To select strains exhibiting antifungal activity against phytopathogenic fungi and having the ability to induce resistance, various rice samples were collected from rice fields at Chonnam National University and Gokseong in the Republic of Korea. The collected rice samples were categorized into leaves, stems, roots, and grains. Each rice sample (10 g) was then mixed with 100 mL of sterile distilled water and blended. After filtration through four layers of cheesecloth, the filtrates of leaves, stems, roots, and grains were diluted by 100-, 40-, 1,000-, and 100-fold, respectively (De Curtis et al., 2010). Subsequently, 100 μL of each dilution was spread onto tryptic soy agar (TSA; Becton, Dickinson and Co., Sparks, MA, United States) plates. The plates were incubated at 30°C for 1–3 days. Single colonies were isolated from TSA plates based on their morphological characters and further incubated on TSA at 30°C for 1 day. A total of 606 strains were isolated. Following this, each strain was incubated in tryptic soy broth (TSB; Becton, Dickinson and Co.) at 30°C with shaking at 150 rpm for 1 day. The strains were stored at −80°C in 30% glycerol until use.

The strains were first incubated on TSA at 30°C for 1 day. A single colony from each strain was then incubated in TSB at 30°C with rotary shaking at 150 rpm for 3 days. The culture broth from each bacterial strain was centrifuged at 13,000 rpm for 5 min to obtain the supernatant. The supernatants containing extracellular compounds were then filtered through a 0.2-μm membrane syringe filter to obtain axenic culture filtrates for antimicrobial activity testing (Han et al., 2022).

A 96-well microtiter plate was used to screen antagonistic bacteria with antifungal activity based on the mycelial growth inhibitory effects of their axenic culture filtrates against two pathogenic strains: F. graminearum KACC 47495 and Z-3639. F. graminearum KACC 47495 was obtained from the Korea Agricultural Culture Collection (Jeonju, Republic of Korea), and F. graminearum Z-3639 was obtained from Prof. Yin-Won Lee of Seoul National University. The two pathogenic strains were incubated in potato dextrose broth (PDB) at 25°C for 5 days and filtered through Miracloth. Following this, the mycelia were washed with sterile distilled water. After weighing the mycelia, sterile distilled water was added at a concentration of 50 mg/mL and the mycelia were homogenized using a homogenizer HG-15D (DAIHAN Scientific, Kangwon, Republic of Korea) at 10,000 rpm for 5 s to prepare a mycelial suspension. Then, the mycelial suspension (1%) was added to PDB. Different concentrations of culture filtrates (0.078–10%) were obtained by performing serial twofold dilutions of the culture filtrates of potential antagonistic strains (Nguyen et al., 2019). PDB was used as a negative control. Each well in the 96-well plate received a final volume of 100 μL of the fungal suspension treated with the culture filtrates of antagonistic bacteria. The plates were incubated at 25°C for 2–3 days. The experiments were conducted in triplicate.

JCK-7158 was selected by screening antagonistic bacteria exhibiting antifungal activity against F. graminearum KACC 47495 and F. graminearum Z-3639. The genomic DNA of JCK-7158 was extracted using the Dneasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and the conserved region of 16S ribosomal ribonucleic acid (rRNA) was amplified by PCR (Han et al., 2022). The PCR thermal profile consisted of an initial denaturation step at 95°C for 5 min; 30 cycles of 95°C for 30 s, annealing at 55°C or 65°C for 30 s, and extension at 72°C for 90 s; and a final extension step at 72°C for 10 min. The amplified fragments were sequenced using the primer 27F/1429R at Genotech Crop (Daejeon, Republic of Korea). The sequence of JCK-7158 16S rRNA was compared for similarity with the reference Bacillus sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST). Phylogenetic analysis was performed on MEGA version 6 using the maximum-likelihood method with 1,000 bootstrapping trials (Han et al., 2022).

To assess the mycelial growth inhibitory activity of the culture broth of JCK-7158 against various phytopathogenic fungi, a dual culture bioassay was performed. The phytopathogenic fungi used in the experiment were as follows: Botrytis cinerea, Colletotrichum coccodes, F. asiaticum, F. graminearum, F. oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, F. verticillioides, Gaeumannomyces graminis, Phytophthora infestans, Pythium ultimum, Rhizoctonia solani AG 2-2(IV) causing brown patch, R. solani AG 2-2(IV) causing large patch, and R. solani AG-4. The phytopathogenic fungi were cultured on PDA at 25°C for 5 days. Agar plugs with mycelia were collected with a 6-mm-diameter cork borer and placed at a point 2 cm away from the edge of the PDA plate. At 5 cm from the pathogen, each strain was streaked for a length of 5 cm. The untreated control groups were inoculated with the pathogens only. The experiments were three times repeated in triplicate. The plates were incubated at 25°C for 5 days. The inhibition rate was the mean of three runs with three replicates.

To assess the mycelial growth inhibitory activity of the culture filtrates against various phytopathogenic fungi, the following fungi were used: B. cinerea, Clarireedia jacksonii, C. coccodes, F. asiaticum, F. graminearum, F. oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, F. verticillioides, G. graminis, P. infestans, R. solani AG 2–2 (IV) causing large patch, and R. solani AG-4. The method was the same as that described for the MIC test.

A 3-day-old TSB culture of JCK-7158 (3 L) was centrifuged at 4,500 rpm for 20 min to obtain the culture supernatant (2.9 L). The culture supernatant was successively extracted twice with ethyl acetate (EtOAc) and butanol (BuOH). The organic solvent extracts were evaporated using a rotary evaporator (N-1110; EYELA Co., Tokyo, Japan) to obtain 594.8 mg of EtOAc extract and 8.1483 g of BuOH extract. Then, the antifungal activity of each solvent extract against F. graminearum KACC 47495 was tested using the 96-well microtiter plate method.

The active antifungal substance was separated from the BuOH layer, which exhibited strong antifungal activity. An aliquot of BuOH extract dissolved in methanol at a concentration of 50 mg/mL was loaded onto a thin layer chromatography (TLC) plate (20 × 20 cm, 0.2 cm thickness; Merck, Darmstadt, Germany) and eluted with chloroform:methanol:water (14:6:1, v/v/v). The TLC plate was then sterilized using UV light and placed on PDA medium incorporated with 1% F. graminearum suspension. The plate was incubated at 25°C, and the mycelial growth inhibitory activity was confirmed. Then, the most active fraction was loaded onto a preparative TLC (prep-TLC) plate (20 × 20 cm, 0.5 cm thickness; Merck, Darmstadt, Germany) and developed using the same solvent system. Five regions were separated, and the silica gel powder was then scraped off. Following this, elution was performed with methanol to obtain five fractions (BF1–BF5). The antifungal activity of the five fractions was tested. The active fraction BF4 was loaded onto the prep-TLC plate and developed using the chloroform:methanol:water (55:36:8, v/v/v) solvent system. Four regions were separated, and the silica gel powder from each region was scraped off. Following this, elution was performed with methanol to obtain four fractions (BF41–BF44). The four fractions and three cyclolipopeptide (CLP) standards (iturin A, fengycin, and surfactin; Sigma-Aldrich, St. Louis, MS, United States) were analyzed by TLC using chloroform:methanol:water (55:36:8, v/v/v). The TLC plate was then developed, observed under UV light at 254 and 365 nm wavelengths, and visualized by spraying p-anisaldehyde and water. The antifungal activity of the four fractions was also tested.

The antifungal metabolites of the partially purified fraction BF4-1 were analyzed by UHPLC-Q-Orbitrap-MS. The UPLC-heated electrospray ionization (HESI)-MS system consisted of the Dionex Ultimate 3,000 UHPLC module and a HESI quadrupole Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) controlled by Xactive Tune 1.1 and Xcalibur 2.2 software (Thermo Fisher Scientific, San Jose, USA), in addition to a C₁₈ reverse-phase column (Acquity UPLC BEH C₁₈ Column, 1.7 μm, 2.1 × 100 mm); it was maintained at 40°C. The mobile phases consisted of solvent A (0.1% formic acid in H₂O) and solvent B (0.1% formic acid in acetonitrile). The flow rate of the two solvents was 300 μL/min, and the injection volume was 2 μL. Gradient elution was performed as follows: 0–10 min, 95% A and 5% B; 10–15 min, 100% B; and 16–20 min, 95% A and 5% B. The conditions of the HESI quadrupole Orbitrap mass spectrometer were as follows: resolution, 70,000; collision energy, 30 V.

To assess the antifungal activity of VOCs produced by JCK-7158, 14 phytopathogenic fungi were used: B. cinerea, Clarireedia jacsonii, C. coccodes, F. asiaticum, F. graminearum, F. oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, F. verticillioides, G. graminis, P. infestans, P. ultimum, R. solani AG 2-2(IV) causing brown patch, R. solani AG 2-2(IV) causing large patch, and R. solani AG-4. The phytopathogenic fungi were cultured on PDA at 25°C for 3–7 days. To block direct contact, a bi-Petri dish with a separate center was used. PDA medium was dispensed on one side of the plate, and TSA medium was dispensed on the other side (Lim et al., 2017). Agar mycelium plugs (6 mm diameter) containing fresh mycelia of the phytopathogens from 3–7 days culture were cut using a sterile cork borer (6 mm diameter) and were inoculated at the center of the PDA medium. The JCK-7158 strain was streaked on the other side. The untreated samples were inoculated with pathogens only, without bacterial inoculum. The experiment was conducted in triplicate. The plates were sealed twice with Parafilm to prevent volatile organic compounds from leaking and incubated at 25°C. Fungal growth was assessed based on the diameter of mycelial growth, and the inhibition rate was calculated as a percentage (%). The experiments were three times repeated in triplicate and the inhibition rate was the mean of three runs with three replicates.

To analyze VOCs produced by the JCK-7158 strain, solid phase microextraction (SPME) GC–MS analysis was performed. The JCK-7158 strain was inoculated into 5 mL of TSB and precultured at 30°C with shaking at 150 rpm for 1 day. An aliquot of the preculture was inoculated into 20 mL of TSB at a concentration of 1% and incubated at 30°C with shaking at 150 rpm for 3 days. VOCs produced by JCK-7158 were collected using SPME fiber (Supelco, Bellefonte, PA, USA) in the headspace at 50°C for 30 min (Xie et al., 2018). The samples were analyzed by GC–MS using Shimadzu GC–MS QP2010 (Shimadzu, Kyoto, Japan) equipped with a DB-5 capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, Agilent). Helium was used as the mobile phase, and the flow rate was maintained at 1.0 mL/min. The injector temperature was maintained at 250°C. The column temperature was maintained at 60°C for 2 min, then increased to 250°C at a rate of 10°C/min, and maintained at 250°C for 20 min. The ionization voltage of the mass spectrometer was 70 eV, and the analysis was performed in the positive ion mode with a scan range of 50–400 m/z at 200°C.

The obtained mass spectrum was identified by comparing it with the data of the WILEY8 Library, and the content of each material was expressed as the area ratio of the total ion chromatogram (TIC) peak. The experiment was conducted in triplicate and uninoculated TSB medium was used as a negative control.

To assess the disease control activity of JCK-7158 against FHB in rice, wettable powder (WP) and suspension concentrate (SC) formulations of JCK-7158 culture broths were prepared. Oxidized starch (Floset Light; Daesang, Seoul, Republic of Korea) was added to a 3-day-old TSB culture broth of JCK-7158 at a concentration of 20% and then spray dried. The spray drying conditions were as follows: inlet temperature, 140°C; air blower, 10 m³/h; deblock, 50; and pump, 10 rpm. WP and SC formulations were prepared using the spray-dried material of JCK-7158 culture broth. The composition of the WP formulation was as follows: spray-dried material of JCK-7158, 10 g; white carbon, 15 g; CR-SDS, 12.5 g; CR-WP100, 12.5 g; and kaolin, 50 g. Each component was added and thoroughly mixed. The composition of the SC formulation was as follows: spray-dried material of JCK-7158, 100 g; CR-NF135B, 30 mL; propylene glycol, 50 mL; xanthan gum 1.0% solution, 100 mL; sodium benzoate, 2 g; CR-SAG672, 1 mL; and distilled water, 719 mL. Each component was added and stirred for 3 h.

Transgenic A. thaliana plants expressing GUS fused with the pathogenesis-related gene 1 (PR1) promoter were used to examine the expression of PR1, a known induced resistance marker gene. The seeds were initially sterilized with 95% ethanol for 30 s and then sterilized with a bleach solution (2% NaOCl, 0.05% Tween 20) for 5 min. The remaining solution was washed thrice with sterile distilled water and stored for 2 days at 4°C under dark conditions (Mishra et al., 2016). The immersed seeds were placed on half-strength Murashige–Skoog (MS) medium (2.2 g MS salt, 10 g sucrose, and 8 g phyto agar/L) containing 50 μg/mL kanamycin (Deng et al., 2021). The seeds were incubated on MS medium for 12 days with a photoperiod of 16 h in a plant growth chamber at 25°C and 80% relative humidity. Various concentrations of culture broth, culture filtrate, and cell suspension of the JCK-7158 strain and two JCK-7158 formulations (SC and WP) were used as samples. The JCK-7158 strain was incubated at 30°C with shaking at 150 rpm for 1 day. All treatment groups were diluted with sterile distilled water by 500-, 1,000-, 2,000-, and 4,000-fold, respectively, following which 2.5 mL of each culture diluent was poured into each well of a 24-well plate. Two 12-day-old A. thaliana plants were placed in each well and maintained at room temperature in an orbital shaker for 48 h. SA (0.1 and 0.01 mM) was used as a positive control, and TSB (0.1%) was used as a negative control. GUS staining was performed using a previously reported method (Kondo et al., 2014). After 48 h, the plants were immersed in 90% acetone at 4°C for 1 h to fix reacted plants and then washed twice with 0.1 M sodium phosphate buffer (pH 7.0), followed by immersion in GUS staining solution [0.1% Triton-X, 2.5 mM ethylenediaminetetraacetic acid, 100 mM sodium phosphate buffer, 2.5 mM potassium ferrocyanide, 2.5 mM potassium ferricyanide, and 2 mM X-GlucA (Duchefa, X1405)] under dark conditions at 4°C. The plates were incubated overnight in a 30°C water bath. The staining solution was then removed and the reaction mixture was fixed in 70% ethanol for 1 h, followed by immersion in 90% ethanol at 1-h intervals to remove chlorophyll. The blue color was observed using a microscope (Stemi 508; Carl Zeiss, Germany).

Rice seeds (Oryza sativa cv. Samkwang) provided by the National Institute of Crop Science (Rural Development Administration, Suwon, Republic of Korea) were used for the rice FHB bioassay. The preparation method of rice plants is described in detail in the Supplementary Information.

A sample was prepared with a 3-day-old TSB culture broth of JCK-7158 (OD₆₀₀ = 0.8). The 3-day-old TSB culture broth of JCK-7158 was then centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.2-μm membrane syringe filter to obtain the culture filtrate. An equal amount of distilled water was added to the pellet to obtain the cell suspension. The culture broth, culture filtrate, and cell suspension of JCK-7158 were further diluted with distilled water by 1,000- and 2,000-fold, following which Tween 20 was added at a concentration of 250 μg/mL. Both SC and WP formulations of JCK-7158 were treated at 1,000- and 2,000-fold dilutions, respectively. Before applying the culture broth, culture filtrate, and cell suspension samples, the rice plants were pretreated with Tween 20 solution (250 μg/mL). The JCK-7158 samples were applied twice by foliar spray at the booting and heading stages at a 1-week interval. Distilled water containing 250 μg/mL of Tween 20 was used as a negative control for the culture broth, culture filtrate, and cell suspension samples. Distilled water was used as a negative control for the formulations. The synthetic fungicide Peulrei (13% propiconazole, 13% difenoconazole; Syngenta Co., Seoul, Republic of Korea) was used as a positive control and was treated at a 2,000-fold dilution a day before inoculation.

The treated rice plants were inoculated with the rice FHB pathogen F. asiaticum 031 at the flowering stage a week after the second application of the JCK-7158 samples. F. asiaticum 031 was isolated from the infected tissue of rice by Prof. Seo Jeong-Ah (Soongsil University, Seoul, Republic of Korea). The pathogen was cultured on PDA at 25°C for 4–5 days. Then, agar plugs containing the mycelia of F. asiaticum 031 were placed in carboxymethyl cellulose broth and incubated at 25°C with shaking at 150 rpm under light conditions for 7 days. The carboxymethyl cellulose broth of F. asiaticum 031 was collected through filtration with four layers of Miracloth to obtain spores (Strange and Smith, 1971). The spore suspension was counted under a microscope (Axio Imager.A2; Carl Zeiss, Yena, Germany) using a hemocytometer. The density of the spore suspension was adjusted to 2.0 × 10⁵ spores/mL, and Tween 20 was then added to the spore suspension at a concentration of 500 μg/mL. The pathogen was inoculated onto rice plants by spraying 1.6 mL spore suspension on each rice panicle. Disease severity was visually evaluated 7 days after pathogen inoculation, with the percentage ranging from 0 to 100%. The experiments were repeated three times in triplicate; each replicate consisted of six panicles. Disease severity, disease incidence, FHB index, and control value of FHB index were calculated using Equations 1–4, respectively. The values were the mean ± standard deviations of three runs with three replicates each.

The control efficacy of JCK-7158 SC against FHB in rice was evaluated in a rice field (O. sativa cv. Samkwang) located at Chonnam National University, Gwangju, Republic of Korea. The replicate plots of the field experiment were 1 × 1 m plots, with a spacing of 1 m between plots. The experiment was conducted in triplicate and using a randomized complete block design. A total of 90 spikes (30 spikes per replicate) were used per treatment. JCK-7158 SC was diluted by 1,000- and 2,000-fold with water. Each sample, approximately 1 L per plot, was treated by foliar spray 2 weeks (booting stage) and 1 week (heading stage) before inoculation. The treatment concentrations and timings were based on the results of in vivo bioassay in this study and our previous paper (Jeon et al., 2022). Distilled water was used as a negative control, and the synthetic fungicide Peulrei was used as a positive control, which was applied a day before pathogen inoculation. F. asiaticum 031 was prepared and inoculated into the rice plants, as described in the in vivo bioassay. Disease severity and disease incidence were assessed thrice at 2-week intervals from 2 weeks after pathogen inoculation. The formulae used to calculate disease severity, disease incidence, and control values are described in the previous section.

Mycotoxin analysis was conducted to determine whether the accumulation of Fusarium mycotoxins, such as NIV and ZEA, in rice grains is reduced on treatment with JCK-7158 SC under the field condition. The grains were harvested 6 weeks after pathogen inoculation and dried for 3 days, following which the dried samples were coarsely ground.

NIV was extracted using a DON-NIV WB column (Vicam, Milford, USA), according to the manufacturer’s recommendations. The extracts were completely dried using nitrogen gas and subsequently redissolved in 1 mL of sterile distilled water:acetonitrile:methanol (90:5:5, v/v/v) solution. The resulting solution was then filtered through a 0.22-μm membrane filter. The experiment was conducted in triplicate. The mycotoxin was quantitively analyzed using the Waters ACQUITY UPLC H-Class System equipped with a PDA detector at 218 nm. The column used was Xselect CSH C₁₈ (2.1 × 100 mm, 2.5 μm); it was maintained at 40°C. The mobile phases consisted of solvent A (distilled water), solvent B (acetonitrile), and solvent C (methanol) (90:5:5, v/v/v). The flow rate was 0.3 mL/min for 10 min, and the injected volume was 10 μL.

