Skip to main content
NCBI home page
The Plant Pathology Journal logoLink to The Plant Pathology Journal
. 2026 Sep 14;42(5):660–672. doi: 10.5423/PPJ.OA.06.2026.0082

Characterization of Streptomyces virginiae JCK-8401 for the Suppression of Tomato Bacterial Wilt

Loan Thi Thanh Nguyen 1,2, Ae Ran Park 3, Ha Hang Le 4, Hang Thi Thu Nguyen 5, Jin-Cheol Kim 3,4,*
PMCID: PMC13655592  PMID: 42642881

Abstract

Bacterial wilt caused by Ralstonia solanacearum is a destructive soil-borne disease affecting numerous crops worldwide. Although synthetic pesticides and antibiotics are commonly used for disease management, their excessive application has raised concerns regarding environmental pollution, human health risks, and the emergence of antibiotic-resistant pathogens. As a sustainable alternative, rhizosphere microorganisms have gained attention as potential biocontrol agents. In our previous study, strain JCK-8401 exhibited strong antibacterial activity against R. solanacearum. In the present study, its biocontrol potential was comprehensively characterized through analyses of physiological and biochemical traits, antagonistic activity against phytopathogens, induction of salicylic acid (SA)-mediated defense responses using Arabidopsis thaliana PR1::GUS reporter line, and in vivo suppression of tomato bacterial wilt. Based on 16S rRNA sequence analysis, JCK-8401 was identified as Streptomyces virginiae. The strain utilized diverse carbon sources and produced hydrolytic enzymes and indole-3-acetic acid. Furthermore, it exhibited antibacterial activity against various phytopathogenic bacteria and oomycetes, though it lacked antifungal activity. Both cell suspension and culture filtrate of the strain induced GUS expression, indicating activation of the SA-mediated defense pathway. In vivo assay demonstrated that seed treatment with JCK-8401 achieved 96.40% control efficacy against tomato bacterial wilt. Additionally, pretreatment with diluted culture filtrates (250-, 500-, and 1000-fold) effectively suppressed disease incidence regardless of the application method, strongly suggesting the activation of induced systemic resistance. Overall, Streptomyces virginiae JCK-8401 is a highly promising, multifunctional biocontrol agent for managing soil-borne diseases through a synergistic combination of antibiosis, bioinoculation, and the induction of host plant defense responses.

Keywords: biocontrol agent, Streptomyces virginiae, tomato bacterial wilt

Tomato bacterial wilt, caused by Ralstonia solanacearum, is a devastating disease worldwide (Kurabachew and Wydra, 2013). This Gram-negative, soil-borne bacterium is characterized by rod-shaped cells and a polar flagellum that enables motility (Balamurugan et al., 2020). The pathogen typically invades plants through root wounds or natural openings, colonizing the vascular system to cause rapid wilting and eventual plant death. R. solanacearum possesses a broad host range, infecting over 200 plant species, including economically critical crops such as tomato, potato, eggplant, and pepper (Lowe-Power et al., 2018). It is widely distributed in the world and possesses a broad host range, infecting more than 200 plant species, including major crops such as tomato, potato, eggplant, and pepper (Ababa, 2024). Due to its prolonged persistence in soil, expansive host range, and high adaptability to diverse environmental conditions, tomato bacterial wilt has emerged as a primary constraint to global crop production (Chachar et al., 2025). Since it was initially reported in Asia and South America, the pathogen has spread globally via infected plant materials, contaminated soil and water, and various agricultural practices (Balamurugan et al., 2020). In many regions, the incidence of the disease continues to increase, resulting in substantial economic losses (Wu et al., 2023).

Current management strategies for bacterial wilt rely on integrated approaches, including the deployment of resistant cultivars, crop rotation, soil fumigation, and chemical treatments (Mamphogoro et al., 2020). However, these methods are frequently limited by the high genetic diversity and adaptability of the pathogen, allowing it to overcome host resistance and persist under shifting environmental conditions (Han et al., 2026). In addition, soil fumigation and chemical control may provide only temporary suppression and are often inconsistent under field conditions (Zhang et al., 2025). Moreover, the repeated application of chemical pesticides and antibiotics has triggered severe concerns regarding environmental contamination, detrimental impacts on non-target organisms, and the accumulation of toxic residues in agricultural ecosystems (Akhter et al., 2024; Zhou et al., 2025). These practices also drive the emergence of resistant bacterial populations, ultimately undermining the efficacy of conventional controls and highlighting the urgent need for sustainable alternatives (Miller et al., 2022). As a result, the management of bacterial wilt remains a significant challenge, highlighting the urgent need for more sustainable and effective alternatives.

The rhizosphere—a biologically dynamic soil zone driven by root exudates—hosts diverse microbial communities that are essential for plant growth and health (Chen and Liu, 2024). Beneficial rhizobacteria support plant development by contributing to nutrient cycling, enhancing soil structure, and improving nutrient bioavailability (Chen et al., 2024). Furthermore, these microorganisms suppress soil-borne pathogens via multiple mechanisms, including niche and nutrient competition, the secretion of antimicrobial compounds, and the modulation of the rhizosphere environment (Alqahtani, 2025). Among these microbes, Streptomyces spp. are renowned for producing a vast array of bioactive secondary metabolites with antibacterial, antifungal, and plant-growth-promoting properties (Khan et al., 2023). Beyond direct antagonism, these bacteria are highly efficient root colonizers, persisting in the rhizosphere to outcompete pathogens while intimately interacting with the host plant (Nazari et al., 2023). Importantly, Streptomyces spp. can enhance plant resistance by activating host defense signaling pathways, providing an additional layer of protection against pathogen infection (Devi and Manhas, 2026; Nguyen et al., 2026). These beneficial microbes may produce various elicitors, including microbial-associated molecular patterns, extracellular enzymes, and bioactive secondary metabolites (Zeng and Soltaniband, 2026). These compounds are recognized by plant immune receptors and stimulate defense-related responses (Pršić and Ongena, 2020). Such interactions can activate complex signaling networks involving salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), leading to the expression of defense-related genes and enhanced disease resistance (Kumar et al., 2025). In particular, SA accumulation and activation of pathogenesis-related (PR) genes, such as PR1, are key components of systemic acquired resistance (SAR), whereas induced systemic resistance is generally associated with JA/ET-dependent signaling induced by beneficial microorganisms (Sharma et al., 2024). Collectively, these traits make them promising candidates for the development of sustainable and plant growth-promoting agents.

