Rice is cultivated on approximately 161 million acres worldwide, yielding 678.7 million tons annually. Of this, Asia accounts for approximately 143 million acres and produces 612 million tons (Asibi et al., 2019). Conversely, wheat production needs to increase to meet the demands of a global population projected to approach nine billion in 2050 (Figueroa et al., 2018). However, Fusarium head blight (FHB) commonly called scab, is a destructive fungal disease affecting major grain crops such as wheat, barley, maize, and rice caused by members of the Fusarium graminearum species complex. The primary causative agents of FHB are F. graminearum and F. asiaticum species (Goswami and Kistler, 2005; Lee et al., 2010; McMullen et al., 2012; Starkey et al., 2007). Infected cereals suffer significant reductions in grain yield and quality, and the contaminated grain is often laden with trichothecene mycotoxins and estrogenic compounds (Kim et al., 2005). Yield losses attributed to FHB can reach up to 80% under severe epidemics (Alisaac and Mahlein, 2023). Among the mycotoxins of great concern are the trichothecenes deoxynivalenol, nivalenol, and HT2/T2, along with the estrogenic mycotoxin zearalenone (Bottalico and Perrone, 2002).
Various strategies have been employed to manage FHB, including agronomic practices, chemical control, and breeding for resistance (Dill-Macky and Jones, 2000; Dweba et al., 2017; Jia et al., 2018; Jouany, 2007; Shah et al., 2018). However, these methods face significant drawbacks: agronomic practices are highly dependent on environmental conditions; chemical control can lead to fungicide resistance and ecological pollution; and breeding programs are time-consuming, often yielding only a limited number of partially resistant cultivars (Anderson et al., 2020; Chen et al., 2021b; Parry et al., 1995; Trail et al., 2002; Wegulo et al., 2015; Zhao et al., 2022). These limitations have spurred interest in biological control agents (BCAs) as environmentally sustainable alternatives. BCAs suppress pathogens through direct mechanisms such as parasitism and antibiosis, as well as indirect mechanisms such as the induction of host resistance, competition for resources, and growth promotion (Legrand et al., 2017). Several antagonist microorganisms can manage FHB, including Bacillus spp., Pseudomonas spp., Trichoderma spp., and Streptomyces spp. (Torres et al., 2019). The control strategies can reduce disease severity and mycotoxin levels, no individual strategy offers significant control under conditions highly conducive to FHB development, indicating that integrated management approaches are regarded as the most effective strategy for mitigating FHB (Shude et al., 2020).
Among BCAs, Streptomyces spp., a group of Gram-positive filamentous actinobacteria have gained significant attention owing to their capacity to produce diverse antimicrobial metabolites and to activate host defense responses (Gopalakrishnan et al., 2020). Previous reports show that numerous Streptomyces sp. strains suppress FHB via direct and indirect mechanisms (Colombo et al., 2019; De Troyer et al., 2025; Jung et al., 2013; Mouloud et al., 2015; Tian et al., 2024). Indirect mechanisms referred to as induced resistance of plants operate mainly via two pathways, including systemic acquired resistance (SAR) and induced systemic resistance (ISR). SAR is typically triggered via pathogen invasion and is mediated via salicylic acid (SA) signaling, leading to the activation of pathogenesis-related genes (Kamle et al., 2020; Pieterse et al., 2014). ISR is triggered via interactions between plants and beneficial rhizosphere microorganisms and is mediated primarily via jasmonic acid and ethylene signaling pathways (Knoester et al., 1999; Li et al., 2004; Pieterse et al., 2014). Previous studies show that the expression level of the pathogenesis-related gene 1 (PR1) gene serves as a reliable marker for the activation of SA-mediated signaling pathways (Niu et al., 2012; Park and Kloepper, 2000).
Following the report of Hazra and Purkait (2019), pesticide formulations are designed to facilitate efficient production, storage, transport, and field application, thereby ensuring safety, convenience, and cost-effectiveness. Therefore, formulation types that combine BCAs with other components represent a promising approach for long-term preservation and quality assurance (Palazzini et al., 2013, 2016). To our knowledge, few studies have systematically explored the application of Streptomyces-mediated induced resistance, field-stable formulation development, and their potential synergy with fungicides in FHB management.
Therefore, this study aims to evaluate the potential of an endophytic Streptomyces strain, JCK-7385, for the sustainable management of FHB. Specifically, we (1) screened its ability to induce plant resistance, (2) assessed its biocontrol efficacy against FHB, (3) developed a stable suspension concentrate (SC) formulation, and (4) investigated its integrated application with a chemical fungicide.
Materials and Methods
Bacterial strain isolation from rice
To isolate strains with antifungal activity against phytopathogenic fungi and potential resistance-inducing mechanisms, rice samples were collected from experimental fields at Chonnam National University and from cultivated fields in Gokseong, Republic of Korea. The collected plant materials were divided into leaf, stem, root, and grain sections. Following the isolation method described by Stoltzfus et al. (1997), with minor modifications, each 10 g subsample was suspended in 100 mL of sterile distilled water (SDW) and homogenized. The mixtures were then filtered through a 4-layer cheesecloth, and the resulting filtrates were diluted (100-, 40-, 1,000-, and 100-fold for leaves, stems, roots, and grains, respectively). From each dilution, 100 μL was spread onto tryptic soy agar plates (TSA; Becton, Dickinson and Co., Sparks, MD, USA) and incubated at 30°C for 1 to 3 days. Single colonies were subsequently cultured in tryptic soy broth (TSB; Becton, Dickinson and Co.) at 30°C with shaking at 150 rpm for 24 h. Each isolate was preserved at −80°C in 30% glycerol for long-term storage (Le et al., 2021). A total of 606 strains were isolated in this study.
Screening of antifungal and plant resistance-inducing activity using an isolated strain
The culture conditions of isolated strains. The strains were first cultured on TSA medium at 30°C for 24 h. A single colony was then transferred into TSB medium to prepare a seed culture (pre-culture) in a shaking incubator at 30°C and 150 rpm. After 24 h of incubation, 1% (v/v) of the pre-culture was inoculated into fresh TSB medium and incubated under the same conditions for 2 days to obtain the main culture. The resulting culture broth (CB) was centrifuged to obtain the cell pellet (Cell) and supernatant. The axenic culture filtrate (CF) was prepared via filtering the supernatant through a 0.2 μm hydrophilic syringe filter (Minisarts, Sartorius, Goettingen, Germany).
Dual culture bioassay
The JCK-7385 strain was tested for its ability to inhibit mycelial growth of F. graminearum and F. asiaticum using a dual-culture assay on agar plates. A 2-day-old JCK-7385 CB was streaked in a straight line 2 cm from the edge of the potato dextrose agar (PDA; Becton, Dickinson and Co.) plate, and a 5-day-old pathogen agar plug was placed on the opposite side, approximately 5 cm away. PDA plates inoculated only with the pathogen served as untreated controls. Plates were incubated at 25°C for 5-7 days (Singh et al., 2019).
Histochemical β-glucuronidase assay in Arabidopsis leaves plants
The plant resistance-inducing activity of isolated strains were conducted using the transgenic Arabidopsis thaliana plants expressing β-glucuronidase (GUS) fused to the PR1 promoter (Park et al., 2020). The plants were prepared and cultured following the methods as described previously (Yeo et al., 2024). The JCK-7385 samples were diluted in SDW at 500-, 1,000-, 2,000-, and 4,000-fold, and each dilution was added to a 24-well plate in triplicate. Subsequently, two 12-day-old Arabidopsis thaliana plants were placed in each well and incubated at 25°C on an orbital shaker for 2 days. SA served as a positive control, and TSB medium as a negative control. Histochemical GUS staining was conducted as described previously (Kondo et al., 2014). After 2 days of incubation, plants were immersed in 90% acetone in 2 mL Eppendorf tubes at 4°C for 1 h to fix the tissues, then washed twice with 0.1 M sodium phosphate buffer (pH 7.0) in 24-well plates. The washed plants were immersed in GUS staining solution containing 0.1% Triton-X, 100 mM sodium phosphate buffer, 2.5 mM potassium ferrocyanide, 2.5 mM potassium ferricyanide, and 2 mM X-GlucA (Duchefa, X1405). The plates were incubated overnight in a water bath at 30°C. The reaction was stopped via incubating the stained plants in 70% ethanol for 1 h, followed by several washing in 90% ethanol for 1 h to remove chlorophyll. The activity was assessed via the presence of blue coloration and observed under an optical microscope (Yeo et al., 2024).
Identification of the JCK-7385 strain
The JCK-7385 strain was incubated in TSB medium at 30°C, with shaking at 150 rpm for 2 days. Genomic DNA was extracted using the i-genomic BYF DNA Extraction Mini Kit (iNtRON Biotechnology, Inc., Seongnam, Korea) according to the manufacturer’s instructions. To identify the JCK-7385 strain, the 16S rRNA and four housekeeping genes including atpD, recA, ropB, and trpB were amplified. Primer pairs are listed in Supplementary Table 1, and amplification conditions were applied as described previously (Guo et al., 2008). Phylogenetic trees were constructed using the neighbor-joining method for the 16S rRNA gene and concentrated sequences of the four housekeeping genes (Saitou and Nei, 1987). Sequence alignment was conducted using MEGA X software, and sequences were manually trimmed at the same position before further analysis and submission to the National Center for Biotechnology Information database (Kumar et al., 2018b).
Biochemical characterization of the JCK-7385 strain
Indole-3-acetic acid production
The JCK-7385 CF strain was used to confirm the production of the plant growth hormone indole-3-acetic acid (IAA). The strain was cultured in TSB medium supplemented with 150 mg/L L-tryptophan at 30°C and 150 rpm for 2 days. The JCK-7385 CB was centrifuged at 13,000 rpm for 5 min at 4°C, and the supernatant was filtered through a 0.2 μm syringe filter. Subsequently, 1 mL of the CF was mixed with 2 mL of Salkowski’s reagent (150 mL H2SO4, 250 mL sterile water, 7.5 mL of 0.5 M FeCl3·6H2O) and incubated in the dark at 25°C for 20 min (De Oliveira-Longatti et al., 2014). TSB medium containing 150 mg/L L-tryptophan served as the negative control. The IAA production was indicated via the appearance of a pink coloration in the mixture.
Siderophore production
An experiment was conducted to verify siderophore production via the JCK-7385 strain using Chrome Azurol S (CAS) agar medium, prepared as previously described (Louden et al., 2011). A 2-day-old CB of the JCK-7385 strain was applied in volumes of 20, 40, and 60 μL onto sterile paper disks placed on CAS agar plates. Sterile TSB medium served as the negative control. The plates were incubated at 30°C for 7 days. Siderophore production was indicated via the formation of an orange halo around the paper disks, and the ratio of halo diameter to colony diameter was calculated as a quantitative measure.
1-aminocyclopropane-1-carboxylic acid deaminase production
1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase production via the JCK-7385 strain was conducted as follows: 1 mL of JCK-7385 CB was cultured in 50 mL of Dworkin and Foster (DF) broth medium and incubated for 24 h at 25 to 30°C with shaking at 200 rpm. The DF medium was prepared as follows: trace elements (10 mg H3BO3, 11.19 mg MnSO4·H2O, 124.6 mg ZnSO4·7H2O, 78.22 mg CuSO4·5H2O, and 10 mg MoO3) were dissolved in 100 mL of SDW and stored in the refrigerator. FeSO4·7H2O (100 mg) was dissolved in 10 mL of SDW and stored in the refrigerator. All of the other ingredients, including 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g MgSO4·7H2O, 2.0 g glucose, 2.0 g gluconic acid, 2.0 g citric acid, 2.0 g (NH4)2SO4, and 0.1 mL of each of the trace element and FeSO4·7H2O solutions, were dissolved in 1 L of distilled water and autoclaved. Moreover, the DF agar medium was prepared with the same materials and included 1.8% Bacto agar (Becton, Dickinson and Co.), then autoclaved and poured into Petri dishes. After 24 h, 1 mL of the culture was transferred to 50 mL of sterile DF minimal salts medium in a 250-mL flask containing 3.0 mM ACC, replacing (NH4)2SO4 as the nitrogen source, under the same conditions (Penrose and Glick, 2003). Following incubation in DF medium supplemented with ACC, the JCK-7385 CB was streaked using a loop onto DF agar plates containing 30 μmol ACC. Sterile TSB medium served as the negative control.
Extracellular enzymes activity
The extracellular enzyme activity of the JCK-7385 strain was evaluated using the clear zone method (Abbasi et al., 2019). The media compositions were as follows: chitinase (1% colloidal chitin, 1.5% agar), cellulase (0.4% carboxymethyl cellulose [CMC] sodium, 1.5% agar), and gelatinase media (10% gelatinase, 1.5% agar). Colloidal chitin was prepared as previously described (Hsu and Lockwood, 1975). Sterile paper disks were placed on each medium, and 20, 40, and 60 μL of JCK-7385 CF obtained from a 2-day-old CB were dispensed onto the disks to determine the extracellular enzyme activity. A sterile TSB medium served as the negative control, and all experiments were conducted in triplicate. The plates were incubated at 30°C for 1 to 7 days. Gelatinase activity was assessed directly, whereas Lugol’s solution (2.5 g/L iodine and 5 g/L potassium iodide) was applied to stain the chitinase and cellulase media plates to visualize the halo zones.
