Bacterial panicle blight (BPB) and seedling rot (BSR) caused by Burkholderia glumae pose serious threats to rice production (Mew et al., 2004; Ortega and Rojas, 2021). The disease disrupts grain filling, leads to empty panicles, and reduces seed quality, while also impairing seedling emergence and stand establishment. The diseases are particularly severe under warm and humid conditions during the flowering stage, making rice-growing regions such as Korea, Japan, and the United States particularly vulnerable, which leads to significant economic losses (Echeverri-Rico et al., 2021; Ham et al., 2011; Nandakumar et al. 2009; Noh et al. 2004). Despite the increasing incidence and economic impact of BSR in major rice-producing areas, effective and sustainable control strategies remain limited.
Biological control using beneficial microorganisms is a promising strategy for eco-friendly and sustainable management of plant diseases. Microbial biological control agents (BCAs), whether applied alone or as part of integrated pest management programs, offer multiple advantages, including environmental safety, specificity to target pathogens, and potential for long-term disease suppression (Lucas et al., 2015; Schisler et al., 2004). Various BCAs have been investigated for their ability to suppress BPB and BSR. For example, pretreatment of rice seeds with an avirulent B. glumae strain, N7503, significantly reduced BSR symptoms caused by virulent isolates (Furuya et al., 1991), while co-inoculation with an avirulent B. gladioli strain effectively controlled BPB in rice panicles (Miyagawa and Takaya, 2000). A genetically engineered Burkholderia strain expressing an N-acyl homoserine lactonase gene also successfully disrupted quorum sensing, reducing BSR severity (Cho et al., 2007). Strains such as Pseudomonas protegens PBL3 and Burkholderia cepacia PBL18 inhibit B. glumae growth and alleviate disease symptoms in rice seedlings (Ortega et al., 2020). Bacillus velezensis IBUN 2755 was reported to reduce B. glumae populations in both roots and shoots across the crop cycle, thereby preventing grain abortion and increasing yield stability (Pedraza-Herrera et al., 2021; Perea-Molina et al., 2022).
The successful field application and commercialization of microbial BCAs for disease control depend largely on the development of stable, effective, and user-friendly formulations. To ensure consistent performance, formulated BCAs must maintain high viability and efficacy under various environmental conditions (Berger et al., 2015). In this regard, endospore-forming Bacillus species offer superior stress tolerance, long shelf life, and multiple beneficial traits. Advances in formulation technologies such as wettable powders, liquid suspensions, and polymer-based delivery systems have further enhanced the practicality and reliability of Bacillus-based biocontrol products (Klein et al., 2016; Schisler et al., 2004).
In this study, we isolated Bacillus velezensis JBCS608 (JBCS608) from rhizosphere soil and evaluated its biocontrol potential against BSR in rice. The strain was characterized for its antagonistic activity against diverse seed-borne bacterial and fungal pathogens and for its plant growth-promoting traits. To facilitate field application, we developed two endospore-based bioformulations (talc-based wettable powder and molasses-humic acid liquid) and assessed their disease suppression efficacy, storage stability, and long-term cell viability under controlled conditions. Our results provide a foundation for the practical application of JBCS608 as an effective and environmentally sustainable BCA for the management of rice diseases.
Materials and Methods
Isolation and identification of strain JBCS608
Rhizosphere soil samples (Iksan, Korea) were collected from rice fields and suspended in 10 volumes of sterile distilled water (DW). The suspension was agitated at 180 rpm for 10 min at room temperature on a rotary shaker. Serial 10-fold dilutions were prepared, and 100 μL aliquots were spread onto Luria-Bertani (LB) agar plates and incubated at 28°C. Colonies with distinct morphological characteristics were subcultured on fresh LB agar and preserved in LB broth containing 20% glycerol at −70°C (Kang et al., 2023). The isolate was previously identified as Bacillus velezensis using whole-genome sequencing and phylogenetic analyses (Dutta et al., 2024).