In our previous work, JCK-8401 was isolated from the tomato rhizosphere and identified as a promising biocontrol candidate because its culture filtrate exhibited strong antibacterial activity against R. solanacearum (Nguyen et al., 2024). However, its physiological characteristics, plant-growth-promoting potential and mechanisms of disease suppression were not investigated. Therefore, present study provides a comprehensive functional characterization of JCK-8401. This study evaluated its physiological and biochemical characteristics, antimicrobial spectrum against phytopathogenic bacteria, fungi, and oomycetes, plant growth-promoting traits, and ability to induce the SA-mediated defense pathway using the Arabidopsis thaliana PR1::GUS reported line. In addition, its biocontrol efficacy against tomato bacterial wilt was assessed under greenhouse conditions.

Materials and Methods

Isolation and culture conditions of JCK-8401

Strain JCK-8401 was previously isolated from tomato roots (Solanum lycopersicum) collected in Sejong, Korea (Nguyen et al. 2024). Its culture filtrate showed antibacterial activity against Ralstonia solanacearum SL341, with minimum inhibition concentration (MIC) ≤10%, and was therefore selected for further investigation. JCK-8401 was inoculated on tryptic soy agar (TSA, Difco; BD, Sparks, MD, USA) at 28°C for 7 days and incubated in tryptic soy broth (TSB, Difco; BD) at with shaking at 180 rpm 28°C for 3 days. For long-term preservation, the strain was kept in 30% glycerol at −80°C freezer.

Morphological, physiological, biochemical and molecular characterization of JCK-8401

The growth of JCK-8401 was evaluated after incubation at 28°C for 7 days on various media, such as tryptone-yeast extract (ISP1), yeast-malt agar (ISP2), oatmeal agar (ISP3), inorganic salt-starch agar (ISP4), glycerol asparagine agar (ISP5), peptone yeast extract iron agar (ISP6), marine agar (MA), Bennet, potato dextrose agar (PDA), and TSA. The compositions of these media were previously described by Shirling and Gottlieb (1966) and Nguyen et al. (2024). Morphological characteristic of JCK-8401 were observed using a scanning electron microscope (SEM) (Quanta™ 250 FEG; FEI Company, Hillsboro, OR, USA).

The strain was tested for growth at various NaCl concentrations (1–9%), temperatures (4–45°C), and pH values (4–10). The pH of the culture media was adjusted using NaOH and HCl and verified with a pH meter. The carbon utilization and enzyme activity of JCK-8401 were evaluated by API 50 CH and API ZYM (bioMérieux, Craponne, France). In addition, the production ability of amylase, cellulase, gelatinase, and protease by JCK-8401 was evaluated based on the methodology described by Hossain et al. (2020). The production of indole-3-acetic acid (IAA) by JCK-8401 also was tested according to the protocol outlined by Gang et al. (2019) and Nguyen et al. (2024).

The genomic DNA of JCK-8401 was extracted using the i-genomic BYF DNA Extraction mini kit (iNtRON Biotechnology, Inc., Seongnam, Korea). The amplification of the 16S rRNA was carried out using the primer pairs 27F (3′-AGAGTTTGATCCTGGCTCAG-5′) and 1492R (3′-GGTTACCTTGTTACGACTT-5′) (Miller et al., 2013). The PCR thermal cycling conditions consisted of 5 steps as follows: 95°C for 5 minutes, 30 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 90 seconds, and 72°C for 10 minutes. DNA sequence similarity was determined using BLAST searches on the National Center for Biotechnology Information (NCBI) database. Phylogenetic relationships were analyzed using the MEGA X software (Kumar et al., 2018), and a phylogenetic tree was constructed using Neighbor-Joining (NJ) algorithms with 1,000 bootstrap replications (Saitou and Nei, 1987).

In vitro evaluation of antimicrobial activity

The antimicrobial activity of the cell-free culture filtrate culture filtrate (CF) of JCK-8401 was evaluated against a broad range of phytopathogens, including soil-borne and air-borne bacteria, fungi, and oomycetes. MIC value was determined using the broth dilution method (Le et al., 2021). Three technical replicates were included in each independent experiment, and the entire experiment was conducted independently three times. Streptomycin sulfate (CAS No. 3810-74-0; Product No. S6501-100G; Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control to validate assay performance. Antibacterial activity was evaluated against the following phytopathogenic bacteria: Acidovorax avenae subsp. cattleyae, Acidovorax konjaci, Agrobacterium tumefaciens, Burkholderia glumae, Clavibacter michiganensis subsp. michiganensis, Erwinia amylovora FB302, Erwinia amylovora TS3128, Erwinia pyrifoliae KI115, Pectobacterium carotovorum subsp. carotovorum, Pectobacterium chrysanthemi, Pseudomonas syringae pv. lachrymans, Ralstonia solanacearum SL341, Xanthomonas arboricola pv. pruni, and Xanthomonas euvesicatoria. Antifungal and anti-oomycetes activities were assessed against Fusarium graminearum, Fusarium oxysporum f.sp. cucumerinum, Phytophthora infestans, Pythium ultimum, Rhizoctonia solani AG 2-2, Rhizoctonia solani AG-4, and Sclerotinia homoeocarpa.

Histochemical analysis of β-glucuronidase activity

The induced resistance activity of JCK-8401 was evaluated using CF and cell suspension (CS) in transgenic Arabidopsis thaliana carrying the pathogenesis-related 1 (PR1) promoter fused to the β-glucuronidase (GUS) (Park et al., 2020). The experiment was performed according to the method described by Nguyen et al. (2024). Briefly, A. thaliana seeds were surface-sterilized and sown on 1/2MS medium. JCK-8401 was cultured in TSB at 28°C with shaking at 180 rpm for 7 days. The culture broth was then separated into CF and CS and diluted 250-fold, 500-fold, and 1,000-fold dilutions. Each treatment was applied to 12-day-old of A. thaliana seedlings, which were then incubated at 25°C for 48 h. After incubation, the seedlings were stained with a chemical solution following the method of Kondo et al., 2014. SA 0.1 mM was used as a positive control, while TSB and sterile distilled water (SDW) served as the negative controls. Each treatment consisted of three biological replicates with two A. thaliana seedlings per replicate (n = 6 seedlings). The entire experiment was independently repeated twice. GUS expression was evaluated using the following scale: 0, no signal; 1, one leaf with signal; 2, two leaves with signal; 3, three leaves with signal; 4, four leaves with signal; 5, entire seedling exhibiting signal (Nguyen et al., 2026).

In planta bioassays

To evaluate the biocontrol efficacy of JCK-8401 as a seed treatment agent, tomato seeds were surface-sterilized and soaked in a JCK-8401 cell suspension (8 × 109 colony forming units [CFU]/mL) for 24 h, followed by overnight drying under a horizontal laminar flow hood. The coated and uncoated seeds were sown in nursery soil (Bunong Horticulture Nursery Soil; Bunong, Gyeongju, Korea) and maintained at 25°C with 75% humidity and 12 hours of daylight. Uncoated plants were treated with a 1,000-fold dilution of buramycin (streptomycin sulfate 20% wettable powder [WP]; FarmHannong Co., Seoul, Korea) 1 day before inoculation (DBI). Uncoated plants treated with SDW were used as the untreated control, while uninoculated control plants were grown from uncoated seeds in non-inoculated pots. Colonization of JCK-8401 in tomato plants was accessed following the method described by Nguyen et al. (2024).

To test the antibacterial activity of JCK-8401, its CF supernatant was diluted with SDW at 10 and 20-fold dilution. These samples were mixed with Tween 20 at 250 μg/mL. A 1,000-fold dilution of buramycin was used as a positive control. The tomato plants were treated with 20 mL of each sample by soil drenching at 1 DBI. The untreated control was applied with TSB mixed with Tween 20 solution, while uninoculated plants grown in non-inoculated pots served as the uninoculated control.

To assess the induced resistance activity of JCK-8401, the CF supernatant of JCK-8401 was applied to tomato plants at the fourth-leaf stage either by soil drenching or foliar spraying. The CF was diluted 250-fold, 500-fold, and 1,000-fold with SDW. CF treatments were supplemented with Tween 20 at 250 μg/mL. For soil drenching (SD), 20 mL of each treatment was applied once to each pot. For foliar spraying (FS), the soil surface was shielded with plastic to prevent unintended soil contact, and the leaves were uniformly sprayed with 10 mL of the treatment solution. Plants treated with TSB containing Tween 20 solution served as untreated control. All treatments were treated at 3 DBI. A 1,000-fold dilution of buramycin was used as a positive control and applied to plant by soil drenching at 1 DBI, while plants grown in uninoculated pots served as the uninoculated control.

All samples were evaluated for their efficacy against tomato bacterial wilt using tomato plants at the fourth-leaf stage. Each treatment consisted of three biological replicates with three tomato seedlings per replicate (n = 3 seedlings). The entire experiment was repeated twice. On the day of bacterial infection, the plants were inoculated with a cell suspension of R. solanacearum SL341, 108 CFU/mL, through soil drenching and kept in the dark for 24 h at 30 ± 2°C. The plants were maintained with 75% humidity and 12 hours of daylight per day. Disease symptoms were assessed 7 days after inoculation. The disease severity on the tomato plants was rated on a scale of 0–4, ranging from 0 (no leaf symptom) to 4 (dead plant), as described by Vu et al. (2017).

The disease severity (DS) was expressed as a percentage of untreated control, along with the standard error. The calculation of the control value was performed using the following equation:

Control value (%)=(DS of untreated control-DS of treatment)DS of untreated control×100%.

Statistical analysis

Statistical analyses of the pot experiment data were conducted using IBM SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA). Differences among treatments were assessed using Student’s t-test, one-way ANOVA, and two-way ANOVA, followed by Tukey’s HSD test. Results are expressed as means ± standard error (SE), and figures were generated using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).

Results

Morphological and molecular characteristics of Streptomyces virginiae JCK-8401

JCK-8401 formed well-developed colonies with distinct radial growth on TSA (Fig. 1A). SEM confirmed the presence of well-developed filamentous structures and bead-like (moniliform) cylindrical spores arranged in long, unfragmented chains along the aerial hyphae, typical of Streptomyces (Fig. 1B). The hyphae were elongated and branched, forming a dense mycelia network. Chains of spore were clearly observed along the aerial hyphae, appearing as bead-like (moniliform) structures. The spores were cylindrical in shape with unsmooth surfaces and arranged in long, unfragmented chains. Phylogenetic analysis of the 16S rRNA gene (GenBank accession: PZ397461) showed 100% sequence similarity to Streptomyces virginiae NRRL ISP-5094T (JOAK01000082) (Fig. 1C). Therefore, based on these results, strain JCK-8401 was identified as Streptomyces virginiae.

Fig. 1.

Fig. 1

Open in a new tab

Morphology characteristics and phylogenetic analysis of JCK-8401. (A) Growth of colony of JCK-8401 on TSA plates after 7 days at 28°C; (B) Spore chain morphology of JCK-8401 by SEM; (C) A neighbor-joining phylogenetic tree based on the 16S rRNA sequences of strain JCK-8401 from bacteria related to the Streptomyces species. Actinomadura madurae ATCC 19425T was used as the outgroup. Bootstrap values (≥50%) based on 1,000 replications are indicated at branch nodes. Bar, the number of substitutions at certain nucleotide positions. TSA, tryptic soy agar; SEM, scanning electron microscope.