Formulation of the JCK-7385 strain
The JCK-7385 strain was formulated for use in the GUS assay and to evaluate its disease control efficacy against FHB in rice. Two types of formulations including wettable powder (WP) and SC formulations were prepared as follows: oxidized starch (Floset light) was added to a 2-day-old TSB CB of JCK-7385 at a concentration of 20% and then spray-dried. Spray drying conditions included: inlet temperature at 140°C, air flow at 10 m3/h, de-block at 50, and pump at 10 rpm. WP and SC formulations were prepared using the spray-dried JCK-7385 material. For the WP formulation (100 g total), the composition included spray-dried JCK-7385 (10 g), white carbon (15 g), sodium dodecyl sulfate (CR-SDS, 12.5 g), naphthalenesulfonic acid (CR-WP100, 12.5 g), and kaolin (50 g). Moreover, to prepare 1 L of JCK-7385 SC, the composition was spray-dried JCK-7385 (100 g), blend of sodium dioctyl sulfosuccinate and TSP ethoxylates (CR-NF135B, 30 mL), propylene glycol (50 mL), 1.0% xanthan gum solution (100 mL), sodium benzoate (2 g), antifoam emulsion (CR-SAG672, 1 mL), and distilled water (719 mL) (Yeo et al., 2024).
Disease control efficacy of JCK-7385 against FHB in rice under greenhouse conditions
Plant preparation and growth conditions of rice
Rice seeds (Oryza sativa cv. Samkwang) were used to evaluate the disease control efficacy of JCK-7385 against FHB under greenhouse conditions. Detailed methods of seed preparation and plant growth conditions are provided in the Supplementary Material.
Single treatment application
The JCK-7385 strain was cultured in TSB medium at 30°C, 150 rpm for 2 days to obtain a CB standardized to OD600 = 0.8. The culture was centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.2 μm membrane filter to obtain the axenic CF. CB, CF, cell suspension, and formulations (WP and SC) of JCK-7385 were diluted 1,000- and 2,000-fold in distilled water containing Tween 20 at a concentration of 500 μg/mL. JCK-7385 treatments were applied twice as foliar sprays at the booting and heading stages, with a 1-week interval (2 weeks before inoculation [2WBI] and 1WBI). Distilled water containing 500 μg/mL Tween 20 served as the negative control. A synthetic fungicide, Peulrei (13% propiconazole + 13% difenoconazole EC; Syngenta, Iksan, Korea), served as a positive control at a 2,000-fold dilution and was applied 1 day before inoculation (DBI).
The pathogen was inoculated at the flowering stage—7 days after the 2WBI treatment of samples and 1 day after the positive control treatment using a Fusarium asiaticum 031 spore suspension isolated from the infected rice tissues. The pathogen was cultured on PDA at 25°C for 5 days. Agar plugs containing F. asiaticum 031 mycelia were transferred to CMC broth and incubated at 25°C and 150 rpm under light for 7 days. After incubation, the spore suspension was collected via filtering using four layers of Miracloth to remove mycelia and agar plugs (Strange and Smith, 1971). Spore concentration was determined using a hemocytometer under a microscope (Axio Imager.A2, Carl Zeiss, Yena, Germany). The density of the spore suspension was adjusted to 2.0 × 105 spores/mL, and then Tween 20 was added at 500 μg/mL concentration. Rice panicles were inoculated via spraying 1.6 mL of the spore suspension per panicle. Disease severity was evaluated 7 days after inoculation using a visible scale from 0-100%. The experiment was conducted in triplicate, with each replicate consisting of six panicles. Disease severity, disease incidence, FHB index, and control value of the FHB index were calculated using the following equations (Yeo et al., 2024):
JCK-7385 SC shelf-life. The JCK-7385 SC formulation was prepared as described above and stored in an oven at 45°C for 18, 15, 12, 9, and 6 weeks, corresponding to 3, 2.5, 2, 1.5, and 1 year of the efficacy warranty period, respectively (National Laws of the Republic of Korea, 2023). The JCK-7385 SC samples were diluted 2,000-fold and supplemented with 500 μg/mL Tween 20, then applied twice as foliar sprays at the booting and heading stages with a 1-week interval (2WBI and 1WBI). Distilled water containing 500 μg/mL Tween 20 served as the negative control. The synthetic fungicide, Peulrei (2,000-fold dilution) was applied 1DBI as the positive control. Pathogen preparation, inoculation, and assessment were described in the previous experiment.
Integrated treatment application
The study by Singh and Chhatpar (2011) performed the integrated treatment of Streptomyces sp. and chemical fungicide to control the Fusarium sp. disease. In this study, the treatment was prepared with modifications: the JCK-7385 SC at a 2,000-fold dilution was applied twice as foliar sprays at the booting and heading stages with a 1-week interval (2WBI and 1WBI). Distilled water containing 500 μg/mL Tween 20 served as the negative control, while the chemical fungicide, Peulrei (2,000-fold dilution) was applied 1DBI as the positive control. Additionally, three integrated treatments containing JCK-7385 SC and Peulrei were tested to control FHB: 2WBI + 1WBI/1DBI; 2WBI/1DBI; and 1WBI/1DBI. Pathogen preparation, inoculation, and assessment were conducted as described in the previous experiment.