ZEA was extracted using a ZearalaTest™ WB column (Vicam, Milford, USA), according to the manufacturer’s recommendations. The extracts were eluted with 1.5 mL of methanol. The methanol solution was mixed with 1.5 mL of sterile distilled water and then filtered through a 0.22-μm membrane filter. The samples were analyzed using UPLC-PDA (Waters, Scotland) in triplicate. The mycotoxin was quantitively analyzed using the Waters ACQUITY UPLC H-Class System equipped with an FLD detector at λ_ex 274 nm and λ_em 440 nm. The column used was Xselect CSH C₁₈ (2.1 × 100 mm, 2.5 μm); it was maintained at 40°C. The mobile phases consisted of solvent A (distilled water), solvent B (acetonitrile), and solvent C (methanol) (43:35:22, v/v/v). The flow rate was 0.3 mL/min for 10 min, and the injected volume was 10 μL.

In rice, the effects of JCK-7158 on the expression of defense-related genes were analyzed to understand the defense mechanism of JCK-7158 against FHB. The culture broth, culture filtrate, and cell suspension of JCK-7158 (1,000-fold dilution) were treated by foliar spray 2 weeks and 1 week before inoculation into rice. Following this, the treated rice samples were inoculated with F. asiaticum spores (2.0 × 10⁵ spore/mL) in Tween 20 solution (250 μg/mL). Tween 20 solution (250 μg/mL) was used as a negative control. Rice leaves were collected 1, 3, and 7 days after the first treatment; 1, 3, and 7 days after the second treatment; and 1 and 2 days after inoculation of the pathogen. The total RNA of rice leaves was extracted using the IQeasy™ Plus Plant RNA Extraction Mini Kit (iNtRON Biotechnology, Seong-nam, Republic of Korea), according to the manufacturer’s recommendations. cDNA was synthesized using the oligo-(dT) primer and SuperScript™ IV reverse transcriptase (Invitrogen Inc., Carlsbad, CA, United States), according to the manufacturer’s instructions. Real-time PCR was performed on a real-time PCR detection system (Bio-Rad CFX 96; Bio-Rad Laboratories, Hercules, CA, United States) using iQ™ SYBR Green Supermix (BioRad Laboratories), according to the manufacturer specifications.

The PCR primers used in this study were synthesized by Genotech (Daejeon, Republic of Korea) and are listed in Supplementary Table S1. The actin gene was used as a reference gene (Jannoey et al., 2017). The phenylalanine ammonia-lyase gene (OsPAL1), rice nonexpressor of PR gene 1 (OsNPR1), PR1, and PR10 were used as SA signaling pathway-related genes (Sathe et al., 2019; Cheng et al., 2020). The lipoxygenase gene (LOX) was involved in the JA signaling pathway (Sathe et al., 2019), and the 1-aminocyclopropane-1-carboxylic acid synthase gene (OsACS1) was involved in the ET signaling pathway (Yoo et al., 2018). The catalase gene (CAT) and alternative oxidase gene (AOX1a) were recognized as genes related to reactive oxygen species (ROS) scavenging (Jannoey et al., 2017; He et al., 2021).

All statistical data were expressed as the mean ± standard deviation (SD) of replicates using SPSS software (version 21.0 for Windows; SPSS, IBM Corp., Armonk, NY, United States). Statistical differences were assessed using one-way ANOVA and determined using Duncan’s test (p < 0.05).

Of the 606 bacterial strains, 10 strains exhibited strong antifungal activity against F. graminearum KACC 47495 and F. graminearum Z-3639 (Table 1). Of these 10 strains, the JCK-7158 strain exhibited the strongest antifungal activity with an MIC value of 1.25%. Therefore, the JCK-7158 strain was further studied.

Based on phylogenetic analysis (Figure 1) and BLAST analysis against the NCBI database using the 16S rRNA sequence of JCK-7158, the strain was identified as B. velezensis. This strain was deposited in the Korean Collection for Type Cultures (KCTC) as KATC15169BP.

JCK-7158 produced extracellular enzymes, such as protease, chitinase, cellulase, and gelatinase (Supplementary Figure S1). Secondary metabolites, including the 2,3-butanediol precursor acetoin and the plant growth hormone IAA, were also released by JCK-7158 (Supplementary Figure S2).