Physiology and functional traits of S. virginiae JCK-8401 associated with its biological potential

A comprehensive analysis of S. virginiae JCK-8401 demonstrated its strong functional potential and adaptability. The strain utilized several carbon sources, including glycerol, D-ribose, D-glucose, and esculin ferric citrate and produced various hydrolytic enzymes including alkaline phosphatase, lipase, trypsin, protease, and gelatinase (Supplementary Tables 1, 2). However, no amylase and cellulase activity were detected. In addition, JCK-8401 produced IAA in the presence of L-tryptophan (Supplementary Fig. 1). The strain exhibited growth across all tested media, with variations in substrate mycelia color (white to gray) and pigment production (dark brown, light brown, or absent) depending on the medium (Fig. 2). Among all tested media, the strain exhibited spore formation and produced the darkest brown pigment on TSA. Furthermore, JCK-8401 showed broad environmental tolerance, growing well at 20–37°C, tolerating up to 5% (w/v) NaCl and sustaining growth across a pH range of 6–10 (Supplementary Table 3).

Fig. 2.

Fig. 2

Open in a new tab

Growth of colony of JCK-8401 on various media after 7 days at 28°C. ISP, International Streptomyces Project; MA, margin agar; PDA, potato dextrose agar. Scale bar is 1.0 mm.

S. virginiae JCK-8401 exhibited significant antibacterial activity but no antifungal activity

The culture filtrate exhibited broad-spectrum antibacterial activity against most tested pathogens, but lacked antifungal activity (Table 1). Notably, it demonstrated targeted antibiotic activity against the oomycete P. infestans (Table 2).

Table 1.

MICs of the fermentation filtrates obtained from selected microorganisms against various plant pathogenic bacteria

Plant pathogenic bacteria JCK-8401 (%) Streptomycin sulfate (μg/mL)
Acidovorax avenae subsp. cattleyae 1.67 ± 0.72 -
Acidovorax kojiaci 5.00 ± 0.00 10.42 ± 3.61
Agrobacterium tumefaciens - 200.00 ± 0.00
Burkholderia glumae 10.00 ± 0.00 20.83 ± 7.22
Clavibacter michiganensis subsp. michiganensis - 25.00 ± 0.00
Erwinia amylovora FB302 5.00 ± 0.00 3.12 ± 0.00
Erwinia amylovora TS3128 4.17 ± 1.44 3.12 ± 0.00
Erwinia pyrifoliae KI115 10.00 ± 0.00 6.25 ± 0.00
Pectobacterium carotovorum subsp. carotovorum 8.33 ± 2.89 12.50 ± 0.00
Pectobacterium chrysanthemi 10.00 ± 0.00 -
Pseudomonas syringae pv. lachrymans - -
Ralstonia solanacerum SL341 8.33 ± 2.89 3.12 ± 0.00
Xanthomonas arboricola pv. pruni 10.00 ± 0.00 12.50 ± 0.00
Xanthomonas euvesicatoria - 16.67 ± 0.00
Open in a new tab

Data are expressed as mean ± standard deviation of three replicates.

MICs, minimum inhibitory concentrations.

Table 2.

MICs of the fermentation filtrates obtained from selected microorganisms against various plant pathogenic fungi and oomycetes

Plant pathogenic fungi and oomycetes JCK-8401 (%)
Fusarium graminearum -
Fusarium oxysporum f.sp. cucumerinum -
Phytophthora infestans 1.25 ± 0.00
Pythium ultimum -
Rhizoctonia solani AG 2-2 -
Rhizoctonia solani AG-4 -
Sclerotinia homoeocarpa -
Open in a new tab

Data are expressed as mean ± standard deviation of three replicates.

MICs, minimum inhibitory concentrations.

Both the cells and culture filtrate of S. virginiae JCK-8401 triggered a positive GUS response in A. thaliana.

In GUS assay, a positive response was indicated by the visible blue coloration of A. thaliana PR1::GUS reporter line, in which GUS expression is driven by a SA-responsive promoter. In the positive control, SA treatment induced strong GUS staining across seedling tissues whereas no color change was observed in the negative controls, TSB and SDW (Fig. 3). Both the bacterial cells and culture filtrate induced blue vein-like staining patterns, although these were less intense than those observed with SA treatment. The GUS index of cell treatment was 1.33, 1.17, and 0.00 whereas the treatment with CF showed higher values of 4.83, 4.67, and 3.33 at 250-, 500-, and 1,000-fold dilutions, respectively (Table 3).

Fig. 3.

Fig. 3

Open in a new tab

GUS activity in transgenic A. thaliana rosette leaves responded to treatment with control, SA, CS, and CF. Control, 0.05% TSB medium and SDW; SA, salicylic acid (0.1 mM); CS, cell suspension of JCK-8401; CF, culture filtrate of JCK-8401; GUS, β-glucuronidase; TSB, tryptic soy broth; SDW, sterile distilled water. Scale bar is 1.0 mm.

Table 3.

GUS assay result of JCK-8401

No Sample Concentration
×250 ×500 ×1,000 0.1 mM
1 JCK-8401 CS 1.33 ± 0.47 1.17 ± 0.37 0.00 ± 37
2 JCK-8401 CF 4.83 ± 0.37 4.67 ± 0.47 3.33 ± 0.47
3 SA 5.00 ± 0.00
4 1% TSB 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
5 SDW 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Open in a new tab

Data are expressed as mean ± standard deviation of three replicates.

JCK-8401 CS, cell suspension of JCK-8401; JCK-8401 CF, culture filtrate of JCK-8055; SA, salicylic acid; TSB, tryptic soya broth; SDW, sterile distilled water.

×250, 250-fold dilution; ×500, 500-fold dilution; ×1,000, 1,000-fold dilution.

S. virginiae JCK-8401 effectively suppressed tomato bacterial wilt via seed treatment, antibacterial activity and the induction of plant resistance

Initially, the colonization of inner root tissues by specific Streptomyces strains was confirmed. The tomato seedling roots were successfully colonized by the selected Streptomyces strain on TSA plates. The morphology and 16S rRNA gene sequence of this strain were similar with that of S. virginiae JCK-8401 (Supplementary Figs. 2, 3). A pot experiment was conducted to evaluate the biocontrol efficacy of JCK-8401 cells against tomato bacterial wilt. Seed treatment resulted in substantial disease suppression, achieving a control value of 96.40%, which was not significantly different from the positive control, buramycin (71.34%, P = 0.0511) (Fig. 4). This was not significantly different from the positive control, buramycin (71.34%, P = 0.0511). There were no symptoms of phytotoxicity.

Fig. 4.

Fig. 4

Open in a new tab

Disease control efficacy of seed treatment with JCK-8401 against tomato bacterial wilt caused by R. solanacearum SL341. The values are presented as the mean ± standard error of three runs, with three replicates each; and ns denotes no significant difference.

Furthermore, the CF supernatant demonstrated high control efficacy against R. solanacearum SL341 (88.51% at 10-fold dilution) (Fig. 5). Buramycin showed 57.58% control value at 1000-fold dilution. One-way ANOVA revealed a significant effect of treatment on disease suppression (P = 0.0464). Post-hoc comparison indicated that the 10-fold dilution showed significantly higher activity than the Buramycin treatment, while lower dilutions exhibited reduced efficacy.

Fig. 5.

Fig. 5

Open in a new tab

Disease control efficacy of pretreatment with the culture filtrate of JCK-8401 against tomato bacterial wilt caused by R. solanacearum SL341. Dilution factors: ×10, 10-fold dilution; ×20, 20-fold dilution; CF, culture filtrate of JCK-8401. The values are presented as the mean ± standard error of three runs, with three replicates each. Asterisks indicate significant differences with *(P < 0.05).

A pretreatment in vivo assay was further performed to assess the efficacy of the CF supernatant of JCK-8401 against tomato bacterial wilt through FS and SD. Both application methods exhibited considerable disease control activity (Fig. 6). CF-FS exhibited the highest control value at 250-fold dilution (71.11%), followed by 1,000-fold (45.56%) and 500-fold dilution (35.78%). In contrast, CF-SD showed peak performance at 500-fold dilution (78.89%), followed by 250-fold (55.56%) and 1,000-fold dilution (52.22%). Two-way ANOVA revealed a significant interaction between application method and dilution level (F(2,12) = 6.776, P = 0.0107), accounting for 42.59% of the total variation. However, the main effects of application method and dilution were not statistically significant (P = 0.1081 and P = 0.2371, respectively).

Fig. 6.

Fig. 6

Open in a new tab

Disease control efficacy of pretreatment with the culture filtrate of JCK-8401 against tomato bacterial wilt caused by R. solanacearum SL341. Dilution factors: ×250, 250-fold dilution; ×500, 500-fold dilution; ×1,000, 1,000-fold dilution; FS, foliar spraying; SD, soil drenching; CF, culture filtrate of JCK-8401. The values are presented as the mean ± standard error of three runs, with three replicates each. Asterisks indicate significant differences with **(P < 0.01).

Discussion

Bacterial wilt, caused by R. solanacearum, is a highly destructive soil-borne disease responsible for massive economic losses in critical agricultural crops (Wang et al., 2023). Since its widespread occurrence worldwide, the disease has become increasingly difficult to manage due to the persistence and adaptation of R. solanacearum in soil and water environments (Chachar et al., 2025). Given the pathogen’s persistence and adaptability, conventional control strategies often yield inconsistent results (Chachar et al., 2025; Wang et al., 2023). Consequently, biological control utilizing rhizobacteria—particularly Streptomyces spp.—has emerged as a highly viable, environmentally friendly alternative (Khan et al., 2023). In this study, Streptomyces virginiae JCK-8401 demonstrated exceptional potential to manage tomato bacterial wilt via a synergistic combination of niche competition, direct antibiosis, and the induction of systemic plant defense responses (Cai et al., 2021; Guo et al., 2004). Among these beneficial microorganisms, Streptomyces spp. are widely recognized as effective biocontrol agents against plant pathogens through their competitive colonization of ecological niches and capacity to produce diverse bioactive compounds (Al-Quwaie, 2024; Nguyen et al., 2026; Shepherdson et al., 2023; Vurukonda et al., 2018). In the present study, Streptomyces virginiae JCK-8401 exhibited strong potential to control tomato bacterial wilt through a combination of niche competition, antibacterial activity, and induction of plant defense responses.

Effective colonization is a prerequisite for successful biocontrol (Al-Quwaie, 2024). Because both Streptomyces spp. and R. solanacearum exploit natural plant openings (e.g., stomata, root hair cracks) to enter host tissues, the early establishment of Streptomyces provides a distinct competitive advantage by preemptively occupying vulnerable infection sites (Lowe-Power et al., 2018; Shimizu, 2011). In addition, Streptomyces spp. secrete various proteins and enzymes that facilitate successful plant colonization and adaptation to the plant environment (Chewning et al., 2019). S. virginiae JCK-8401’s production of diverse hydrolytic enzymes (protease, lipase, and gelatinase), utilization of various carbon sources, and robust tolerance to fluctuations in temperature, salinity, and pH underscore its exceptional physiological flexibility. The strain also demonstrated the ability to utilize diverse carbon sources and exhibited physiological flexibility, reflected by its growth across various media and tolerance to wide range of temperature, salinity, and pH conditions. These traits are critical for maintaining field performance under variable environmental conditions. Furthermore, the strain’s ability to synthesize IAA directly promotes root development and nutrient uptake, positioning JCK-8401 as a dual-action bioinoculant for both disease suppression and crop enhancement.

Streptomyces spp. can suppress plant disease through both directly and indirectly, including the production of antagonistic metabolites against the pathogens and the induction of the host defense responses (Nguyen et al., 2024, 2025). Our findings reveal that JCK-8401 predominantly secretes metabolites targeting bacterial pathogens, as evidenced by its strong MICs ranging from 1.25% to 10% against various phytopathogenic bacteria, with an interesting cross-over activity against the soil-borne oomycete P. infestans. This selective antimicrobial spectrum suggests that the strain predominantly produces metabolites targeting bacterial pathogens. The observed activity against the soil-borne oomycete P. infestans (MIC = 1.25%) indicated that certain metabolites may act on cellular targets distinct from those in true fungi, thereby expanding its potential application to different type of plant pathogens. Beyond direct antibiosis, JCK-8401 successfully triggered SA-mediated defense pathways in transgenic Arabidopsis, suggesting that the secretion of specific elicitor metabolites is a key component of its mode of action. Therefore, Streptomyces virginiae JCK-8401 may suppress plant diseases, particularly bacterial diseases, by both direct antibacterial activity and the induction of plant defense responses.

The in planta assays demonstrated that the disease-control efficacy of JCK-8401 culture filtrates varied with dilution level and application method. At lower dilutions (10- and 20-fold), control efficacy increased in a dose-dependent manner, suggesting that direct antibacterial activity contributed substantially to disease suppression at these concentrations (Nguyen et al., 2024). In contrast, the comparable efficacy observed at higher dilutions (250- and 1000-fold), regardless of whether the culture filtrate was applied by foliar spraying or soil drenching, suggests that host defense activation may also contribute to disease suppression (Nguyen et al., 2025). However, the variable efficacy among dilution levels and application methods indicates that disease control is likely influenced by multiple factors, including the concentration, stability, and bioavailability of active metabolites, rather than dilution alone. In the present study, we did not investigate the effects of culture age, cell density, or metabolite concentration on the biocontrol efficacy of the culture filtrate. These factors may affect the production of bioactive compounds and should be systematically evaluated in future studies to optimize the application of JCK-8401. Collectively, these findings suggest that JCK-8401 may suppress tomato bacterial wilt through a combination of direct antibacterial activity and host defense induction, although the relative contribution of each mechanism requires further investigation. The ability of JCK-8401 to potentially suppress disease through both direct antagonism and activation of plant defense responses suggests a multifunctional mode of action that may contribute to its effectiveness as a biocontrol agent.

Seed treatment of JCK-8401 provided 96.40% disease control, with successful re-isolation of the strain confirming root colonization and early protection against R. solanacearum. Previous studies reported that combined seed treatment with Pseudomonas fluorescens Pf3 and Trichoderma longibrachiatumUNS11, as well as seed coating experiment with Streptomyces sp. JCK-8055, provided up to 62% control efficacy against tomato bacterial wilt under greenhouse conditions (Konappa et al., 2020; Nguyen et al., 2024). Similar findings have been reported for several beneficial Streptomyces strains, in which efficient rhizosphere colonization was closely associated with stable disease suppression and enhanced plant protection. For example, S. exfoliatus FT05W and S. cyaneus ZEA17I effectively colonized plant roots and suppressed soil-borne pathogens through niche competition and persistence in the rhizosphere (Chen et al., 2016). These finding suggests that the strong colonization ability of JCK-8401 may play a critical role in its high biocontrol efficacy by enabling stable establishment in the rhizosphere and providing continuous protection against pathogen invasion.

The present study has several limitations. Although JCK-8401 was successfully re-isolated from tomato roots, its colonization within internal plant tissues was not confirmed, and further investigation using more precise methods is required. In addition, the antibacterial metabolites and defense-inducing compounds responsible for the biocontrol activity of JCK-8401 have not yet been identified, and their mechanisms of action remain unclear. Future studies should focus on bioassay-guided fractionation, purification, and structural characterization of these bioactive metabolites to elucidate the mechanisms underlying both direct antibacterial activity and plant defense induction. Moreover, the disease control efficacy of JCK-8401 has not yet been validated under field conditions. Therefore, future research should also evaluate the long-term efficacy and stability of JCK-8401 under diverse field environments.

This study firmly establishes Streptomyces virginiae JCK-8401 as a highly effective, multifunctional biocontrol agent for suppressing bacterial plant diseases, particularly tomato bacterial wilt. Its robust efficacy is driven by a synergistic combination of broad-spectrum antibacterial activity and the induction of SA-mediated plant defense responses. These mechanisms are further bolstered by the strain’s metabolic versatility, hydrolytic enzyme production, and plant growth-promoting traits, making it a prime candidate for the development of novel, commercial bioagents. Although S. virginiae JCK-8401 exhibited promising biocontrol efficacy under greenhouse conditions, further studies are required to identify the active antibacterial and defense-inducing compounds, optimize formulation development, evaluate storage stability, and validate efficacy under diverse field conditions before its commercial application can be supported.

Footnotes

Conflicts of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgments

We would like to express our gratitude to Dr. S. D Lee (Rural Development Administration), Prof. S.-W. Lee (Dong-A University), and Prof. Y-G Ko (Suncheon National University) for generously providing us with the phytopathogenic bacteria and fungi used in this study.

This work was supported by a grant (Project No. RS-2025-02215543) from Rural Development Administration, Republic of Korea.

Electronic Supplementary Material

Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).

References

  1. Ababa G. Pathogenic diversity, ecology, epidemiology, and management practices of the potato bacterial wilt (Ralstonia solanacearum) disease. Cogent Food Agric. 2024;10:2407953. [Google Scholar]
  2. Akhter S., Bhat M. A., Ahmed S., Siddiqui W. A. Antibiotic residue contamination in the aquatic environment, sources, and associated potential health risks. Environ. Geochem. Health. 2024;46:387. doi: 10.1007/s10653-024-02146-5. [DOI] [PubMed] [Google Scholar]
  3. Alqahtani F. S. The utilization of microorganisms for biological control of soil-borne plant pathogens: a sustainable strategy for managing plant diseases - a comprehensive review. J. Plant Pathol. 2025;107:1815–1839. [Google Scholar]
  4. Al-Quwaie D. A. The role of Streptomyces species in controlling plant diseases: a comprehensive review. Australas Plant Pathol. 2024;53:1–14. [Google Scholar]
  5. Balamurugan A., Sakthivel K., Gautam R. K., Sharma S. K., Kumar A. Ralstonia solanacearum: biology and its management in solanaceous vegetable crops. In: Sharma S. K., Singh U. B., Sahu P. K., Singh H. V., Sharma P. K., editors. Rhizosphere Microbes: Soil and Plant Functions. Springer; Singapore: 2020. pp. 259–289. [Google Scholar]
  6. Cai Q., Zhou G., Ahmed W., Cao Y., Zhao M., Li Z., Zhao Z. Study on the relationship between bacterial wilt and rhizospheric microbial diversity of flue-cured tobacco cultivars. Eur. J. Plant Pathol. 2021;160:265–276. [Google Scholar]
  7. Chachar Z., Xue X., Fang J., Chen M., Jiarui C., Chen W., Ahmed N., Chachar S., Narejo M.-N., Ahmed N., Fan L., Lai R., Qi Y. Key mechanisms of plant-Ralstonia solanacearum interaction in bacterial wilt pathogenesis. Front. Microbiol. 2025;16:1521422. doi: 10.3389/fmicb.2025.1521422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen L., Liu Y. The function of root exudates in the root colonization by beneficial soil rhizobacteria. Biology. 2024;13:95. doi: 10.3390/biology13020095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Q., Song Y., An Y., Lu Y., Zhong G. Soil microorganisms: Their role in enhancing crop nutrition and health. Diversity. 2024;16:734. [Google Scholar]
  10. Chen X., Pizzatti C., Bonaldi M., Saracchi M., Erlacher A., Kunova A., Berg G., Cortesi P. Biological control of lettuce drop and host plant colonization by rhizospheric and endophytic Streptomycetes. Front Microbiol. 2016;20:714. doi: 10.3389/fmicb.2016.00714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chewning S. S., Grant D. L., O’Banion B. S., Gates A. D., Kennedy B. J., Campagna S. R., Lebeis S. L. Root-associated Streptomyces isolates harboring melC genes demonstrate enhanced plant colonization. Phytobiomes J. 2019;3:165–176. [Google Scholar]
  12. Devi S., Manhas R. K. Harnessing the multifunctional bioagent Streptomyces sp. SP5 for Fusarium wilt suppression, metabolite-mediated antioxidant activity, and plant growth promotion. Sci. Rep. 2026;16:13147. doi: 10.1038/s41598-026-42641-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gang S., Sharma S., Saraf M., Buck M., Schumacher J. Analysis of indole-3-acetic acid (IAA) production in Klebsiellaby LC-MS/MS and the Salkowski method. Bio. Protoc. 2019;9:e3230. doi: 10.21769/BioProtoc.3230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guo J.-H., Qi H.-Y., Guo Y.-H., Ge H.-L., Gong L.-Y., Zhang L.-X., Sun P.-H. Biocontrol of tomato wilt by plant growth-promoting rhizobacteria. Bio. Control. 2004;29:66–72. [Google Scholar]
  15. Han M., Liu X., Li G., Li P. Analysis of bacterial wilt management strategies from the dynamic perspective of environmental adaptation approaches of Ralstonia solanacearum. Environ. Microbiol. Rep. 2026;18:e70335. doi: 10.1111/1758-2229.70335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hossain T. J., Chowdhury S. I., Mozumder H. A., Chowdhury M. N. A., Ali F., Rahman N., Dey S. Hydrolytic exoenzymes produced by bacteria isolated and identified from the gastrointestinal tract of Bombay duck. Front. Microbiol. 2020;11:2097. doi: 10.3389/fmicb.2020.02097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Khan S., Srivastava S., Karnwal A., Malik T. Streptomyces as a promising biological control agents for plant pathogens. Front. Microbiol. 2023;14:1285543. doi: 10.3389/fmicb.2023.1285543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Konappa N., Krishnamurthy S., Arakere U. C., Chowdappa S., Ramachandrappa N. S. Efficacy of indigenous plant growth-promoting rhizobacteria and Trichoderma strains in eliciting resistance against bacterial wilt in a tomato. Egypt. J. Biol. Pest Control. 2020;30:106. [Google Scholar]
  19. Kondo Y., Ito T., Nakagami H., Hirakawa Y., Saito M., Tamaki T., Shirasu K., Fukuda H. Plant GSK3 proteins regulate xylem cell differentiation downstream of TDIF-TDR signalling. Nat. Commun. 2014;5:3504. doi: 10.1038/ncomms4504. [DOI] [PubMed] [Google Scholar]
  20. Kumar P., Pandey S., Pati P. K. Interaction between pathogenesis-related (PR) proteins and phytohormone signaling pathways in conferring disease tolerance in plants. Physiol. Plant. 2025;177:e70174. doi: 10.1111/ppl.70174. [DOI] [PubMed] [Google Scholar]
  21. Kumar S., Stecher G., Li M., Knyaz C., Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018;35:1547–1549. doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kurabachew H., Wydra K. Characterization of plant growth promoting rhizobacteria and their potential as bioprotectant against tomato bacterial wilt caused by Ralstonia solanacearum. Biol. Control. 2013;67:75–83. [Google Scholar]
