Antagonistic Activity and Genomic Insights of Bacillus velezensis JE250 against Erwinia amylovora

Article information

Plant Pathol J. 2025;41(6):723-735

Publication date (electronic) : 2025 December 1

doi : https://doi.org/10.5423/PPJ.OA.03.2025.0037

Jueun Lee ¹^,^†, Won-Kwon Jung ²^,^†, Hee-Young Jung ³, Yong Chull Jeun ⁴

, Yongho Jeon ¹, Hyong Woo Choi ¹^,

¹Department of Plant Medicals, GyeongKuk National University, Andong 36729, Korea

²Gyeongsangbuk-do Agricultural Research & Extension Services, Daegu 41404, Korea

³Department of Plant Medicine, Kyungpook National University, Daegu 41566, Korea

⁴Sustainable Agriculture Research Institute, Jeju National University, Jeju 63243, Korea

^*Corresponding author. Phone) +82-54-820-6224, FAX) +82-54-820-6320, E-mail) hwchoi@anu.ac.kr

†These authors contributed equally to this work.Handling Editor : Youn-Sig Kwak

Received 2025 March 7; Revised 2025 September 22; Accepted 2025 September 22.

Abstract

Fire blight, caused by Erwinia amylovora, is a highly destructive bacterial disease that affects apple and pear orchards worldwide, leading to significant economic losses. In this study, we isolated and characterized endophytic bacterial strains from apple trees in Gyeongsangbuk-do, South Korea, to identify potential biocontrol agents against E. amylovora. Among the five antagonistic strains identified, Bacillus velezensis JE80 and JE250 exhibited the strongest inhibitory effects. Further analysis using culture filtrates (CFs) from these strains demonstrated that the CFs of JE80 and JE250 not only suppressed E. amylovora growth in a growth-phase-dependent manner but also significantly impaired bacterial motility and biofilm formation. Notably, in planta assays revealed that JE250 effectively reduced fire blight symptoms in apple blossoms, performing comparably to streptomycin sulfate. Whole-genome sequencing of JE250 identified biosynthetic gene clusters associated with the production of antimicrobial compounds, including difficidin, fengycin, bacillaene, macrolactin, bacillibactin, and bacilysin, further supporting its strong antagonistic potential. These findings suggest that B. velezensis JE250 is a promising biocontrol agent for sustainable fire blight management. Future research should focus on optimizing formulation methods for field application, characterizing specific antimicrobial compounds, and evaluating its long-term efficacy in orchard environments.

Keywords: antimicrobial compounds; Bacillus velezensis; biological control; Erwinia amylovora; fire blight

Fire blight, caused by Erwinia amylovora, is one of the most destructive bacterial diseases affecting apple and pear orchards worldwide. The disease results in severe economic losses due to rapid wilting, necrosis, and eventual tree death, particularly in warm and humid conditions that favor pathogen spread (Ham et al., 2025; Piqué et al., 2015; Song et al., 2024). Current fire blight management strategies primarily rely on chemical control measures, such as copper-based bactericides and antibiotics (e.g., streptomycin and oxytetracycline), which have been used to suppress E. amylovora infections (Ham et al., 2024; McGhee and Sundin, 2011; Ryu et al., 2023; Stockwell and Duffy, 2012). However, excessive reliance on these chemicals has raised concerns regarding the emergence of antibiotic-resistant E. amylovora strains, potential toxicity to non-target organisms, and negative environmental impacts (Ham et al., 2022; Sundin and Wang, 2018). These concerns highlight the urgent need for sustainable alternatives to conventional fire blight control methods.

E. amylovora employs several virulence strategies to infect host plants, including flagella-mediated motility and biofilm formation. Swimming and swarming motilities allow the bacterium to migrate on plant surfaces and penetrate host tissues, facilitating systemic infection (Koczan et al., 2011; Yuan et al., 2022). Biofilm formation further enhances its ability to adhere to host cells and protect itself from environmental stress and plant defense responses (Koczan et al., 2011; Piqué et al., 2015). Therefore, targeting these virulence factors can be an effective strategy in controlling fire blight progression.

Among the most promising alternatives to synthetic pesticides and chemical-based disease management are biological control agents (BCAs), which leverage beneficial microorganisms to enhance plant health and suppress plant pathogens through various mechanisms (Droby et al., 2009; Kim et al., 2022, 2024b; Kong et al., 2021; Lee et al., 2024a, 2024b). In particular, the genus Bacillus has gained significant attention due to its strong biocontrol potential. Bacillus spp. are well known for their ability to form stress-resistant endospores and produce a diverse range of bioactive secondary metabolites, including lytic enzymes, cyclic peptides, polyketides, nonribosomal peptides, siderophores, volatile organic compounds, and antimicrobial toxins (Caulier et al., 2019; Wu et al., 2015). These metabolites allow Bacillus spp. to directly inhibit the growth of bacteria, fungi, viruses, and nematodes, or indirectly suppress pathogens by stimulating plant immune responses, induced systemic resistance (Pršić and Ongena, 2020). The primary biocontrol mechanisms of Bacillus species involve disrupting pathogen cell membranes, degrading cell walls, inhibiting essential cellular processes, and modulating microbial interactions within the plant microbiome (Fira et al., 2018). Several Bacillus species, including B. subtilis, B. thuringiensis, B. cereus, B. velezensis, and B. licheniformis, have been extensively studied for their antimicrobial properties and are increasingly being developed as commercial biopesticides (Caulier et al., 2019).

In this study, we isolated and characterized endophytic bacterial strains from apple orchards in Gyeongsangbuk-do, South Korea, to identify potential biocontrol agents against E. amylovora. Among the five antagonistic strains identified from 287 bacterial isolates, Bacillus velezensis JE80 and JE250 exhibited the strongest inhibitory effects. Further analysis revealed that culture filtrates (CFs) from these strains suppressed E. amylovora growth in a growth-phase-dependent manner and significantly impaired bacterial motility and biofilm formation. Notably, in planta assays demonstrated that JE250 effectively reduced fire blight symptoms and disease incidence in apple blossoms, performing comparably to streptomycin sulfate. Whole-genome sequencing of JE250 identified biosynthetic gene clusters (BGCs) associated with antimicrobial compound production, further supporting its strong antagonistic potential. These findings highlight B. velezensis JE250 as a promising biocontrol agent for sustainable fire blight management, warranting further research on its field efficacy and formulation for practical application.

Materials and Methods

Isolation of endophytic microorganisms from apple tissue

To isolate endophytic microorganisms from apple tissues, fruit, leaf, and branch samples were surface-sterilized using a sequential treatment of 70% ethanol for 3 min, followed by 1% sodium hypochlorite (NaOCl) for 3 min, and a final rinse with sterile distilled water (SDW). These tissues, including the peel, were sectioned into 2 cm² pieces and homogenized in 1 mL of SDW using a Silamat S5 mixer (Ivoclar Vivadent, Liechtenstein, Germany) with glass beads. Following homogenization and serial dilution, 100 μL of the supernatant was spread onto nutrient agar medium (KisanBio, Seoul, Korea). Each isolate was grown in fresh nutrient broth medium and stored at −80°C with 10% glycerol for further use. The unknown bacterial strains were screened for their antagonistic activity against E. amylovora S59/5 (ATCC no. 15580).

Screening and test of antagonistic bacteria

The selection of antibacterial microorganisms against E. amylovora S59/5 was performed using an agar plate assay, as previously described (Koo et al., 2023). A total of 287 unknown microorganisms were isolated from different apple orchards in Gyeongsangbuk-do province (Supplementary Table 1). Each bacterium and E. amylovora was cultured in 10 mL of tryptic soy broth (TSB; KisanBio) at 28°C for 2 days in a shaking incubator. To prepare the inoculum, the optical density at 600 nm (OD₆₀₀) of E. amylovora S59/5 was adjusted to 0.2 in SDW, and 100 μL of the inoculum was spread onto tryptic soy agar (TSA).

For the disc diffusion assay, sterile paper discs (6 mm in diameter, Whatman, Maidstone, UK) were placed on the surface of E. amylovora S59/5-inoculated TSA medium and inoculated with 20 μL of each unknown bacterial culture. The formation of a clear zone around the disc indicated antimicrobial activity due to the diffusion of antimicrobial substances from the unknown bacteria. Microorganisms demonstrating antimicrobial activity were selected for further testing. The diameter of the clear zone (mm) was measured by subtracting the diameter of the bacterial colony from the total clear zone diameter.

Five bacterial strains identified from the initial screen (JE31, JE72, JE80, JE170, and JE250) were further evaluated using a dual streak assay. In this assay, E. amylovora S59/5 was streaked onto TSA medium alongside colonies of the five candidate strains and incubated at 28°C. The width of E. amylovora colonies was measured to assess the antagonistic activity of each candidate strain. Inhibition efficacy was quantified using the following formula:

Bacterial growth inhibition (%)=[1-(Width of E. amylovora with treatment/Width of E. amylovora) without treatment]×100

Molecular identification

The 16S rRNA gene sequencing was performed to identify the five candidate bacteria (JE31, JE72, JE80, JE170, and JE250). Genomic DNA (gDNA) was extracted using the HiGene Genomic DNA Prep Kit (BIOFACT, Daejeon, Korea). PCR amplification was conducted in a 50 μL reaction mixture comprising 5 μL of 2× Taq buffer, 5 μL of 5× band doctor, 1 μL of dNTP mix, 0.25 μL of Taq polymerase, 32.75 μL of distilled water, 2 μL of DNA template, and 2 μL of primers. The 16S rRNA region was amplified using the primers 27F (5′-AGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GTTACCTTGTTACGACTT-3′). The PCR protocol included an initial denaturation at 95°C for 2 min, followed by 33 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 40 s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. The 16S rRNA gene sequences were analyzed by Solgent sequencing services (Solgent, Daejeon, Korea) and compared with sequences in National Center for Biotechnology Information (NCBI)'s GenBank (http://www.ncbi.nlm.nih.gov) to identify the closest species. Phylogenetic trees were constructed using the maximum likelihood method in MEGA 11 software (Kumar et al., 2018).

Antagonistic activity of CFs of Bacillus spp

CFs were prepared to test for antimicrobial activity. Each Bacillus strain was cultured in 500 mL of TSB and incubated at 28°C in a shaking incubator for either 1 or 4 days. The cultures were then transferred to 50 mL conical tubes and centrifuged at 12,000 rpm for 20 min. The supernatant was carefully collected using a 10 mL syringe and filtered through a 0.2 μm syringe filter (Merck Millipore, Burlington, MA, USA) to remove bacterial cells. The CFs were either used immediately or stored at 4°C for further experiments.

The agar plate assay was performed as described above. Twenty microliters of CFs from cultures incubated for 1 to 7 days was applied to paper discs, and the diameter of the clear zone was measured to assess antagonistic activity. To compare antibacterial efficacy, CFs from 1- and 4-day-old cultures were mixed with E. amylovora S59/5 (OD₆₀₀ = 0.2) and incubated overnight at 28°C. After incubation, the mixtures were serially diluted and plated on TSA medium to measure the bacterial population. Bacterial growth was also monitored by measuring OD₆₀₀ using a microplate reader (SpectraMax iD5, Molecular Devices, San Jose, CA, USA).

The effects of Bacillus CFs on the swimming and swarming motility of E. amylovora S59/5 were assessed as previously described (Koo et al., 2023). Different concentrations of CFs were mixed with Luria-Bertani (LB) medium (KisanBio), and 0.3% and 0.6% agar were added for swimming and swarming motility assays, respectively. The medium was dried for one day to solidify completely. A 2 μL drop of E. amylovora (OD₆₀₀ = 0.05) was inoculated at the center of the soft LB-agar medium, and colony diameter was measured 48 h after incubation at 28°C.

A biofilm formation assay was conducted as previously described (Koo et al., 2023). The OD₆₀₀ of E. amylovora S59/5 culture was adjusted to 0.2 with TSB, then mixed with TSB containing different CF concentrations (final concentrations = 0, 5, 10, 25, and 50%). Each mixture (100 μL) was incubated in 24-well plates (SPL Life Sciences, Pocheon, Korea) at 28°C for 48 h under stationary conditions. After incubation, the liquid medium was gently removed, and the biofilms were stained with 1 mL of 0.1% crystal violet for 50 min. The wells were washed three times with SDW and treated with 1 mL of 95% ethanol. Biofilm formation was quantified by measuring absorbance at 590 nm using a microplate reader (SpectraMax iD5, Molecular Devices).

Antagonistic activity of JE250 against E. amylovora in apple blossoms

In vivo test with detached apple blossoms was performed as previously described with minor modifications (Kim et al., 2024b). Freshly harvested apple blossoms were spray-inoculated with E. amylovora Ea385 (OD₆₀₀ = 0.01, approximately 5 × 10⁶ colony-forming unit [CFU]/mL). One hour after inoculation, the apple blossoms were treated with either sterile water (negative control), 0.025% streptomycin sulfate (STP; positive control), or JE250 (OD₆₀₀ = 0.2, approximately 10⁸ CFU/mL). Disease incidence was assessed 7 days post-inoculation (dpi) by monitoring the presence of bacterial ooze at the floral stalk. The growth of E. amylovora was also measured at 1, 24, and 48 hours post-inoculation.

Quantification of E. amylovora in apple blossoms

The number of E. amylovora Ea385 cells in apple blossoms was measured as previously described (Kim et al., 2024b; Kunz, 2006). Briefly, four apple blossoms without petals were washed with 8 mL of SDW (2 mL per blossom), and the washing fluids containing bacterial cells were collected and directly used as PCR templates without DNA extraction. Quantitative PCR was performed with the primer pair (p29TF 5′-CAC TGA TGG TGC CGT TG-3′ and p29TR 5′-CGC CAG GAT AGT CGC ATA-3′) using a QuantiFast SYBR Green kit (Qiagen, Hilden, Germany) on a Bio-Rad CFX Duet system (Salm and Geider, 2004). Amplification conditions followed Hinze et al. (2016), and bacterial cell numbers were quantified using an external standard curve.

Whole genome sequencing

Whole genome sequencing of JE250 was performed as previously described (Lee et al., 2024a). Briefly, gDNA was extracted using the Maxwell RSC Pure Food GMO and Authentication Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The gDNA was quantified using an Epoch spectrophotometer (BioTek, Winooski, VT, USA).

For sequencing, library preparation was performed using the NEBNext Ultra II FS DNA Library Prep Kit (NEB, Ipswich, MA, USA) and NEBNext Multiplex Oligos, Dual Index 3 (NEB). The prepared library was quantified with a Qubit 3.0 fluorometer (Invitrogen, Waltham, MA, USA) and sequenced using an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) by CJ Bioscience (Seoul, Korea). The Illumina sequencing data underwent quality control using Trimmomatic-0.36, and PhiX sequences were removed using BBMap 38.32. The preprocessed data were assembled using SPAdes 3.15.3 (Algorithmic Biology Lab, St. Petersburg Academic University of the Russian Academy of Sciences). Genome contamination was assessed by comparing 16S rRNA gene fragments, and no contamination was detected. Gene prediction and functional annotation of the assembled genome were performed using the EzBioCloud genome database. Protein-coding sequences (CDSs) were identified using Prodigal 2.6.2 (Hyatt et al., 2010). Genes encoding tRNA were detected using tRNAscan-SE 1.3.1 (Schattner et al., 2005), while rRNA and other non-coding RNAs were identified through a covariance model search against the Rfam 12.0 database (Nawrocki and Eddy, 2013). The CDSs were classified into functional categories based on orthologous groups using the EggNOG 4.5 database (http://eggnogdb.embl.de) (Powell et al., 2014). The Clusters of Orthologous Genes (COG) database was used for phylogenetic classification of proteins from microbial genomes (Tatusov et al., 1997), with results displayed as a bar chart. Additional functional annotation was performed by comparing predicted CDSs against the Kyoto Encyclopedia of Genes and Genomes database (Kanehisa et al., 2014). Secondary metabolite analysis was conducted using antiSMASH v7.0.0 with the parameter set to "relaxed" to identify potential BGCs (Blin et al., 2023) (https://antismash.secondarymetabolites.org/). Orthologous average nucleotide identity (OrthoANI) was calculated by comparing homologous genes between Bacillus velezensis B-41580, Bacillus siamensis KCTC13613, Bacillus amyloliquefaciens DSM7, and Bacillus nakamurai B419091 to construct a phylogenetic tree (Lee et al., 2016). The whole-genome sequence of B. velezensis JE250 has been deposited in the NCBI GenBank database under the accession number CP193498.

Results

Screening of antagonistic bacteria against E. amylovora

A total of 287 unknown bacterial strains were isolated from different apple orchards in Gyeongsangbuk-do province and screened for antagonistic activity. Among these, five strains (JE31, JE72, JE80, JE170, and JE250) exhibited antagonistic activity against E. amylovora (Fig. 1A). Notably, JE80 and JE250 showed the strongest inhibitory effects, with clear zone diameters of 21.0 ± 2.2 mm and 18.7 ± 1.1 mm, respectively (Fig. 1B). The dual streak assay further confirmed the highest antagonistic activity of JE80 and JE250, with inhibition rates of 66.7 ± 8.9% and 70.8 ± 6.7%, respectively (Fig. 1C and D). Phylogenetic analysis based on the 16S rRNA sequence identified JE31, JE72, JE80, JE170, and JE250 as B. velezensis (Fig. 1E). As JE80 and JE250 demonstrated the highest antagonistic potential in both assays, these isolates were selected for further experiments.

Fig. 1

Isolation and identification of Bacillus spp. as antagonistic bacteria against Erwinia amylovora. (A) Representative images showing the antagonistic activity of different bacterial strains (JE31, JE72, JE80, JE170, and JE250) against E. amylovora in a disc diffusion assay. (B) Clear zone diameters induced by different bacterial strains against E. amylovora. (C) Representative images showing the antagonistic activity of different bacterial strains (JE31, JE72, JE80, JE170, and JE250) against E. amylovora in a dual streak assay. (D) Growth inhibition rate of E. amylovora induced by different bacterial strains. Data represent the mean ± standard error (n = 3). Bars with different letters indicate significant differences according to the least significant difference test (P < 0.05). (E) Phylogenetic tree analysis of 16S rRNA gene of JE31, JE72, JE80, JE170, and JE250. MEGA11 software was used to construct phylogenetic trees using the maximum-likelihood method. Bootstrap values based on 1,000 replicates were shown at each node. Lacticaseibacillus paracasei ATCC 25302 (NR 117987.1) was used as an outgroup.

Antibacterial activity of CF of JE80 and JE250

To assess the antibacterial activity of JE80 and JE250 against E. amylovora, CFs were tested using a disc diffusion assay (Fig. 2A and B). Interestingly, CFs prepared from 1-day-old cultures of JE80 and JE250 failed to induce a clear inhibition zone. However, CFs from 2- to 7-day-old cultures exhibited distinct antibacterial activity.

Fig. 2

Antagonistic activity of culture filtrates (CFs) from selected Bacillus spp. against Erwinia amylovora. (A) Representative images showing the growth inhibition of E. amylovora in a disc diffusion assay using CFs collected at different time points (1 to 7 days) after bacterial culture. (B) Clear zone diameters induced by CFs from Bacillus spp. (C) Representative images showing E. amylovora colonies. E. amylovora (10⁵ CFU/mL) was incubated with the indicated CFs for 24 h, serially diluted, and 5 μL of each dilution was plated onto tryptic soy agar medium. (D) Quantitative analysis of E. amylovora (E.a.) growth in the absence or presence of the indicated CFs. Data represent the mean ± standard error (n = 6). Bars with different letters indicate significant differences according to the least significant difference test (P < 0.05).

Similarly, a growth assay conducted in the presence of 50% CFs from JE80 and JE250 revealed that CFs from 1- and 4-day-old cultures significantly inhibited E. amylovora growth (Fig. 2C and D). Monitoring OD₆₀₀ further confirmed that the addition of 50% CFs from 4-day-old cultures of JE80 and JE250 completely suppressed E. amylovora growth (Fig. 3). These findings suggest that JE80 and JE250 produce and secrete antimicrobial compounds capable of inhibiting E. amylovora growth.

Fig. 3

Inhibition of Erwinia amylovora growth by culture filtrates (CFs) from JE80 and JE250. E. amylovora was grown in the absence (control) or presence of CFs obtained from 1-day (1d) or 4-day (4d) cultures of JE80 or JE250. Data represent the mean ± standard error (n = 6).

Inhibitory effects of CFs of JE80 and JE250 on motility and biofilm formation of E. amylovora

To investigate whether the CFs of JE80 and JE250 could inhibit key virulence traits of E. amylovora, such as motility and biofilm formation, we performed swimming and swarming assays, as well as biofilm quantification assays. Both CFs significantly reduced swimming and swarming motility (Fig. 4A–D). Notably, CF from JE250 exhibited the strongest inhibitory effect, with 50% CF inhibiting E. amylovora swimming and swarming motility by 99.9% and 97.27%, respectively. In addition, CFs from JE80 and JE250 suppressed biofilm formation by E. amylovora (Fig. 4E and F). The addition of 50% CFs from JE80 and JE250 reduced biofilm formation by 52.9% and 62.6%, respectively.

Fig. 4

Antagonistic activity of culture filtrates (CFs) from JE80 and JE250 against Erwinia amylovora. (A, C) Inhibition of swimming (A) and swarming motility (C) of E. amylovora by CFs from JE80 and JE250. (B, D) Inhibition rates of swimming (B) and swarming (D) motility of E. amylovora induced by CFs from JE80 and JE250. (E) Inhibition of E. amylovora biofilm formation by CFs from JE80 and JE250. (F) Quantification of biofilm formation by E. amylovora in the presence of different concentrations of CFs from JE80 and JE250. CF conc., the concentration of culture filtrate used in the assay; N.C., negative control containing only tryptic soy broth medium without bacterial inoculation. Data represent the mean ± standard error (n = 4). Bars with different letters indicate significant differences according to the least significant difference test (P < 0.05).

Biocontrol activity of JE250 against E. amylovora in apple blossoms

Given its strong and consistent antagonistic activity, JE250 was selected for further biocontrol experiments. Apple blossoms inoculated with E. amylovora and treated with SDW (negative control) developed severe disease symptoms (Fig. 5A and B). The negative control exhibited a disease incidence of 69.4% at 7 dpi (Fig. 5C). However, apple blossoms inoculated with E. amylovora and treated with either 0.025% STP or JE250 (10⁸ CFU/mL) showed significantly reduced disease symptoms and disease incidence. Consistently, the population of E. amylovora was significantly lower in apple blossoms treated with STP or JE250 compared to the negative control (Fig. 5D). These results indicate that JE250 exhibits significant biocontrol activity against fire blight in apple blossoms.

Fig. 5

Antagonistic activity of JE250 against Erwinia amylovora in apple blossoms. (A) Disease symptoms on apple blossoms at 7 days post-inoculation (dpi) with E. amylovora. Each treatment (Ctrl, untreated control; STP, sprayed with 0.025% streptomycin sulfate; JE250, sprayed with 10⁸ colony-forming unit [CFU]/mL of JE250) was applied to apple blossoms inoculated with E. amylovora (5 × 10⁶ CFU/mL). (B) Representative images of asymptomatic and symptomatic apple blossoms at 7 dpi. Bacterial ooze droplets, observed only in symptomatic apple blossoms, are indicated by red arrows. (C, D) Disease incidence (C) and E. amylovora growth (D) in apple blossoms left untreated (Ctrl) or treated with streptomycin sulfate (STP) and B. velezensis JE250. Data represent the mean ± standard error (n = 3). Bars with different letters indicate significant differences according to the least significant difference test (P < 0.05).

Whole genome sequence analysis of JE250

Whole-genome sequencing provided comprehensive genomic insights into B. velezensis JE250 (Fig. 6A). The sequencing achieved a genome coverage of 660.15×, sufficient to generate a high-quality draft genome assembly. The JE250 genome consists of 3,887,661 bp with a GC content of 46.4% and contains 3,748 CDSs distributed across 16 contigs. Phylogenetic analysis of the whole genome sequence revealed that JE250 clustered closely with B. velezensis B-41580 (Fig. 6B). EggNOG/COG analysis identified 78 genes in JE250 related to secondary metabolite biosynthesis, transport, and catabolism (Fig. 6C). To identify BGCs associated with secondary metabolite production, the JE250 genome was analyzed using antiSMASH v7.0. A total of 12 predicted BGCs were identified, of which six showed 100% similarity with previously reported BGCs, including those responsible for the biosynthesis of difficidin, fengycin, bacillaene, macrolactin, bacillibactin, and bacilysin (Fig. 6D).

Fig. 6

Whole-genome sequence analysis of Bacillus velezensis JE250. (A) Circular genome map of JE250. The circular map displays coding sequences in the forward and reverse directions, rRNA and tRNA genes, a GC skew graph, and a GC ratio graph. (B) Phylogenetic analysis of the whole-genome sequence of Bacillus velezensis JE250. (C) EggNOG/COG analysis of the JE250 genome. (D) Identification of secondary metabolite biosynthetic gene clusters in JE250 using antiSMASH analysis.

Discussion

The increasing demand for biocontrol products over traditional pesticides underscores the growing need for sustainable agricultural practices (Haq et al., 2024; Kim et al., 2024a; Tadesse Mawcha et al., 2025). Consequently, the identification and selection of effective BCAs are crucial for reducing dependence on chemical control while ensuring effective disease suppression. In this study, we aimed to isolate and characterize bacterial strains with antagonistic activity against E. amylovora, the causal agent of fire blight in apples, which remains one of the most devastating bacterial diseases worldwide.

Screening of endophytic bacteria from apple plants using an agar plate assay identified five bacterial strains (JE31, JE72, JE80, JE170, and JE250) with significant antagonistic activity against E. amylovora. Among these, JE80 and JE250 exhibited the strongest inhibitory effects. Phylogenetic analysis revealed that JE80 and JE250 belongs to B. velezensis. These findings are consistent with previous studies demonstrating that Bacillus species, particularly B. subtilis and B. velezensis, are among the most effective bacterial BCAs against various plant pathogens due to their ability to produce a wide array of antimicrobial compounds and their adaptability to diverse environmental conditions (Caulier et al., 2019; Koo et al., 2023; Qin et al., 2025; Yang et al., 2023).

Assays using CFs provided insights into the antibacterial properties of JE80 and JE250. Interestingly, CFs from early-stage cultures (1-day-old) exhibited limited antibacterial effects, whereas CFs from 2- to 7-day-old cultures significantly inhibited the growth of E. amylovora. This suggests that antimicrobial compound production in JE80 and JE250 is growth-phase-dependent, potentially regulated by quorum sensing or other metabolic control mechanisms (Raaijmakers et al., 2010; Zhang et al., 2023). Similar findings have been reported in previous studies, where Bacillus spp. exhibited differential production of antimicrobial compounds depending on growth stage and nutrient availability (Luo et al., 2022; Wu et al., 2015). Furthermore, growth suppression assays demonstrated that CFs from 4-day-old cultures completely inhibited E. amylovora growth, indicating that optimal metabolite production occurs at this stage.

The inhibitory effects of JE80 and JE250 extended beyond bacterial growth suppression. Both strains significantly impaired bacterial motility and biofilm formation, which are critical virulence factors for E. amylovora. In particular, CFs from JE250 exhibited the strongest inhibition of swimming and swarming motility (99.9% and 97.27%, respectively), further underscoring its biocontrol potential. These findings align with previous research demonstrating that microbial secondary metabolites can interfere with flagellar function and quorum sensing, ultimately disrupting bacterial motility and biofilm formation (Rütschlin and Böttcher, 2020).

Bacterial motility plays a crucial role in the pathogenicity of E. amylovora, facilitating host surface colonization, infection, and dispersal (Koczan et al., 2011; Pedroncelli and Puopolo, 2024; Peng et al., 2021; Piqué et al., 2015). While swimming motility refers to the movement of individual cells, swarming motility involves the coordinated movement of bacterial clusters (Kearns, 2010). E. amylovora primarily utilizes flagella-driven motility to migrate within host tissues and spread systemically (Hossain and Tsuyumu, 2006; Yuan et al., 2022). The ability of JE80 and JE250 to suppress these motility traits suggests that they interfere with critical components of E. amylovora pathogenesis, potentially by targeting flagellar assembly, chemotaxis, or quorum sensing pathways. Especially, the observed suppression of motility and biofilm formation was measured using sub-inhibitory concentrations of JE250 CF (≤10%) (Supplementary Fig. 1). This suggests that the effects are not solely attributable to growth inhibition but may involve specific phenotypic alterations affecting virulence-related pathways. Such responses have been observed in other studies involving Bacillus-derived secondary metabolites acting as signaling disruptors or modulators of bacterial behavior (Kim et al., 2024b; Naga et al., 2023).

To evaluate the biocontrol efficacy of JE250 under more realistic agricultural conditions, we conducted in planta assays using apple blossoms, one of the primary infection sites of E. amylovora (Schnyder et al., 2022). Treatment with JE250 significantly reduced disease incidence compared to the negative control and performed comparably to STP, a commonly used antibiotic for fire blight control (Shade et al., 2013). These findings highlight the potential of JE250 as a viable alternative to chemical antibiotics for managing fire blight in apple production systems. Moreover, JE250 was originally isolated as an endophytic bacterium from apple fruits, which may confer it with an ecological advantage for colonization and persistence within the host plant. Endophytic origin suggests a higher likelihood of compatibility with the plant microbiome, allowing the strain to establish more effectively. This host-specific adaptation may enhance its long-term biocontrol efficacy in apple orchards compared to non-endophytic or rhizospheric Bacillus strains.

Among the tested isolates, B. velezensis JE250 demonstrated more consistent and pronounced antagonistic activity against E. amylovora than B. velezensis JE80. Based on these results, JE250 was selected for genome analysis. Whole-genome sequencing of JE250 provided valuable insights into its biocontrol potential at the genetic level. Genome analysis identified BGCs associated with secondary metabolite biosynthesis, transport, and catabolism, including difficidin, fengycin, bacillaene, macrolactin, bacillibactin, and bacilysin. These antimicrobial compounds have been widely reported for their ability to inhibit bacterial pathogens by targeting membrane integrity, protein synthesis, or metabolic pathways (Luo et al., 2022; Wu et al., 2015). In particular, difficidin and bacillaene exhibit broad-spectrum antibacterial activity, while fengycin and bacilysin contribute to the suppression of bacterial and fungal pathogens (Fan et al., 2017). Several of these compounds have been reported to exhibit broad-spectrum antibacterial activity. For instance, difficidin and bacillaene are known to inhibit a wide range of Gram-negative bacteria, including E. amylovora (Chen et al., 2009; Wu et al., 2015). In particular, Chen et al. (2009) demonstrated that difficidin and bacilysin produced by B. amyloliquefaciens effectively suppressed E. amylovora growth and reduced fire blight symptoms. Fengycin and bacilysin have also been implicated in the suppression of bacterial motility and biofilm formation in other phytopathogenic bacteria (Luo et al., 2022; Rütschlin and Böttcher, 2020). While more specific studies are needed to elucidate their direct roles against E. amylovora virulence traits, the presence of these BGCs in JE250 strongly supports its observed antagonistic activity against E. amylovora and further confirms its potential as a biocontrol agent.

Similar results have been reported for other Bacillus species, demonstrating their effectiveness in reducing fire blight severity by outcompeting the pathogen, producing antimicrobial compounds, and enhancing host resistance (Caulier et al., 2019; Chen et al., 2009; Pršić and Ongena, 2020; Wu et al., 2015). Compared to previously reported Bacillus-based BCAs, JE250 displays several distinct advantages. First, JE250 exhibited not only strong inhibition of E. amylovora growth but also significant suppression of motility and biofilm formation, which are key virulence traits of the pathogen. These multifaceted effects may enhance its overall biocontrol efficacy. Second, genome analysis revealed that JE250 possesses a diverse set of BGCs, including difficidin, fengycin, bacillaene, and macrolactin, many of which were identified with high similarity to known functional clusters. This suggests that JE250 may produce a broader or more potent combination of antimicrobial metabolites than other Bacillus strains. Finally, in planta assays showed that JE250’s performance was comparable to streptomycin sulfate, indicating its strong potential as an eco-friendly alternative to antibiotics in agricultural settings.

STP has been widely used as a chemical control agent for fire blight management due to its strong antibacterial activity against E. amylovora. However, its prolonged use has led to several issues, including the emergence of streptomycin-resistant strains of E. amylovora in various countries (McManus et al., 2002; Stockwell and Duffy, 2012). In addition, the use of antibiotics in agriculture raises environmental and public health concerns, prompting regulatory restrictions or bans in many regions (Vidaver, 2002). These limitations highlight the urgent need for effective and environmentally sustainable alternatives. In this context, B. velezensis JE250 presents a promising biocontrol strategy, as it suppresses key virulence traits like motility and biofilm formation and harbors diverse BGCs for antibacterial compound production. Unlike STP, the use of JE250 poses minimal risk for resistance development and aligns with current trends in sustainable plant disease management.

Collectively, our findings demonstrate that B. velezensis JE250 exhibits strong antagonistic activity against E. amylovora through multiple mechanisms, including suppression of motility and biofilm formation and the production of antimicrobial secondary metabolites. Its efficacy in reducing fire blight symptoms in apple blossoms further supports its potential application as a biocontrol agent. While this study provides promising insights, future research should focus on identifying the specific antimicrobial compounds responsible for its activity, optimizing formulation methods, and evaluating its field performance under diverse environmental conditions. Additionally, further genetic studies will be necessary to fully elucidate the regulatory networks controlling secondary metabolite production and bacterial interactions. Investigating how B. velezensis JE250 interacts with other microbial communities in the orchard ecosystem will also be critical for developing robust and sustainable fire blight management strategies. Given the growing concerns about antibiotic resistance and the environmental impact of chemical pesticides, biological control approaches such as those offered by B. velezensis JE250 represent an essential step toward integrated and eco-friendly disease management in apple production.

Notes

Conflicts of Interest

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

Acknowledgments

This work was supported by a Research Grant of Gyeongsangbuk-do Basic Project (One Team-One Professor Collaborative Research; LP0052262024).

Electronic Supplementary Material

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

PPJ-OA-03-2025-0037-Supplementary-Fig-1.pdf

PPJ-OA-03-2025-0037-Supplementary-Table-1.pdf

References

Blin K., Shaw S., Augustijn H. E., Reitz Z. L., Biermann F., Alanjary M., Fetter A., Terlouw B. R., Metcalf W. W., Helfrich E. J. N., van Wezel G. P., Medema M. H., Weber T.. 2023;antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic Acids Res 51:W46–W50.

Caulier S., Nannan C., Gillis A., Licciardi F., Bragard C., Mahillon J.. 2019;Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front. Microbiol 10:302.

Chen X. H., Scholz R., Borriss M., Junge H., Mögel G., Kunz S., Borriss R.. 2009;Difficidin and bacilysin produced by plant-associated Bacillus amyloliquefaciens are efficient in controlling fire blight disease. J. Biotechnol 140:38–44.

Droby S., Wisniewski M., Macarisin D., Wilson C.. 2009;Twenty years of postharvest biocontrol research: is it time for a new paradigm? Postharvest Biol. Technol 52:137–145.

Fan B., Blom J., Klenk H.-P., Borriss R.. 2017;Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an “operational group B. amyloliquefaciens” within the B. subtilis species complex. Front. Microbiol 8:22.

Fira D., Dimkić I., Berić T., Lozo J., Stanković S.. 2018;Biological control of plant pathogens by Bacillus species. J. Biotechnol 285:44–55.

Ham H., Lee S.-W., Lee Y. H.. 2025;Genetic diversity and genotype distribution of Erwinia amylovora in Korea. Plant Pathol. J 41:88–99.

Ham H., Oh G.-R., Lee Y. H., Lee Y. H.. 2024;Comparison of resistance acquisition and mechanisms in Erwinia amylovora against agrochemicals used for fire blight control. Plant Pathol. J 40:525–536.

Ham H., Oh G.-R., Park D. S., Lee Y. H.. 2022;Survey of oxolinic acid-resistant Erwinia amylovora in Korean apple and pear orchards, and the fitness impact of constructed mutants. Plant Pathol. J 38:482–489.

Haq I. U., Rahim K., Yahya G., Ijaz B., Maryam S., Paker N. P.. 2024;Eco-smart biocontrol strategies utilizing potent microbes for sustainable management of phytopathogenic diseases. Biotechnol. Rep 44:e00859.

Hinze M., Köhl L., Kunz S., Weißhaupt S., Ernst M., Schmid A., Voegele R. T.. 2016;Real-time PCR detection of Erwinia amylovora on blossoms correlates with subsequent fire blight incidence. Plant Pathol 65:462–469.

Hossain M. M., Tsuyumu S.. 2006;Flagella-mediated motility is required for biofilm formation by Erwinia carotovora subspcarotovora. J. Gen. Plant Pathol 72:34–39.

Hyatt D., Chen G.-L., Locascio P. F., Land M. L., Larimer F. W., Hauser L. J.. 2010;Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11:119.

Kanehisa M., Goto S., Sato Y., Kawashima M., Furumichi M., Tanabe M.. 2014;Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res 42:D199–D205.

Kearns D. B.. 2010;A field guide to bacterial swarming motility. Nat. Rev. Microbiol 8:634–644.

Kim B., Lee S. Y., Park J., Song S., Kim K.-P., Roh E.. 2024a;Bacteriophage cocktail comprising Fifi044 and Fifi318 for biocontrol of Erwinia amylovora. Plant Pathol. J 40:160–170.

Kim S., Shin Y. H., Weißhaupt S., Kunz S., Jeun Y. C.. 2024b;Protection efficacy of antibacterial strains against fire blight caused by Erwinia amylovora on apple blossom. Plant Pathol. J 40:633–640.

Kim Y. S., Ngo M. T., Kim B., Han J. W., Song J., Park M. S., Choi G. J., Kim H.. 2022;Biological control potential of Penicillium brasilianum against fire blight disease. Plant Pathol. J 38:461–471.

Koczan J. M., Lenneman B. R., McGrath M. J., Sundin G. W.. 2011;Cell surface attachment structures contribute to biofilm formation and xylem colonization by Erwinia amylovora. Appl. Environ. Microbiol 77:7031–7039.

Kong H. G., Ham H., Lee M.-H., Park D. S., Lee Y. H.. 2021;Microbial community dysbiosis and functional gene content changes in apple flowers due to fire blight. Plant Pathol. J 37:404–412.

Koo Y. M., Heo A. Y., Choi H. W.. 2023;Isolation and identification antagonistic bacterium Paenibacillus tianmuensis YM002 against Acidovorax citrulli. Front. Plant Sci 14:1173695.

Kumar S., Stecher G., Li M., Knyaz C., Tamura K.. 2018;MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol 35:1547–1549.

Kunz S.. 2006;Fire blight control in organic fruit growing: systematic investigation of the mode of action of potential control agents. Mitt. Biol. Bundesanst. Land Forstwirtsch 408:249–253.

Lee I., Kim Y. O., Park S.-C., Chun J.. 2016;OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int. J. Syst. Evol. Microbiol 66:1100–1103.

Lee J., Jung W.-K., Ahsan S. M., Jung H.-Y., Choi H. W.. 2024a;Identification of Pantoea ananatis strain BCA19 as a potential biological control agent against Erwinia amylovora. Front. Microbiol 15:1493430.

Lee S. Y., Yun M., Yang S., Roh E.. 2024b;Construction of microbial consortium FireFighter-C for controlling fire blight disease and its control effect. Res. Plant Dis 30:402–408.

Luo L., Zhao C., Wang E., Raza A., Yin C.. 2022;Bacillus amyloliquefaciens as an excellent agent for biofertilizer and biocontrol in agriculture: an overview for its mechanisms. Microbiol. Res 259:127016.

McGhee G. C., Sundin G. W.. 2011;Evaluation of kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of kasugamycin resistance potential in Erwinia amylovora. Phytopathology 101:192–204.

McManus P. S., Stockwell V. O., Sundin G. W., Jones A. L.. 2002;Antibiotic use in plant agriculture. Annu. Rev. Phytopathol 40:443–465.

Naga N. G., El-Badan D. E., Ghanem K. M., Shaaban M. I.. 2023;It is the time for quorum sensing inhibition as alternative strategy of antimicrobial therapy. Cell Commun. Signal 21:133.

Nawrocki E. P., Eddy S. R.. 2013;Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29:2933–2935.

Pedroncelli A., Puopolo G.. 2024;This tree is on fire: a review on the ecology of Erwinia amylovora, the causal agent of fire blight disease. J. Plant Pathol 106:823–837.

Peng J., Schachterle J. K., Sundin G. W.. 2021;Orchestration of virulence factor expression and modulation of biofilm dispersal in Erwinia amylovora through activation of the Hfq-dependent small RNA RprA. Mol. Plant Pathol 22:255–270.

Piqué N., Miñana-Galbis D., Merino S., Tomás J. M.. 2015;Virulence factors of Erwinia amylovora: a review. Int. J. Mol. Sci 16:12836–12854.

Powell S., Forslund K., Szklarczyk D., Trachana K., Roth A., Huerta-Cepas J., Gabaldón T., Rattei T., Creevey C., Kuhn M., Jensen L. J., von Mering C., Bork P.. 2014;eggNOG v4.0: nested orthology inference across 3686 organisms. Nucleic Acids Res 42:D231–D239.

Pršić J., Ongena M.. 2020;Elicitors of plant immunity triggered by beneficial bacteria. Front. Plant Sci 11:594530.

Qin X., Xiao Y., Xiong Q., Kong W.-L., Borriss R., Gao Z., Fan B.. 2025;Four antimicrobial compounds and ISR induction are involved in biocontrol of crown gall disease by the plant beneficial rhizobacterium Bacillus velezensis FZB42. Plant Dis 109:1760–1769.

Raaijmakers J. M., De Bruijn I., Nybroe O., Ongena M.. 2010;Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol. Rev 34:1037–1062.

Rütschlin S., Böttcher T.. 2020;Inhibitors of bacterial swarming behavior. Chemistry 26:964–979.

Ryu D. K., Adhikari M., Choi D. H., Jun K. J., Kim D. H., Kim C. R., Kang M. K., Park D. H.. 2023;Copper-based compounds against Erwinia amylovora: response parameter analysis and suppression of fire blight in apple. Plant Pathol. J 39:52–61.

Salm H., Geider K.. 2004;Real-time PCR for detection and quantification of Erwinia amylovora, the causal agent of fire blight. Plant Pathol 53:602–610.

Schattner P., Brooks A. N., Lowe T. M.. 2005;The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33:W686–W689.

Schnyder A., Eberl L., Agnoli K.. 2022;Investigating the biocontrol potential of the natural microbiota of the apple blossom. Microorganisms 10:2480.

Shade A., Klimowicz A. K., Spear R. N., Linske M., Donato J. J., Hogan C. S., McManus P. S., Handelsman J.. 2013;Streptomycin application has no detectable effect on bacterial community structure in apple orchard soil. Appl. Environ. Microbiol 79:6617–6625.

Song S., Kim B., Kim K.-P., Roh E.. 2024;Novel detection protocol for Erwinia amylovora in orchard soil after removal of infected trees. Plant Pathol. J 40:282–289.

Stockwell V. O., Duffy B.. 2012;Use of antibiotics in plant agriculture. Rev. Sci. Tech 31:199–210.

Sundin G. W., Wang N.. 2018;Antibiotic resistance in plant-pathogenic bacteria. Annu. Rev. Phytopathol 56:161–180.

Tadesse Mawcha K., Malinga L., Muir D., Ge J., Ndolo D.. 2025;Recent advances in biopesticide research and development with a focus on microbials. F1000Res 13:1071.

Tatusov R. L., Koonin E. V., Lipman D. J.. 1997;A genomic perspective on protein families. Science 278:631–637.

Vidaver A. K.. 2002;Uses of antimicrobials in plant agriculture. Clin. Infect. Dis 34Suppl 3. :S107–S110.

Wu L., