DNA damage in plant pathogens, further impairing their virulence and infectivity (Almeida et al., 2022; Raio, 2024). These findings suggest that VOCs exert their biocontrol effects by modulating key genes involved in pathogenicity, motility, and oxidative stress responses, offering a promising strategy for pathogen management.
In this study, we selected VOC-producing bacterial strains, P. koreensis KF32 and P. fitomaticsae KF45, which inhibit the growth of R. pseudosolanacearum and suppress bacterial wilt in tomato plants. We also elucidated the effect of VOCs produced by these strains on the transcriptome of R. pseudosolanacearum. The bacterial VOCs were identified using gas chromatography-mass spectrometry (GC-MS) analysis, and each VOC’s activity was evaluated through an I-plate and plant assay. Our findings contribute to the development of sustainable biocontrol strategies targeting bacterial wilt and highlight the promising role of VOC-producing Pseudomonas strains in plant disease management.
Materials and Methods
Bacterial strains
A total of 45 strains were isolated from the soil in Gangneung, Gangwon-do, Republic of Korea in 2024 (37°47′41″N, 128°53′42″E). Soil samples (1 g) were suspended in 9 mL of sterile 10 mM MgSO4 solution. The soil suspension was diluted serially, and then smeared on tryptic soy agar (TSA; Difco, Sparks, MD, USA) amended with cycloheximide (50 μg/mL). Three days after incubation at 28°C, the morphological distinct colony was pick up and cultured in TSA. The single colony was transferred into 3 mL of tryptic soy broth (TSB; Difco) and cultured. After overnight growth, cultures were mixed with sterile glycerol to 20% (v/v) and stored at −80°C before use.
Selection of antibacterial VOC-producing bacteria in I-plate assay and bacterial identification
The 45 strains were preliminarily screened by I-plate assay for selection of VOCs-producing bacteria having inhibitory activity of R. pseudosolanacearum. In the I-plate assay, one compartment with TSA for cultivation of the 45 strains, while the opposite compartment with tetrazolium chloride (TZC) agar for incubation of R. pseudosolanacearum. For bacterial treatments, 45 bacteria and the pathogen, were cultured in 5 mL of TSB, and TZC for 48 h at 28°C respectively, and centrifuged at 3,000 rpm for 10 min. The collected bacterial pellet was suspended in 10 mM MgSO4 solution and adjusted OD600 = 0.2 by a spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan). The 45-cell suspension were smeared in one compartment with TSA and then 1 day later, cell suspension of R. pseudosolanacearum was smeared on TZC agar at 28°C. Two days later, the pathogen’s growth with red and white color was measured. When only two strains among 45 strains, were cultured in a compartment in I-plates, the pathogen’s growth was not observed. Therefore, the two strains were selected and used in further assay. For quantitatively measuring antibacterial activity of VOC-producing bacteria, serially diluted R. pseudosolanacearum was smeared in the I-plate system and then the colony-forming units were measured 48 hours after incubation.
For the identification of anti-Ralstonia pseudosolanacearum VOC-producing KF32 and KF45, 16S rRNA was amplified by using universal primers. The 16S rRNA genes sequence was searched for homology with the type strain using the EzTaxon database (http://www.ezbiocloud.net/) and aligned using MAFFT (multiple alignments using fast Fourier transform) version 7 with type strain (Katho et al., 2019). Phylogenetic analysis was performed based on the 16S rRNA gene sequences and conducted by the Neighbor-joining method with the Maximum Composite Likelihood method in MEGA12 (Kumar et al., 2024). The percentage of replicate trees that grouped the associated taxa together in the bootstrap test based on 1,000 replicates is shown next to the branches.
Disease suppression of VOCs produced by KF32 and KF45 and VOC-producing bacteria in tomato plants
For assessment of disease suppression by VOCs produced by KF32 and KF45 in tomato plants, we performed a VOC cage assay using an assembled magenta box (bottom) and an insect-breeding box (top). The VOC cage assay was a modified version of the pot-jar assembly method (Sheikh et al., 2023). All tomato experiments (Solanum lycopersicum, ‘Superdotaerang’, Koregon, Anseong, Korea) were cultivated with a 16/8 h (light/dark) photoperiod at 25 ± 5°C and 50 ± 5% relative humidity in a plant growth chamber. Tomato seeds were germinated in plastic pots containing commercial soil mixture (Bunong, Seoul, Korea), and three weeks later, three seedlings were transferred into each insect-breeding box containing potting mixture. Bacterial strains KF32 and KF45 were incubated for 48 h on TSA at 28°C, and the agar plates were placed in the magenta boxes to allow tomato plants to be exposed to VOCs produced by KF32 and KF45 for 7 days. R. pseudosolanacearum was incubated on TZC medium for 48 h at 28°C, and was suspended in 10 mM MgSO4 solution and adjusted OD600 = 0.2 using a spectrophotometer. One week after treatment, 4 mL of the R. pseudosolanacearum suspension was inoculated into soil in each VOC cage. Assessment of disease incidence by R. pseudosolanacearum was performed 5 days after inoculation, and the experiment was conducted twice with four replications, each containing three plants per replication.
For the evaluation of biocontrol activity by VOC-producing bacterial strains in tomato plants, bacterial cell suspensions were prepared according to the description above. These bacterial strains were cultured on 10 mL of TSB for 48 h with shaking at 150 rpm, 28°C (OD600 = 0.2). The suspensions were drenched onto three-week-old tomato plants. Benzothiadiazole (BTH, 0.1 mM) was drenched as a positive control. One week after treatment, tomato plants were inoculated with R. pseudosolanacearum (OD600 = 0.2, 1 mL/g of the potting mixture). Disease incidence was assessed 5 days after inoculation, and the experiment was performed three replications of three plants each.
Tomato root colonization of R. pseudosolanacearum exposure to VOCs of KF32 and KF45
Tomato seeds were sterilized using 70% ethanol for 1 min followed by 1% sodium hypochlorite for 15 min and then washed five times with sterile distilled water. The sterilized seeds were placed on sterile filter paper moistened with sterile distilled water in a tissue culture plate and incubated in the dark for 5 days. After germination, seedlings with similar root lengths were transferred to MS medium (4.4 g/L Murashige and Skoog, 30 g/L sucrose, pH 5.8) in square dish. R. pseudosolanacearum suspension was dropped on tomato roots (20 μL, OD600 = 0.2). For VOCs exposure, strains KF32 and KF45 were pre-cultured on TSA medium for 24 h at 28°C in square dish. Each TSA medium was placed face-to-face with the MS medium (tomato and R. pseudosolanacearum dropping). The plates were sealed with parafilm to prevent VOCs leakage and incubated for 3 days at 28°C. After the exposure period, tomato roots were cut into 2 cm segments and homogenized in sterile distilled water. Serial dilutions of the homogenate were smeared on TZC medium and the colony-forming units were measured 48 h after incubation.
Transcriptome analysis
Total RNA was isolated from R. pseudosolanacearum cells exposed to VOCs produced by strains KF32 and KF45 using the easy-spin Total RNA Extraction Kit (iNtRON, Seongnam, Korea). Ribosomal RNA was removed using the NEBNext rRNA Depletion Kit (NEB, Ipswich, MA, USA), and libraries were constructed using the Illumina TruSeq Standed mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA). RNA-seq was performed on the Illumina NovaSeq X platform by Macrogen Incorporated. The reference genome sequence and gene annotation data were downloaded from the NCBI RefSeq database. Transcript abundance was quantified with HTSeq (v0.10.0) to obtain raw counts, FPKM (fragments per kilobase of transcript per million mapped reads), and TPM (transcripts per million) values. Differential expression analysis was conducted using DESeq2 (v1.38.3), with RLE normalization and Wald test for significance (|fold change| ≥ 2, P < 0.05). Principal component analysis and multidimensional scaling plots were generated to assess sample clustering, and significant genes were subjected to hierarchical clustering. Gene ontology (GO) enrichment was performed using topGO, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted using an in-house KEGG Mapper script.
For validation of RNA-seq results, total RNA was isolated again using the same RNA extraction kit, and RNA quality and purity were analyzed using a NanoDrop spectrophotometer (ND-1000, Thermo Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using TOPscript RT DryMIX (Enzynomics, Daejeon, Korea). Quantitative real-time PCR was performed in a 20 μL of reaction mixture containing 1 μL of each primer (10 pmol), and 10 μL of 2× SYBR reaction buffer using TOPreal SYBR Green qPCR High-ROX PreMix (Enzynomics). The amplification was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and the PCR conditions were 95°C for 10 min, followed by 40 cycles of 15 s at 95°C, 20 s at 58°C, and 18 s at 72°C. The primer sequences are listed in Supplementary Table 1. The relative expression of target genes was calculated according to the 2−ΔΔCT method (Livak and Schmittgen, 2001).
Identification of VOCs produced by strains KF32 and KF45 and the single compound’s disease suppressive activity
To identify the VOCs produced by KF32 and KF45, we performed GC-MS analysis through NICEM (National Instrumentation Center for Environmental Management, Korea). GC-MS analysis was performed using a Thermo Scientific TRACE 1300 gas chromatograph with an ISQ single quadrupole mass spectrometer (Thermo Scientific). The temperature program was initially set at 50°C for 5 min, then increased to 320°C at a rate of 7°C/min and held for 5 min, with a total run time of 65.08 min. The injection port was maintained at 280°C in split mode (split ratio 10:1). We screened the effects of six identified VOCs on R. pseudosolanacearum using an I-plate assay. One compartment was treated with VOCs at concentrations 0, 10, and 100 μg/mL, while the opposite compartment was filled with TZC agar medium and inoculum of R. pseudosolanacearum. Two days later, the growth of the pathogen was evaluated based on the appearance of red and white colonies.
For assessment of disease suppression by VOCs in tomato plants, we performed the VOC cage assay. Three VOCs that showed effective antibacterial activity against R. pseudosolanacearum (2-decanone, 2,4,6-trimethyl pyridine, and 2-methyl-3-isopropylpyrazine) were treated at concentrations of 1 and 10 μg/mL to filter paper placed in 6 cm Petri dish, which was then located in a magenta box. Tomato plants were exposed to VOCs for one week and inoculated with R. pseudosolanacearum. Assessment of disease incidence by R. pseudosolanacearum was performed 5 days after inoculation, and this experiment was repeated three times with eight replications, each containing three plants.
Statistical analysis
All experiments were conducted in at least three independent biological replicates from two experiments. Data are presented as the mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism version 8.4.2 (GraphPad Software, San Diego, CA, USA). Differences between treatment groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test, or using a two-tailed Student’s t-test. A P-value < 0.05 was considered statistically significant.
Results
The VOCs produced by two Pseudomonas strains, KF32 and KF45, have antibacterial activity against Ralstonia pseudosolanacearum
We selected VOCs-producing strains KF32 and KF45, which significantly (p < 0.0001) reduced the population of R. pseudosolanacearum by 99.1% [log(CFU) = 1.3 ± 1.3] and 99.3% [log(CFU) = 1.0 ± 1.0], respectively, compared to control [log(CFU) = 148.0 ± 22.3] in I-plate assay (Fig. 1A and B). For identification of the selected bacterial strains, we amplified the 16S rRNA sequences and phylogenetic analysis was performed using type strains of various Pseudomonas species (Fig. 1C, Supplementary Table 2). Strain KF32 showed 99.72% similarity to Pseudomonas koreensis (Ps 9-14T, AF468452), and the sequence of strain KF45 was identical to that of the type strain Pseudomonas fitomaticsae (FIT81T, MZ773500).
Disease suppressive activity by KF32 and KF45 on tomato bacterial wilt caused by R. pseudosolanacearum
To determine whether VOCs produced by these strains could similarly decrease disease incidence, we conducted a plant assay system utilizing bacterial VOCs (Sheikh et al., 2023). VOCs produced by strains KF32 and KF45 significantly reduced the incidence of tomato bacterial wilt to 62.5% and 68.75%, respectively, following a 7-day exposure to its volatile (P = 0.019 and P = 0.007), compared to control (Fig. 2A and C). In other hands, strains KF32 and KF45, when the bacterial cell suspensions were drenched, were examined for biocontrol activity against bacterial wilt caused by R. pseudosolanacearum in tomato plants. When the drench application, both KF32 and KF45 significantly (P < 0.05) reduced disease incidence by 36% and 55%, respectively, in tomato plants compared to the control (disease incidence = 91.7%) (Fig. 2B).
Effect of VOCs produced by KF32 and KF45 on root colonization of R. pseudosolanacearum in tomato
We investigated that VOCs produced by KF32 and KF45 affected colonization of R. pseudosolanacearum in tomato root (Fig. 3A). The root colonization of R. pseudosolanacearum was significantly (P < 0.0001) reduced in the KF32 exposed root [log(CFU/cm) = 5.7 ± 0.1] compared to the control [log(CFU/cm) = 7.1 ± 0.2], whereas the KF45 exposed root showed a similar bacterial population to the control [log(CFU/cm) = 6.9 ± 0.2] (Fig. 3B). Colony morphology was observed differently on TZC medium, when the pathogens was isolated from KF32 exposed root. Colonies from the control and KF45 exposed root exhibited irregular shapes, moist colony, a pink center, and a large white edge. In contrast, the R. pseudosolanacearum, which was isolated from root exposed to KF32 was round, dry and red in the middle of colony and a narrow or no white edge (Fig. 3C).
Transcriptome analysis of R. pseudosolanacearum by VOCs of KF32 and KF45
To investigate the responses of R. pseudosolanacearum to VOCs treatment produced by strains KF32 and KF45, RNA sequencing analysis was performed on VOC-exposed and non-exposed bacterial cells. A total of 1,124 differentially expressed genes (DEGs) were identified, including 280 upregulated and 466 downregulated genes in the VOC produced by KF32-treated group compared to the control, and 441 upregulated and 589 downregulated genes in the VOC produced by KF45-treated group compared to the control (Fig. 4A). To further characterize their functional roles, KEGG pathway enrichment analysis was performed. A total of 721 DEGs were mapped to 149 KEGG pathways. However, only 26 pathways were significantly enriched (P < 0.05). Compared with the control treatment, R. pseudosolanacearum exposed to VOCs produced by strains KF32 and KF45 exhibited a marked downregulation of genes associated with cell motility, including bacterial chemotaxis and flagellar assembly (Table 1, Fig. 4B). Genes involved in the two-component system and quorum sensing were also downregulated than upregulated. Notably, VOCs exposure led to the downregulation of genes related to xenobiotic degradation, including benzoate degradation, glutathione metabolism, and fatty acid degradation (Table 1).
To validate the RNA-seq data, eight genes related to cell motility and xenobiotic degradation were randomly selected for quantitative PCR (qPCR) analysis. The genes flgA, flgK, fliF, and fliI were involved in flagellar assembly. The other genes, 60501288, 60500500, 60502177, and 60503159, were possibly related to xenobiotic degradation gene; however, these gene functions have not been clearly identified and are described only as putative or probable. The results obtained by qPCR analysis using gene-specific primers confined similarity to the RNA-seq results (Fig. 5, Supplementary Fig. 1).
Identification of VOCs produced by strains KF32 and KF45, the single compound’s antibacterial activity against R. pseudosolanacearum and disease suppressive activity on tomato bacterial wilt
We analyzed the VOCs produced by strains KF32 and KF45 using SPME-GC-MS to identify what compounds present (Supplementary Fig. 2). Among these, 2,4,6-trimethyl-pyridine was produced by both strains KF32 and KF45; 2-decanone was produced by strain KF32, while other VOCs were produced by strain KF45 (Table 2). These VOCs were evaluated for their antibacterial activity against R. pseudosolanacearum (Fig. 6). Three VOCs, 2-undecanone, acetophenone, and thymol, exhibited no antibacterial activity against R. pseudosolanacearum at any tested concentration. 2,4,6-Trimethyl-pyridine exhibited that reduced the colony-forming units of R. pseudosolanacearum at 10 μg/mL, but however the reduction was not statistically significant (P = 0.0574). In contrast, 2-methyl-3-isopropylpyrazine decreased the growth of R. pseudosolanacearum at 10 μg/mL (P = 0.039); however, its effect at 100 μg/mL was nearly identical to that at 0 μg/mL. 2-Decanone significantly inhibited the growth of R. pseudosolanacearum, reducing it by more than 50% at both 10 μg/mL and 100 μg/mL (P < 0.01).
Three single compounds, 2-decanone, 2-methyl-3-isopropylpyrazine, and 2,4,6-trimethyl-pyridine, were evaluated to determine whether they suppress the incidence of bacterial wilt in tomato (Fig. 7). The results showed that 2-methyl-3-isopropylpyrazine and 2,4,6-trimethyl-pyridine exhibited no significant disease suppressive activity of tomato bacterial wilt. Only 2-decanone at 10 μg/mL reduced the incidence of tomato bacterial wilt to 37.1% compared to control (P = 0.0061).
Discussion
We investigated that the VOCs produced by P. koreensis KF32 and P. fitomaticsae KF45 exhibit antibacterial activity against R. pseudosolanacearum, both in vitro and in planta. In I-plate assays, VOCs produced by these strains drastically reduced the bacterial population of R. pseudosolanacearum, while tomato plants exposed to the VOCs significantly decreased disease incidence. Notably, transcriptomic analysis revealed that the VOCs commonly led to the downregulation of genes involved in cell motility and xenobiotic degradation, indicating that VOCs impair multiple essential functions of R. pseudosolanacearum. Among the identified VOCs, 2-methyl-3-isopropylpyrazine, 2,4,6-trimethylpyridine, and 2-decanone showed inhibitory activity in vitro, however, 2-decanone exhibiting significantly disease reduction in tomato plant compared to control. These results suggest that VOCs-producing bacteria, strains KF32 and KF45 should serve as promising biocontrol agents by suppressing pathogen growth and disrupting key biological pathways involved in virulence and survival.
Many Pseudomonas strains are well known for their roles in promoting plant growth and exhibiting antimicrobial activity (Dimkić et al., 2022; Kipgen et al., 2021). The Pseudomonas fluorescens complex produced a variety of VOCs that inhibited pathogen growth, motility, and biofilm formation, and also ISR in plants (Raio, 2024). In this study, the VOCs emitted by Pseudomonas strains KF32 and KF45 exhibited clear antagonistic effects against R. pseudosolanacearum as well as the biocontrol activity by drench treatment of the two strains. Root colonization of R. pseudosolanacearum was only significantly reduced in the KF32-exposed compared to control. As well as colony morphology differed that the control group colonies were irregular and a pink center and a large white edge on TZC medium but many colonies from KF32-exposed root were observed round and red center and a narrow or no white edge. Previous studies have reported that the colony of virulent R. solanacearum strain was irregular, mucoid, a pink center and a large white edge, while avirulent colony was round, dry, dark red and a narrow or no white edge (Zheng et al., 2019, 2022). These results suggest that the VOCs produced by KF32 may contribute to the reduction of root colonization and to the conversion of virulent strains to avirulent strains. These findings underscore the potential of bacterial volatile as non-contact, diffusible agents capable of limiting pathogen viability and spread. The consistent reduction in bacterial wilt through both in vitro and in planta assays suggests that VOCs may function as an effective mode of biocontrol, offering a sustainable alternative to chemical bactericides.
R. pseudosolanacearum is a soilborne pathogen capable of invading host roots, and inducing systemic wilting through the coordinated action of virulence factors such as motility, and type III secretion system (Rana et al., 2024; Wang et al., 2023). As a result of transcriptome analysis of R. pseudosolanacearum exposed to the VOCs from Pseudomonas strains KF32 and KF45, multiple virulence-related pathways of R. pseudosolanacearum were commonly affected. Among these genes expression of cell motility involved in flagellar assembly and chemotaxis were downregulated, suggesting reduced capacity for root colonization and host invasion. In fact, R. solanacearum initiates infection by attaching to plant roots, guided by chemotaxis to amino acids and malate in root exudates and flagellar motility is essential for root colonization and disease establishment (Corral et al., 2020; Tahir et al., 2023; Vailleau and Genin, 2023). In the results of transcriptome analysis of R. pseudosolanacearum, exposure to VOCs reduced genes involved in xenobiotic degradation. The suppression of genes related to xenobiotic degradation including glutathione metabolism and benzoate degradation suggests a compromised ability to detoxify plant-derived defense compounds (Baroukh et al., 2023; Wangsanut and Pongpom, 2022). Such a compromised detoxification system may render the pathogen more vulnerable to reactive oxygen species and other plant immune responses (Vailleau and Genin, 2023). In various organisms, downregulation of xenobiotic degradation has been shown to cause intracellular accumulation of toxic metabolites and elevated oxidative stress, leading to reduced capacity for stress tolerance, survival, and virulence under host-induced conditions (Gullner et al., 2018; Hartman et al., 2021; Shi et al., 2023). Glutathione S-transferase (GST) and glutathione reductase (GR) play key roles in detoxifying xenobiotics (Gupta and Gupta, 2021). Suppression of GST genes has been shown to reduce resistance to microbial pathogens in plants (Gullner et al., 2018), and GR-deficient Acidithiobacillus caldus exhibited increased sensitivity to heavy metals and oxidative stress, while overexpression enhanced tolerance (Shi et al., 2023). These transcriptomic changes demonstrate that VOCs can disrupt pathogen virulence through multiple mechanisms, including transcriptional regulation, metabolic disruption, motility functions, and host immune priming (Raio, 2024). To validate RNA-seq results, we performed qPCR for gene related to cell motility and xenobiotic degradation. The qPCR results were consistent with RNA-seq results, supporting the accuracy of the observed transcriptional changes. In particular, flgA and fliI exhibited significant downregulation in both RNA-seq and qPCR analysis. flgA and fliI are essential components involved in the early stages of bacterial flagellar assembly. flgA encodes a chaperone protein that is essential for the assembly of the P-ring in the flagellar basal body and fliI act as an ATPase within the flagellar type III secretion system, supplying the energy required to export flagellar components (Matsunami et al., 2016; Minamino and Kinoshita, 2023). Other genes related possible gene of xenobiotic degradation such as glutathione metabolism and benzoate degradation were also reduced in qPCR analysis, but these genes have not yet been identified accurate function. Therefore, further study is required to elucidate the potential contribution of the downregulation of xenobiotic degradation genes to pathogenicity attenuation in bacterial pathogen responses to VOC exposure.
Several bacterial VOCs, such as 2-nonanone, 2-undecanone, 2-decanone, 2,3 butanediol, and dimethyl disulfide have been previously reported to inhibit plant pathogens (Jeong et al. 2022, Raio 2024; Wang et al. 2024, 2025). In this study, we identified six VOCs produced by strains KF32 and KF45. Among them, 2-decanone, 2-undecanone, and thymol have been reported as antimicrobial compounds in various bacterial biocontrol strains (Jeong et al., 2022; Kumari et al., 2018; Wang et al., 2025). Our results showed that 2-decanone and 2-methyl-3-isopropylpyrazine exhibited significant antibacterial activity against R. pseudosolanacearum, whereas 2-undecanone and thymol did not show inhibitory effects. Notably, 2,4,6-trimethylpyridine was produced by both KF32 and KF45 but did not exert significant inhibition alone. In the I-plate assay, exposure to individual VOC compounds resulted in higher CFU counts of R. pseudosolanacearum compared to exposure to strains KF32 and KF45, indicating weaker antibacterial activity. Similarly, VOCs produced by Bacillus amyloliquefaciens SQR-9 significantly inhibited the growth of R. solanacearum on agar medium and soil, and nine VOCs individually exhibited 1-11% antibacterial activity; however, the VOC mixture reduced the growth of R. solanacearum by 70% (Raza et al., 2016). This suggests that the stronger inhibition observed with bacterial VOCs may be due to the combined or synergistic action of multiple VOCs. Furthermore, the concentration of each VOC likely plays a critical role in determining the overall antimicrobial efficacy. The single compound of VOC was exposed to tomato and analyzed incidence of bacterial wilt. 2-decanone which was reported antimicrobial activity (Guardado-Fierros et al., 2025; Jayakumar et al., 2021; Wang et al., 2025), produced by P. koreensis KF32 and suppressed tomato bacterial wilt. P. koreensis B17-12 has been reported to directly suppress several tomato diseases caused by Botrytis cinerea, Phytophthora infestans, and Alternaria solani through VOCs and stable root colonization (Wei et al., 2024). However, 2-decanone was not detected in the VOC of strain B17-12, while 25 different VOCs were detected, including 2-nonanone, 2-undecanone, and tetradecane, which have been previously reported to have antimicrobial activity (Wei et al., 2024; Zhao et al., 2023). These results suggest that VOC combinations, rather than single compounds, may contribute to biocontrol efficacy. Nevertheless, in this study, we observed that 2-decanone suppressed bacterial wilt as single compound in tomato plants, supporting its potential as an effective biocontrol agent.
Collectively, our findings demonstrate that VOCs produced by Pseudomonas koreensis KF32 and P. fitomaticsae KF45 can effectively suppress R. pseudosolanacearum by impairing essential biological functions, particularly those related to virulence, and motility. The identification of 2-decanone as a potent inhibitory compound adds to the repertoire of candidate molecules for biocontrol applications. To further advance the practical application of bacterial VOCs, subsequent investigations will focus on evaluating their mechanism in plant, and elucidating the underlying plant physiological change. Strain KF32 exposure suppressed plant growth in vitro on MS medium, but no inhibitory effect on plant growth was observed in soil. These results may be explained by the buffering capacity of soil, and under in vitro conditions, plants are directly exposed to high concentrations of VOCs, leading to suppressed plant growth. Perhaps the VOCs interfered with early developmental stage of tomato seedlings. Additionally, assessing the impact of VOC exposure on plant growth and physiological responses will be essential to ensure their compatibility with plant health. These efforts will contribute to a more comprehensive understanding of plant-microbe interactions mediated by bacterial volatiles and support the development of targeted, sustainable strategies for the biological control of bacterial wilt. This study expands our understanding of how bacterial volatiles influence plant-pathogen interactions at both phenotypic and transcriptomic levels, offering new perspectives for sustainable management of bacterial wilt disease.
PDF Links
PubReader
ePub Link
Full text via DOI
Download Citation
Supplement1
Print