Disease control efficacy of JCK-7385 against FHB in wheat under greenhouse conditions
Eunpamil wheat seeds (Triticum aestivum L.) were used to evaluate the disease control efficacy of JCK-7385 SC against FHB under greenhouse conditions. To sterilize the seeds, they were placed in an ice bucket and treated with the synthetic fungicide Spotak (25% prochloraz EC, Syngenta) at a 2,000-fold dilution for 2 h. Following sterilization, the seeds were immersed in water overnight in a 30°C incubator for germination. Wheat seeds were sown in a 105-well tray filled with autoclaved rice nursery soil at three seeds per well. The trays were maintained in a cooling chamber at 10 ± 2°C for 3 weeks under a 16 h/8 h light/dark cycle. After 3 weeks, the seedlings were transplanted into plastic pots (nine seedlings per pot) containing a 1:1 mixture of nursery soil (Punong Co., Ltd., Gyeongju, Korea) and rice nursery soil (Chungnong Co., Ltd., Suncheon, Korea). The wheat plants were then cultivated in a greenhouse (20 to 25°C) for further experiments.
JCK-7385 SC containing 500 μg/mL Tween 20 was applied twice via foliar spray at booting and heading stages, with a 1-week interval (2WBI and 1WBI). Distilled water containing 500 μg/mL Tween 20 served as a negative control. The synthetic fungicide, Peulrei was applied at a 2,000-fold dilution as the positive control, treated 1DBI. Additionally, three combination treatments of JCK-7385 SC and Peulrei were tested: 2WBI + 1WBI/1DBI; 2WBI/1DBI; and 1WBI/1DBI.
The pathogen F. graminearum strain Z-3639 was cultured on PDA at 25°C for 4-5 days. Agar plugs containing actively growing mycelia were then transferred to CMC broth and incubated at 25°C under light conditions with shaking at 150 rpm for 7 days (Cappellini and Peterson, 1965). After incubation, the mycelium and agar plugs were filtered out with 4 layers of Miracloth. The resulting spore suspension was quantified using a hemocytometer under a microscope. The density of the spore suspension was adjusted to 5.0 × 105 spores/mL, and Tween 20 was added to a final concentration of 500 μg/mL.
Inoculation was conducted via pipetting 10 μL of the spore suspension onto the middle spikelet of each wheat spike (Feng et al., 2018). Disease severity was visually evaluated 7-14 days post-inoculation on a scale of 0-100%. The experiment was conducted in triplicate, with each replicate consisting of six heads. Disease severity, disease incidence, FHB index, and the control value of the FHB index were calculated as previously described in the rice experiment.
Disease control efficacy of JCK-7385 against FHB in rice field
The control efficacy of JCK-7385 SC against rice FHB was evaluated in a field experimental located at Chonnam National University, Gwangju, South Korea. The experiment was arranged in a randomized complete block design with triplicate plots (1 m × 1 m) separated by 1 m. Each block contained 90 spikes, with 30 spikes per replicate.
F. asiaticum 031 was prepared and inoculated into treated plants following the procedure described in the greenhouse experiments. Disease severity and incidence were evaluated three times at 2-week intervals, beginning 2 weeks after inoculation (WAI). Disease severity, disease incidence, FHB index, and the control value of the FHB index were calculated as previously described in the rice greenhouse experiments.
In 2023, a single treatment was applied for FHB control. JCK-7385 SC was diluted 1,000- and 2,000-fold with water, and Tween 20 was added at a concentration of 500 μg/mL. Each sample (2 L) was applied as a foliar spray 2WBI at the booting stage and 1WBI at the heading stage. Distilled water containing 500 μg/mL Tween 20 served as the negative control, while a chemical fungicide (Peulrei) served as the positive control, applied 1DBI of the pathogen.
In 2024, two approaches were tested for FHB control: a single treatment with JCK-7385 SC (2,000-fold dilution) and an integrated treatment combining JCK-7385 SC (2,000-fold dilution) with the chemical fungicide (Peulrei). The JCK-7385 SC sample was prepared following the same method as in the previous year. The single JCK-7385 SC treatment (2 L) was applied as a foliar spray 2WBI at the booting stage and 1WBI at the heading stage. The integrated treatment was applied separately at 2WBI with JCK-7385 SC and 1DBI with Peulrei. Distilled water containing 500 μg/mL Tween 20 served as the negative control, while a synthetic fungicide (Peulrei) served as the positive control, applied 1DBI of the pathogen.