The MIC test was performed to examine the in vitro antifungal activity of JCK-7158 against various phytopathogenic bacteria. The JCK-7158 strain exhibited the strongest antifungal activity with MIC values of 1.25% against C. jacksonii and F. graminearum. It also exhibited antifungal activity against B. cinerea, C. coccodes, and G. graminis, among others, with MIC values of 5%. These results confirmed that the JCK-7158 strain exhibited strong antifungal activity against various phytopathogenic fungi.

A dual culture bioassay was also performed to assess the mycelial growth inhibitory activity of JCK-7158 against various phytopathogenic fungi. The JCK-7158 strain exhibited more than 50% inhibitory activity against most pathogens used in the experiment. In particular, it exhibited strong mycelial growth inhibitory activity against B. cinerea, F. oxysporum f. sp. cucumerinum, and F. asiaticum (Table 2; Supplementary Figure S3).

The culture filtrate of JCK-7158 was extracted with EtOAc and BuOH to isolate the antifungal metabolites. The BuOH extract of JCK-7158 exhibited antifungal activity against F. graminearum KACC 47495, as assessed by bioautography (Supplementary Figure S4A). The BuOH extract of JCK-7158 showed an MIC value of 125 μg/mL against F. graminearum KACC 47495. Among the five fractions obtained from prep-TLC, the BF4 fraction was the most active against F. graminearum KACC 47495. To isolate pure substances from the BF4 fraction, prep-TLC was performed and four fractions were obtained. Of these, BF4-1, BF4-2, and BF4-3 were found to contain iturin A as a major substance (Supplementary Figure S4B). BF4-1 exhibited antifungal activity against F. graminearum KACC 47495 with an MIC value of 125 μg/mL. LC–MS analysis of the BF4-1 fraction revealed an active metabolite, iturin A, with a molecular weight of 1043.5495 in the positive ion mode. Moreover, the chemical composition of another active compound having a peak molecular weight of 1036.68757 in the positive ion mode was C₅₃H₉₃O₁₃N₇, which was consistent with that of surfactin (Ma et al., 2016; Figure 2). In vitro, the antifungal activity of BF4-1 was assessed against various phytopathogenic fungi using the MIC method. BF4-1 exhibited the lowest MIC value of 31.25 μg/mL against C. coccodes, followed by C. jacksonii, R. solani AG 2–2 (IV) causing large patch, and R. solani AG-4 (Supplementary Table S2).

JCK-7158 not only produced antifungal compounds but also generated VOCs that inhibited the growth of various phytopathogenic fungi (Supplementary Figure S5). The inhibition efficacy against B. cinerea was the strongest at approximately 51%, followed by F. graminearum at approximately 49% (Table 3).

VOCs produced by JCK-7158 inhibited the growth of various phytopathogenic fungi. Therefore, GC–MS analysis was performed to identify VOCs produced by JCK-7158. Five VOCs were detected within 7 min (Supplementary Table S3). By comparing the mass spectra of the substances in the library, the chemical structures of the five substances were found to be 6-methylheptan-2-one (34.89%), heptane-2-one (24.06%), 5-methyl-2-heptanone (20.75%), 2,5-dimethylpyrazine (15.94%), and 5-methylhexan-2-one (4.36%). Of these, the JCK-7158 strain produced 6-methylheptan-2-one as a major volatile substance.

All JCK-7158 treatments, such as the culture broth, culture filtrate, cell suspension, and SC and WP formulations, showed positive reactions with regard to PR1::GUS expression. As shown in Figure 3, a blue color appeared along the leaf veins in transgenic A. thaliana leaves in all treatments. However, the untreated control did not show any blue color. The positive control SA also showed a very strong blue color in the treated leaves.

The culture broth, culture filtrate, and cell suspension of JCK-7158 effectively controlled the development of FHB in rice (Table 4; Figure 4). At 1,000- and 2,000-fold dilutions, the disease control values were 50 and 35% for the culture broth, 44 and 36% for the culture filtrate, and 48 and 42% for the cell suspension, respectively, which were similar to the findings in the positive control group. Meanwhile, out of the two JCK-7158 formulations (WP and SC) under greenhouse conditions, the 1,000-fold diluted SC formulation most effectively reduced the development of FHB in rice with a control value of 65.5%; this value was higher than that in the positive control group (44.9%) (Table 5; Figure 5). The SC formulation exhibited higher disease control efficacy than the WP formulation.

To assess the disease control efficacy of JCK-7158 SC under field conditions, 1,000- and 2,000-fold dilutions of JCK-7158 SC were applied 2 weeks and 1 week before pathogen inoculation. At 2, 4, and 6 weeks after pathogen inoculation, treatment with 1,000-fold diluted JCK-7158 SC exhibited control values of 54, 55, and 44%, respectively, while treatment with 2,000-fold diluted JCK-7158 SC exhibited control values of 24, 20, and 21%, respectively. The control values of the positive control Peulrei were 37, 39, and 28% after 2, 4, and 6 weeks, respectively. Thus, the 1,000-fold diluted JCK-7158 SC formulation controlled FHB in rice more effectively than the synthetic fungicide Peulrei (Table 6; Figure 6).

Rice yield was measured by weighing 30 spikes with husk. Compared with the untreated control, the weight of rice grains increased on treatment with JCK-7158 SC at a 1,000-fold dilution. The yield of the untreated control rice was 97.47 g, while that of rice treated with 1,000-fold diluted JCK-7158 SC was 107.91 g. Thus, 1,000-fold diluted JCK-7185 SC significantly increased rice yield compared with the untreated control (Table 7; Supplementary Figure S6).

Mycotoxins, such as NIV and ZEA, were quantitatively analyzed in the rice grains treated with JCK-7158 SC. Only NIV was detected in all the samples. The NIV content of rice treated with 1,000-fold diluted JCK-7158 SC was 169.29 μg/kg, while that of the untreated control rice was 285.09 μg/kg (Table 8), indicating a 40.62% decrease in NIV accumulation in rice grains on treatment with 1,000-fold diluted JCK-7158 SC.

To assess the effect of JCK-7158 on the expression of plant defense-related genes, qRT-PCR was performed using JCK-7158-treated rice leaves. Compared with the untreated control group, the expression level of PAL1 was upregulated by 2.3- and 4.57-fold in leaves treated with the culture broth and culture filtrate of JCK-7158, respectively, in the 1 T1 group (1 day after the first treatment). In cell suspension-treated leaves, the expression level of PAL1 was significantly upregulated in the 1 T3 group (3 days after the first treatment) and the 2 T1 group (1 day after the second treatment) (Figure 7A). Compared with the untreated control group, the expression level of NPR1 was upregulated by 5.06-, 5.68- and 2.85-fold on treatment with the culture broth, culture filtrate, and cell suspension of JCK-7158, respectively, in the 2 T1 group (Figure 7B). Moreover, compared with the untreated control group, the expression level of PR1 was upregulated by 3.81-, 3.60-, and 2.40-fold on treatment with the culture broth, culture filtrate, and cell suspension of JCK-7158, respectively, in the 1 T1 group (Figure 7C). Compared with the untreated control group, the expression level of PR10 was also upregulated by 12.16-, 23.65-, and 24.43-fold on treatment with the culture broth, culture filtrate, and cell suspension of JCK-7158, respectively, in the 2 T1 group (Figure 7D).

A gene involved in the JA-related pathway, LOX, was upregulated by 3.22- and 2.70-fold in leaves treated with the culture broth and culture filtrate of JCK-7158, respectively, in the 1 T1 group. Moreover, LOX expression was significantly upregulated in leaves treated with the cell suspension of JCK-7158 in the 2 T3 group (Figure 7E). A gene involved in ET-related pathways, OsACS1, was significantly upregulated following all treatments in the 2 T7 and I2 groups (Figure 7F). Of the genes involved in ROS-related pathways, AOX1 was significantly upregulated by 2.36-, 2.66-, and 1.54-fold on treatment with the culture broth, culture filtrate, and cell suspension of JCK-7158, respectively, in the 2 T1 group (Figure 7G). CAT was upregulated following all treatments in 1 T1–I1 groups (Figure 7H). Thus, JCK-7158 enhanced the expression of all of the SA, JA, and ET signaling pathway-related genes and ROS-scavenging genes tested in this study.