Preparation of B. glumae-infected rice seeds
Rice seeds were artificially infected with the B. glumae strain KACC17230 following the method of Fang et al. (2009) with minor modifications. Briefly, healthy rice seeds were surface-sterilized with 2% sodium hypochlorite for 2 min and rinsed thoroughly with sterile DW. B. glumae cultures grown in LB broth at 28°C were centrifuged at low speed, and the pellets were resuspended in sterile DW containing 0.2% carboxymethyl cellulose (CMC; Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 1 × 108 colony-forming unit (CFU)/mL. Surface-sterilized seeds were immersed in a bacterial suspension (1:10, w/v) and incubated at 25°C with agitation (100 rpm) for 12 h. The inoculated seeds were air-dried at room temperature for 12 h before use (Kang et al., 2023).
Application of JBCS608 to B. glumae-infected rice seeds
JBCS608 cells cultured in LB broth at 28°C for 24 h were harvested and resuspended in sterile DW containing 0.2% CMC at final concentrations of 1 × 106, 107, or 108 CFU/mL. Infected rice seeds were incubated in the JBCS608 suspension at 28°C for 1 h with gentle shaking (100 rpm) to facilitate bacterial attachment. Seeds were then placed on sterile filter paper and air-dried at room temperature for 1 h. Treated seeds were sown in pots filled with nursery bed soil (Hungnong Bio Co., Seoul, Korea) and maintained in a growth chamber (28°C, 16 h light/8 h dark). The plants were watered daily without fertilization. Controls included seeds treated with sterile DW containing 0.2% CMC and an agrochemical seed disinfectant, prochloraz-copper chloride-tebuconazole (12.5 + 12.5%) suspension concentrate (2,000 times dilution for treatment) that was applied following the manufacturer’s instructions. Each treatment was performed in triplicate (10 seeds/pot) with additional large-scale tests using 100 seeds/plastic box. Disease incidence was evaluated 21 days after sowing and scored on a scale of 0-4: 0 = healthy seedlings, 1 = pale yellow leaves, 2 = chlorosis and stunting, 3 = death post-germination, 4 = rooted without germination. Disease severity was calculated using the following formula: disease index = [(0n0 + 1n1 + 3n2 + 5n3 + 7n4)/7N] × 100, where n0-5 is the number of leaves in each degree (0 to 4) and N is the total number of seedlings investigated. Experiments were repeated three times.
Application of JBCS608 to healthy rice seeds
Surface-sterilized, healthy rice seeds were treated with JBCS608 suspensions as described above and sown in nursery soil. The plants were grown at 30°C and >70% relative humidity. At 21 days after sowing, fresh and dry weights were measured. Each treatment consisted of three replicates of 50 seeds/pot, and the experiments were repeated three times.
Antagonistic activity assay of JBCS608 against bacterial and fungal pathogens
The antagonistic activity of JBCS608 was evaluated against bacterial pathogens, including B. glumae, B. plantarii KACC18964, B. gladioli KACC19137 (bacterial grain rot of rice), Acidovorax avenae KACC18468 (bacterial stripe), and Xanthomonas oryzae pv. oryzae KACC10332 (bacterial leaf blight). LB agar plates were prepared with each pathogen at a concentration of 1 × 106 CFU/mL. JBCS608 cells adjusted to 1 × 107 CFU/mL and inoculated (20 μL) onto 8 mm diameter paper disks (8 mm) placed on the LB agar. The plates were incubated at 28°C for 2 days and inhibition zones were measured (Kang et al., 2023).
The antifungal activity of JBCS608 was tested against Bipolaris oryzae (brown spot), Fusarium moniliforme (bakanae disease), Fusarium graminearum (Fusarium blight), Pyricularia grisea (rice blast), and Rhizoctonia solani (sheath blight), which were isolated and stored in our lab, using a dual culture technique. Fungal agar plugs (8 mm) from the margins of 5-7-day-old cultures were placed in the center of potato dextrose agar plates. The JBCS608-inoculated paper disks were positioned 2.5 cm away from the fungal agar plug. After incubation at 25°C for 7 days, inhibition zones were recorded. All experiments were conducted three replicates and repeated independently.
Production of antagonistic and plant growth-promoting compounds
Siderophore production by JBCS608 was determined using the chrome azurol S (CAS) agar method (Schwyn and Neilands, 1987). JBCS608 cultures (20 μL) were spotted on CAS agar and incubated at 30°C for 4 days; orange halos indicated positive siderophore production. Phosphate solubilization was tested using Pikovskaya agar (Pikovskaya, 1948) and silicate solubilization using glucose media (Lee et al., 2019). The JBCS608 cultures were inoculated onto sterile paper disks in each medium. They incubated at 30°C for 3 days and the formation of a clear zone around the colony was considered positive. hydrogen cyanide (HCN) production was determined using LB agar supplemented with glycine (Millar and Higgins, 1970). JBCS608 was streaked onto the medium, and a filter paper soaked in 0.5% (w/v) picric acid and 2% sodium carbonate was placed on the lid of the Petri dish. After incubation at 30°C, a color change of the filter paper indicated HCN production. Protease activity was assessed using skim milk agar (Smibert and Krieg, 1994). Clear zones indicated protease activity. All assays were performed three times.
Phytohormone production
For indole-3-acetic acid (IAA) production, JBCS608 was cultured in nutrient broth (OD600 = 0.5) and incubated at 30°C and 180 rpm for 48 h in the dark. After centrifugation (13,000 ×g, 10 min), the supernatant was mixed with Salkowski’s reagent (Sigma-Aldrich) (3:2, v/v) and incubated in the dark for 30 min. IAA concentration was measured spectrophotometrically at 530 nm (Apine and Jadhav, 2011). Cytokinin production was assessed using M9 medium supplemented with 0.2% casamino acids, 0.01% thiamine, and 2 pg/L biotin. JBCS608 (1 mL) was added to 100 mL M9 and incubated at 30°C, 180 rpm for 3 days. Absorbance at 665 nm was used for cytokinin quantification (Akiyoshi et al., 1987; Patel and Saraf, 2017). For gibberellic acid production, supernatants were acidified (pH 2.5), extracted with ethyl acetate (1:3, v/v), and absorbance was measured at 254 nm (Pandya and Desai, 2014). All experiments were performed three times.
Endospore production
Overnight cultures of JBCS608 in LB broth were spread on Schaeffer’s sporulation media (Sorokulova et al., 2008) and incubated at 37°C for 5 days to promote sporulation, and then cooled at 4°C for 30 min. Spores were collected by flooding the surface of the culture with cold sterile DW and gently scraping with a cell spreader. The spore suspension was heat-treated at 65°C for 30 min, centrifuged at 3,500 ×g at 4°C for 6 min, and washed vigorously five times with cold sterile DW. The final spore suspension was adjusted to 1 × 1010 spores/mL and stored at 4°C. Endospore formation was confirmed by malachite green staining and microscopic examination.
Preparation of powder and liquid formulations
A talc-based powder formulation of JBCS608 was prepared following the method of Vidhyasekaran et al. (1997) with minor modifications. Briefly, 100 g of sterilized talc powder was mixed with 1 g of CMC and 1.5 g of calcium carbonate. After autoclaving on two consecutive days for 30 min each, 50 mL of JBCS608 spore suspension (1.0 × 1010 spores/mL) was aseptically incorporated into the talc mixture and thoroughly mixed to form a paste. The mixture was air-dried in a laminar airflow cabinet at 25°C, pulverized using a mortar and pestle, and stored in sterile aluminum zipper bags (Labkom, Seoul, Korea) (Fig. 1). For liquid formulation, JBCS608 spores were suspended in a sterile mixture of 20% molasses and 1% humic acid (Sigma-Aldrich) (Myo et al., 2019; Wong et al., 2019). The formulation was thoroughly mixed and stored in sterile polyethylene bottles (Daejin Plastic, Seoul, Korea) for subsequent experiments.
Biocontrol assay with formulated products
B. glumae-infected rice seeds were treated with either powder or liquid formulations of JBCS608 adjusted to a final concentration of 1 × 108 CFU/mL with 0.2% CMC for 4 h under gentle agitation. Treated seeds were air-dried in the dark at room temperature for 30 min and sown in pots containing nursery bed soil. The plants were grown in a plant growth room (28°C, 16 h light/8 h dark) and watered daily. Control treatments included a carrier formulation without bacterial cells and an agrochemical seed disinfectant (prochloraz-copper chloride-tebuconazole). Each treatment was conducted with three replicates of 50 seeds/plastic box, and the experiment was repeated. Disease severity was assessed 21 days after sowing as described above.
Storage and viability of formulations
The wettable powder and liquid formulations of JBCS608 were packaged in sterile aluminum zipper bags and polyethylene flasks, respectively and stored at two different temperatures, 4°C and 15°C. For powder formulation, three replicate samples collected from two independently stored packages at each temperature were rehydrated in sterile DW (1:100, w/v) and mixed thoroughly. For liquid formulation, the stored flasks were shaken vigorously to ensure homogeneity, and an aliquot of suspension was diluted in sterile DW (1:9, v/v). The mixtures were then vortexed for 1 min and allowed to settle for 5 min at room temperature. Subsequently, 10-fold serial dilutions of each sample were prepared and the appropriate dilutions were plated on LB agar. The plates were incubated at 28°C for 48 h and colony numbers were enumerated. Results were expressed as CFU per gram for the powder formulation and CFU per milliliter for the liquid formulation. Viability was assessed at regular intervals for a year with the initial measurement (time zero) within 24 h of formulation.
Results
Biocontrol efficacy of JBCS608 against BSR
Optimizing the concentration of a BCA is essential for maximizing its efficacy under field conditions and enabling cost-effective large-scale applications. To determine the optimal dose, we evaluated the effects of bacterial cell densities of JBCS608 on the suppression of BSR in rice. Disease severity decreased significantly with increasing JBCS608 concentrations, with disease indices of 43.3%, 27.0%, and 20.0% at 1 × 106, 1 × 107, and 1 × 108 CFU/mL, respectively, compared to 60.0% in the untreated control (Fig. 2). Based on these results, 1 × 108 CFU/mL was selected as the optimal concentration for large-scale evaluation. Under these conditions, JBCS608 reduced disease severity to 18.0%, corresponding to a control efficacy of 68.9%. The incidence of BSR was reduced to 10.0% by agrochemical control (procloraz-copper chloride-tebuconazole). These results indicate that JBCS608 is an effective and potent biocontrol agent for the management of BSR in rice plants.
Antagonistic activity of JBCS608 against rice pathogens
To elucidate the potential mechanisms underlying the biocontrol activity of JBCS608, its antagonistic effects were assessed against major bacterial and fungal pathogens of rice. JBCS608 inhibited the growth of B. glumae, B. gladioli, B. plantari, and A. avenae, with inhibition zones ranging from 14 to 20 mm, while no inhibition was observed against X. o. pv. oryzae (Table 1, Supplementary Fig. 1). Antifungal assays revealed that JBCS608 suppressed the mycelial growth of multiple pathogens, including F. moniliforme, F. graminearum, B. oryzae, R. solani, and P. grisea, indicating broad-spectrum antagonistic activity (Supplementary Fig. 2). These results suggest that JBCS608 possesses a diverse range of antimicrobial properties that contribute to its biocontrol efficacy.
Plant growth-promoting effects of JBCS608
During the biocontrol assay, we observed growth promotion in rice plants as well as disease suppression by the treatment of JBCS608. Hence, we evaluated the growth-promoting activities of JBCS608 using healthy rice seeds. Seed coating with JBCS608 led to significant increases in both fresh and dry biomass, by 8.6% and 6.0%, respectively, compared with those of untreated control (Fig. 3). These results indicate that JBCS608 not only protects against B. glumae, but also promotes early-stage plant development, highlighting its dual functionality as a biocontrol and growth-promoting agent.
Production of antagonistic and plant growth-promoting compounds
To elucidate the functional traits associated with the biocontrol and plant growth-promoting activities, we evaluated the biochemical activities of JBCS608. The strain was found to produce siderophores and protease, which are known to limit pathogen growth through iron sequestration and lytic activity. JBCS608 also solubilized inorganic phosphate but did not exhibit silicate solubilization or HCN production (Supplementary Fig. 3). Among the phytohormones, IAA production was observed, whereas gibberellin and cytokinin production were not detected. These results support the multifaceted role of JBCS608 in antagonizing pathogens and stimulating plant growth.
Development of JBCS608 formulations and their biocontrol efficacy
The development of a stable formulation is crucial for field application and commercialization of BCAs. To facilitate field applicability, we developed two formulations, talc-based wettable powder and molasses humic acid liquid, using JBCS608 endospores. Both formulations significantly suppressed BSR in rice seedlings, reducing the disease incidence to 17.0% (powder) and 16.0% (liquid), compared with 50.0% in the untreated control (Fig. 4). Their biocontrol efficacy was comparable to that of freshly cultured cells (68.0% control efficacy), indicating that the formulations retained biological activity and are suitable for practical use in BSR management.
Storage stability and viability of JBCS608 formulations
Long-term viability is essential for the storage and distribution of microbial products. We assessed the shelf-life of both formulations at 4°C and 15°C over 12 months. Initial viable counts were 2.4 × 109 CFU/g in the powder formulation and 1.4 × 109 CFU/mL in the liquid formulation (Fig. 5). The viability of JBCS608 cells in both formulations gradually decreased over time at both storage temperatures. After 2 months of storage, viable cell counts in the powder formulation remained above 9.0 × 108 CFU/g at both temperatures, and were maintained at >1.0 × 108 CFU/g up to 6 months. In the liquid formulation, cell numbers were >7.0 × 108 CFU/mL at 2 months of storage and >1.0 × 108 CFU/mL at 6 months after storage. At 12 months, viable cell counts declined to 1.93 × 107 and 1.58 × 107 CFU/g (powder) and 1.05 × 107 and 5.8 × 106 CFU/mL (liquid) at 4°C and 15°C, respectively. These results confirm that both formulations maintain acceptable viability for at least 6 months under cold storage, although longer storage leads to suboptimal levels of viability.
Discussion
The BPB and BSR caused by B. glumae are recognized as major seed-borne threats to rice production (Naughton et al., 2016). As average global temperature rises, outbreaks of BPB are becoming more frequent and severe, underscoring the increasing relevance of the pathogen in both temperate and tropical rice-growing regions (Echeverri-Rico et al., 2021; Ham et al., 2011; Suárez-Moreno et al., 2019). Since B. glumae is predominantly seed-transmitted and rapidly proliferates in rice nurseries, the use of pathogen-free or disinfected healthy seeds is imperative to prevent early-stage infections that can compromise crop establishment and yield (Tsushima, 2011). Despite growing awareness of its importance, control strategies for BPB and BSR remain inadequate. Although breeding for resistance has yielded promising lines (Mizobuchi et al., 2013), such varieties are not yet widely adopted or stable across diverse environments. Similarly, molecular diagnostic tools (Cha et al., 2001; Fang et al., 2009) have enhanced early detection but do not offer curative benefits. Chemical controls, including oxolinic acid, have shown efficacy, but suffer from resistance, phytotoxicity, and environmental concerns (Ham et al., 2024; Hikichi, 1993; Maeda et al., 2004; Tsushima, 2011). Consequently, there is a need to develop reliable and environmentally-friendly strategies for the control of the disease.
Biological control using plant-beneficial microorganisms is a promising, environmentally friendly strategy. Seed priming with BCAs reduces seed-borne inoculum, and enhances seedling vigor and resilience. The JBCS608 isolated from rhizosphere soil demonstrated potent efficacy in suppressing BSR under growth room conditions. Disease suppression was dose-dependent, with the optimal concentration identified as 1 × 108 CFU/mL. This is in line with previous findings, where seed priming with high-density beneficial bacteria (e.g., avirulent B. glumae, Pseudomonas, or Bacillus strains) led to significant reductions in disease incidence (Furuya et al., 1991; Pedraza-Herrera et al., 2021). These results indicate the importance of delivering sufficient viable cells for the robust colonization and suppression of pathogens.
Biocontrol mechanisms, such as antibiosis, parasitism, induced resistance, and competition for space and nutrients, have been reported (Kakembo and Lee, 2019; Yu and Lee, 2015; Zhang et al., 2023). The mode of action of JBCS608 appears to be multifaceted. Broad-spectrum antagonism was observed against a wide array of phytopathogenic bacteria (B. glumae, B. gladioli, B. plantarii, A. avenae) and fungi (F. moniliforme, F. graminearum, B. oryzae, R. solani, and P. grisea). Although the specific metabolites responsible for inhibition remained unidentified in this study, previous reports have suggested that B. velezensis produces various lipopeptides (e.g., surfactin, iturin, and fengycin) and secondary metabolites (e.g., bacillaene and difficidin) that contribute to pathogen suppression (Dutta et al., 2024; Liu et al., 2024). Culture filtrates of Pseudomonas protegens or Bacillus strains directly suppressed B. glumae growth and reduced symptom development (Chung et al., 2015; Ortega et al., 2020). In addition, niche exclusion through competition, as demonstrated by B. velezensis IBUN2755 (Pedraza-Herrera et al., 2021; Perea-Molina et al., 2022), may also contribute to the antagonistic effects of JBCS608.
JBCS608 also exhibited plant growth-promoting properties by enhancing biomass accumulation in rice seedlings. The strain produced IAA, solubilized inorganic phosphate, and synthesized siderophores, all of which are well-established traits of plant growth-promoting rhizobacteria (Bhattacharyya et al., 2015; Zhang et al., 2023). These mechanisms likely contribute to improved root development and nutrient acquisition, although their exact contributions and microbial interactions require further investigation (Lugtenberg and Kamilova, 2009).
To facilitate commercialization, microbial BCAs must be formulated to ensure their viability, ease of application, and consistent performance. To this end, JBCS608 was successfully formulated as a talc-based wettable powder and molasses humic acid-based liquid, both using endospore preparations to enhance long-term stability. Talc-based formulations are widely used owing to their low moisture content, inert nature, and ability to protect microbial viability (Martínez-Álvarez et al., 2016; Vidhyasekaran et al., 1997). Previous studies have reported the successful stabilization of P. fluorescens and P. aeruginosa in similar talc-based or kaolin-based carriers (Kandasamy et al., 2009; Wong et al., 2019). Molasses and humic acid in liquid formulations have been shown to enhance microbial survival and activity during storage and application (Myo et al., 2019; Wong et al., 2019). The viability and performance of Streptomyces fradiae formulated as a liquid formulation using 1% humic acid and 20% molasses were enhanced (Myo et al., 2019). The efficacy of biocontrol formulations closely correlates with the concentration of viable cells and sustained microbial stability during storage (Gotor-Vila et al., 2019; Schisler et al., 2004). In general, microbial formulations must maintain at least 107 CFU/mL or CFU/g for 6-12 months at room or refrigerated temperatures (Berninger et al., 2018). Both formulations of JBCS608 retained viable cell densities for 6 months at 4°C and 15°C, which meets the commonly accepted threshold for shelf-stable commercial microbial products (Berninger et al., 2018), while maintaining disease suppression efficacy comparable to that of freshly cultured cells. Although viability declined slightly after extended storage, disease control remained effective, indicating the robustness of the formulations for practical use.
Taken together, JBCS608 showed multiple beneficial traits, such as broad-spectrum antagonism, plant growth promotion, and formulation versatility, which indicating its strong potential as a BCA for managing BPB and BSR in rice. The consistent suppression of B. glumae through both direct and indirect mechanisms indicate the potential of JBCS608 as an effective and sustainable alternative to chemical pesticides. Further validation under field conditions, detailed metabolomic profiling, and assessment of its compatibility with native soil microbiota will strengthen its application in rice disease management. The application of JBCS608 may significantly reduce chemical pesticide dependence, lower environmental risks, and enhance crop resilience under increasingly variable climatic conditions.
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