  23. Le K. D., Kim J., Nguyen H. T., Yu N. H., Park A. R., Lee C. W., Kim J. C. Streptomyces sp. JCK-6131 protects plants against bacterial and fungal diseases via two mechanisms. Front. Plant Sci. 2021;12:726266. doi: 10.3389/fpls.2021.726266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lowe-Power T. M., Khokhani D., Allen C. How Ralstonia solanacearum exploits and thrives in the flowing plant xylem environment. Trends Microbiol. 2018;26:929–942. doi: 10.1016/j.tim.2018.06.002. [DOI] [PubMed] [Google Scholar]
  25. Mamphogoro T. P., Babalola O. O., Aiyegoro O. A. Sustainable management strategies for bacterial wilt of sweet peppers (Capsicum annuum) and other Solanaceous crops. J. Appl. Microbiol. 2020;129:496–508. doi: 10.1111/jam.14653. [DOI] [PubMed] [Google Scholar]
  26. Miller C. S., Handley K. M., Wrighton K. C., Frischkorn K. R., Thomas B. C., Banfield J. F. Short-read assembly of full-length 16S amplicons reveals bacterial diversity in subsurface sediments. PloS One. 2013;8:e56018. doi: 10.1371/journal.pone.0056018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Miller S. A., Ferreira J. P., LeJeune J. T. Antimicrobial use and resistance in plant agriculture: a one health perspective. Agriculture. 2022;12:289. [Google Scholar]
  28. Nazari M. T., Schommer V. A., Braun J. C. A., dos Santos L. F., Lopes S. T., Simon V., Machado B. S., Ferrari V., Colla L. M., Piccin J. S. Using Streptomyces spp. as plant growth promoters and biocontrol agents. Rhizosphere. 2023;27:100741. [Google Scholar]
  29. Nguyen L. T. T., Park A. R., Im H. W., Le V. V., Nguyen H. T. T., Le Dang Q., Hoa T. T. N., Yeo Y. J., Le H. H., Nguyen V. T., Hwang I., Kim J. C. Biological control of tomato bacterial wilt and apple fire blight through the induced resistance of azomycin derived from Streptomyces sp. JCK-8368. Front. Plant Sci. 2025;16:1654826. doi: 10.3389/fpls.2025.1654826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nguyen L. T. T., Park A. R., Van Le V., Hwang I., Kim J. C. Exploration of a multifunctional biocontrol agent Streptomyces sp. JCK-8055 for the management of apple fire blight. Appl. Microbiol. Biotechnol. 2024;108:49. doi: 10.1007/s00253-023-12874-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nguyen L. T. T., Park A. R., Oh C. W., Im H. W., La V. H., Kim J. C. Biological control of tomato bacterial wilt and apple fire blight using n-docosane, a plant resistance inducer from Streptomyces sp. JCK-8055. Pestic. Biochem. Physiol. 2026;220:107081. doi: 10.1016/j.pestbp.2026.107081. [DOI] [PubMed] [Google Scholar]
  32. Park A. R., Jeong S. I., Jeon H. W., Kim J., Kim N., Ha M. T., Mannaa M., Kim J., Lee C. W., Min B. S., Seo Y. S., Kim J. C. A diketopiperazine, Cyclo-(L-Pro-L-Ile), derived from Bacillus thuringiensis JCK-1233 controls pine wilt disease by elicitation of moderate hypersensitive reaction. Front. Plant Sci. 2020;11:1023. doi: 10.3389/fpls.2020.01023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pršić J., Ongena M. Elicitors of plant immunity triggered by beneficial bacteria. Front. Plant Sci. 2020;11:594530. doi: 10.3389/fpls.2020.594530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  35. Sharma S., Kumar P., Singh A. Systemic acquired resistance vs induced systemic resistance: a review. Agric. Rev. 2024;45:410–419. [Google Scholar]
  36. Shepherdson E. M., Baglio C. R., Elliot M. A. Streptomyces behavior and competition in the natural environment. Curr. Opin. Microbiol. 2023;71:102257. doi: 10.1016/j.mib.2022.102257. [DOI] [PubMed] [Google Scholar]
  37. Shimizu M. Endophytic actinomycetes: biocontrol agents and growth promoters. In: Maheshwari D. K., editor. Bacteria in Agrobiology: Plant Growth Responses. Springer; Berlin, Germany: 2011. pp. 201–220. [Google Scholar]
  38. Shirling E. B., Gottlieb D. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 1966;16:313–340. [Google Scholar]
  39. Vu T. T., Kim H., Tran V. K., Vu H. D., Hoang T. X., Han J. W., Choi Y. H., Jang K. S., Choi G. J., Kim J. C. Antibacterial activity of tannins isolated from Sapium baccatum extract and use for control of tomato bacterial wilt. PLoS One. 2017;12:e0181499. doi: 10.1371/journal.pone.0181499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vurukonda S. S. K. P., Giovanardi D., Stefani E. Plant growth promoting and biocontrol activity of Streptomyces spp. as endophytes. Int. J. Mol. Sci. 2018;19:952. doi: 10.3390/ijms19040952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wang Z., Luo W., Cheng S., Zhang H., Zong J., Zhang Z. Ralstonia solanacearum – A soil borne hidden enemy of plants: research development in management strategies, their action mechanism and challenges. Front. Plant Sci. 2023;14:1141902. doi: 10.3389/fpls.2023.1141902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wu S., Su H., Gao F., Yao H., Fan X., Zhao X., Li Y. An insight into the prevention and control methods for bacterial wilt disease in tomato plants. Agronomy. 2023;13:3025. [Google Scholar]
  43. Zeng Q., Soltaniband V. Biological control microorganisms that induce plant defense responses. Annu. Rev. Phytopathol. 2026;64 doi: 10.1146/annurev-phyto-011325-023109. In press. [DOI] [PubMed] [Google Scholar]
  44. Zhang Y., Wang Y., Chen J., Zhang Y. A review on the mechanism of soil flame disinfection and the precise control technology of the device. Agriculture. 2025;15:2447. [Google Scholar]
  45. Zhou W., Li M., Achal V. A comprehensive review on environmental and human health impacts of chemical pesticide usage. Emerg. Contam. 2025;11:100410. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PPJ-OA-06-2026-0082-Supplementary-Fig-1,2.pdf (408.7KB, pdf)
PPJ-OA-06-2026-0082-Supplementary-Fig-3.pdf (87.6KB, pdf)
PPJ-OA-06-2026-0082-Supplementary-Table-1.pdf (89.9KB, pdf)
PPJ-OA-06-2026-0082-Supplementary-Table-2,3.pdf (116.2KB, pdf)

Articles from The Plant Pathology Journal are provided here courtesy of The Korean Society of Plant Pathology

RESOURCES