Results
JCK-7385 strain selection
A dual culture bioassay was conducted to evaluate the mycelial growth inhibition of JCK-7385 against F. graminearum and F. asiaticum pathogens. However, no direct antifungal activity was observed against either pathogen (Supplementary Fig. 1A). Therefore, the CB of strain JCK-7385 was employed to investigate its ability to induce expression of the defense-related gene PR1 in transgenic A. thaliana plants using the GUS reporter assay. The results showed that JCK-7385 CB treatments triggered GUS expression, characterized by blue fluorescence along the leaf veins in the treated plants (Supplementary Fig. 1B). Moreover, the JCK-7385 strain also effectively controlled the development of FHB in rice in a screening experiment using inducing-resistance strains under greenhouse conditions (data not shown). According to these results, the JCK-7385 strain was selected to be a candidate for further experiments in this study.
Identification of the JCK-7385 strain
Supplementary Fig. 2 shows the JCK-7385 activated morphology strain on TSA agar. JCK-7385 strain identification was conducted as described previously (Nguyen et al., 2021). JCK-7385 strain was taxonomically characterized using 16S rRNA gene sequencing (Genbank accession no. PQ621117) and concatenated sequence analysis of four housekeeping genes, atpD (PQ618555), trpB (PQ650606), recA (PQ650607), ropB (PQ650608). The 16S rRNA gene sequence analysis showed that strain JCK-7385 had the highest similarity to S. showdoensis ISP 5504, S. viridobrunneus LMG 20317.1, and S. cinereoruber subsp. cinereoruber JCM 4205 (Fig. 1A). However, multilocus sequence analysis phylogeny based on housekeeping genes revealed a different topology to the 16S rRNA gene counterpart, wherein strain JCK-7385 formed S. vietnamensis GIM4.001 with 94% bootstrap value (Fig. 1B). Consequently, it could be confirmed that the JCK-7385 strain belongs to the genus Streptomyces.
Biochemical characterization of JCK-7385
Plant growth-promoting functions of JCK-7385
Strain JCK-7385 was found to produce IAA, as indicated by the color change of its filtrate to pink, whereas the untreated control retained the original medium color (Supplementary Fig. 3A). According to the experiment results, JCK-7385 also produced siderophore on CAS agar. The halo zone increased owing to the treatment sample size of 14.1, 19.3, and 21.4 mm for 20, 40, and 60 μL, respectively (Supplementary Fig. 3B). Additionally, ACC deaminase activity was confirmed via the JCK-7385 strain on DF agar medium (Supplementary Fig. 3C).
Extracellular enzyme activity
JCK-7385 also exhibited extracellular enzyme activities, including chitinase, cellulase, and gelatinase. Consequently, quantitative analysis showed that the clear zone diameters increased between 3 and 7 days of incubation. After 7 days with a 60 μL sample, the parameters for chitinase, cellulase, and gelatinase were 23.88, 24.76, and 14.47 mm, respectively (Supplementary Fig. 4).
PR1 gene expression by β-glucuronidase assay
All JCK-7385 treatments including CB, CF, cell suspension, SC, and WP induced GUS expression with blue fluorescence along the leaf veins in Arabidopsis thaliana. The intensity was slightly weaker than that in the SA control sample. Conversely, no fluorescence was detected in the untreated plant (Fig. 2). Therefore, JCK-7385 treatments can induce PR1 expression, serving as a marker of SAR pathway activation in A. thaliana.
Disease control efficacy of JCK-7385 against FHB in rice under greenhouse conditions
Single treatment application
Experimental results demonstrated that the JCK-7385 samples inhibited FHB development in rice. FHB control efficacy was higher in the treatment at a 2,000-fold dilution for JCK-7385 CB, CF, and cell suspension by 35.01%, 31.75% and 42.43%, respectively (Fig. 3, Supplementary Table 2).
Conversely, SC and WP formulations at 1,000- and 2,000-fold dilutions were tested to assess the disease control activity in rice under greenhouse conditions. The results showed that the JCK-7385 SC at 2,000-fold dilution most effectively reduced FHB development by 52.5%, followed by the positive control (42.08%) and JCK-7385 WP at 2,000-fold dilution treatment (38.25%) (Fig. 4, Supplementary Table 3).
JCK-7385 SC shelf-life
To investigate the stability and effectiveness of JCK-7385 SC, samples stored at 45°C for 0-18 weeks were tested for FHB control in rice. The experiment results demonstrated that the sample stored for 6 weeks showed the highest efficacy, reducing FHB development by 54.4%. The following values were observed on positive treatment, 9-, 0-, and 12-week stored samples, ranging from 45-52% for FHB control. Moreover, the control efficacy declined rapidly in the 15- and 18-week samples, reaching only approximately 40% (Fig. 5, Supplementary Table 4).
Integrated treatment application
The integrated treatment combining JCK-7385 SC with the chemical fungicide (Peulrei) showed significantly higher effectiveness in FHB control than that in single treatments (JCK-7385 SC or Peulrei only). The control efficacies were specifically 62.29%, 62.26%, and 57.86% for the 2WBI/1DBI; 2WBI, 1WBI/1DBI; and 1WBI/1DBI treatment, respectively. Moreover, the control efficacy of single treatments was approximately 45% in FHB management (Fig. 6, Supplementary Table 5).
Disease control efficacy of JCK-7385 against FHB in wheat under greenhouse conditions
The JCK-7385 SC was further evaluated for its efficacy against FHB in wheat caused by F. graminearum Z-3639 under greenhouse conditions. A single application at a 2,000-fold dilution reduced disease severity by 47.04%, comparable to the positive control (Peulrei, 49.82%). In addition, the combined application of JCK-7385 SC (2,000-fold dilution) with Peulrei achieved significantly higher inhibition on the disease development, ranging from 66.5-71.6% (Fig. 7, Supplementary Table 6).
Disease control efficacy of JCK-7385 against FHB in rice field
In 2023, based on greenhouse experiment results, JCK-7385 SC was tested under rice field conditions at 1,000- and 2,000-fold dilutions. The 2,000-fold dilution also showed a higher FHB disease control efficacy than the 1,000-fold dilution and positive control (Peulrei) treatments. The 2,000-fold dilution treatment showed control values of 45.0%, 40.3%, and 40.1% at 2WAI, 4WAI, and 6WAI, respectively. Additionally, the 1,000-fold dilution yielded 28.7%, 15.9%, and 23.2% (2WAI, 4WAI, and 6WAI, respectively) while the control values of the Peulrei sample were 42.4%, 36.6%, and 38.8% (Fig. 8, Supplementary Table 7).
In 2024, the integrated treatment of JCK-7385 SC at a 2,000-fold dilution with Peulrei achieved the highest control efficacy against FHB than that of other treatments. The control values were 65.1%, 53.8%, and 52.0% for 2WAI, 4WAI, and 6WAI, respectively. Conversely, single treatments maintained control values of approximately 45% at 6WAI (Fig. 9, Supplementary Table 8).
Discussion
Fusarium asiaticum is predominantly found in rice, barley, and wheat, exhibiting FHB symptoms with a prevalence of 95% in Korea (Jang et al., 2019). The Fusarium graminearum species complex is frequently associated with severe FHB epidemics in many regions. Globally, FHB is regarded as the most devastating fungal disease of wheat worldwide, causing significant grain yield and quality loss, reduced harvest and marketability, as well as accumulating trichothecene mycotoxins (Xu et al., 2022). Therefore, F. asiaticum and F. graminearum were selected for the in vivo assays to evaluate FHB in rice and wheat, respectively. While many studies globally have examined the use of Streptomyces species as a biocontrol agent against FHB in wheat, comparable research in rice remains limited. To our knowledge, this is the first report demonstrating the successful control of FHB in rice using a Streptomyces sp. strain.
In this study, unlike many Streptomyces strains with antifungal activity, JCK-7385 did not inhibit F. graminearum and F. asiaticum pathogen development on PDA agar dual-culture plates (Supplementary Fig. 1A). Therefore, following Park et al. (2020), transgenic Arabidopsis plants carrying the PR1 promoter fused to β-GUS serve as a model for a system to screen bacterial inducers that enhance disease resistance mechanisms in various plant species. The experiment result showed that JCK-7385 CB treatments induced GUS expression, visible as blue fluorescence along the leaf veins in Arabidopsis thaliana leaves (Supplementary Fig. 1B), suggesting activation of the SA-dependent SAR pathway, consistent with previous findings (Ali et al., 2018; Molinari et al., 2014).
The JCK-7385 strain was identified as Streptomyces sp. based on the phylogenetic analysis of the 16S rRNA and four housekeeping gene sequences (Fig. 1). It exhibited plant growth-promoting traits, including IAA and ACC deaminase production (Supplementary Fig. 3A and C). Igarashi et al. (2002) demonstrate that these metabolites are essential for the growth-enhancing properties of Streptomyces spp. IAA is considered a key determinant of root development, promoting cell elongation and division (Nascimento et al., 2020). ACC deaminase degrades ACC, the immediate precursor of ethylene, thereby inhibiting ethylene biosynthesis and enhancing plant tolerance to unfavorable conditions. The JCK-7385 strain also showed siderophore production on the CAS agar plate (Supplementary Fig. 3B). The siderophore compounds facilitate plant nutrient uptake, providing a direct mechanism for promoting plant growth. Moreover, they contribute to plant defense by competing with pathogens for iron (Chen et al., 2021a; Etesami and Maheshwari, 2018). Conversely, the JCK-7385 strain produced extracellular enzymes, including chitinase, cellulase, and gelatinase (Supplementary Fig. 4). These enzymes are involved in plant defense mechanisms, enhancing activity and responsiveness to pathogens, indicating the potential to suppress plant diseases (Kumar et al., 2018a; Saberi Riseh et al., 2024; Shimoi et al., 2010).
Experiment results showed that JCK-7385 treatments induced the PR1 expression in transgenic Arabidopsis plants. The CB, CF, and cell suspension of JCK-7385 at a low concentration (2,000-fold dilution) showed a positive response in the GUS bioassay (Fig. 2). Moreover, these treatments also inhibited FHB development in rice under greenhouse conditions (Figs. 3 and 4, Supplementary Tables 2 and 3). These findings indicate that JCK-7385 controls FHB in rice primarily via an induced resistance mechanism. Therefore, further research is needed to identify the plant-inducing compounds from JCK-7385 samples, especially in the cells of the JCK-7385 strain.
Formulations of BCAs enhance product management and application, via preserving the organisms during manufacturing, transportation, and storage, protecting them from environmental stress, and enhancing their activity. The primary goals of formulation include stabilizing the active ingredient, facilitating ease of use, and minimizing applicator exposure risks (Leggett et al., 2011). SC is a liquid formulation containing solid particles suspended in a liquid medium, offering stability and ease of application (Copping and Menn, 2000). In this study, the SC formulation was prepared, including several beneficial ingredients. Specifically, propylene glycol served as a solvent, emulsifier, and humectant, aiding in the distribution and stabilization of active ingredients while enhancing the ability of the product to spread and absorb (Gupta et al., 2024). Xanthan gum is commonly used in pesticide formulations for various purposes, such as enhancing spray characteristics and enhancing pesticide effectiveness (Zhang and Liu, 2011). Conversely, sodium benzoate functions primarily as a preservative, preventing microbial growth in the pesticide formulation and ensuring its stability and shelf life (Johnston et al., 2008). The JCK-7385 SC sample showed stable efficacy in controlling FHB in rice, as confirmed via the SC shelf-life experiment. Therefore, the JCK-7385 SC sample should be used within 2 years to ensure optimal efficacy (Fig. 5, Supplementary Table 4). According to Australian Agvet Chemical Regulator, samples that demonstrate sufficient stability at temperatures ranging from 40°C to 54°C are expected to remain stable for at least 2 years under standard storage conditions (Australian Agvet Chemical Regulator, 2020).
FHB is a devastating disease in cereal crops, leading to significant yield losses and mycotoxin contamination in grains. The absence of effective biorational control strategies renders sporadic FHB outbreaks a significant risk to global food security and safety. To reduce crop failure and yield loss associated with this destructive fungal disease, integrated management strategies leveraging existing knowledge are essential (Chen et al., 2022). Importantly, the integrated treatment of JCK-7385 SC and a chemical fungicide (Peulrei) achieved significantly higher FHB control efficacy than single treatments under greenhouse and field conditions in this study (Figs. 6, 7, and 9, Supplementary Tables 5, 6, and 8). Many studies demonstrate the significant effectiveness of combining biological and chemical agents in enhancing plant disease control and represent an effective method in sustainable agriculture, thereby reducing the reliance on chemical pesticides (Di Francesco and Mari, 2014; Duraisamy et al., 2022; Omar et al., 2006; Ons et al., 2020). Taken together, previous reports have shown the disease control efficacy of Streptomyces sp. isolated from rice against Fusarium species and others through its indirect, direct mechanisms, and plant growth-promoting abilities (Awla et al., 2017; Chaiharn et al., 2020; Jung et al., 2013; Yang et al., 2024). Therefore, future research should focus on detecting the JCK-7385 second metabolites to determine the action mechanism and apply it to other plant diseases.
In summary, the Streptomyces sp. JCK-7385 strain exhibited plant growth-promoting activity and produced extracellular enzymes in an in vitro bioassay. JCK-7385 treatments also induced expression of the defense-related marker gene PR1 in transgenic Arabidopsis (GUS assay) and, at low concentrations, inhibited FHB development in vivo, suggesting that its activity may involve an induced resistance mechanism. In future research, it will be essential to isolate and identify the metabolites of the JCK-7385 strain that induce the plant resistance pathway. Additionally, JCK-7385 SC treatment exhibited significant control efficacy against FHB under greenhouse and field conditions. Moreover, the integrated treatment of JCK-7385 SC with the chemical fungicide (Peulrei) effectively controlled FHB development, outperforming either application alone. To our knowledge, this is the first report of FHB control in rice using a Streptomyces strain. These findings indicate that the JCK-7385 strain can be developed as a biological fungicide.
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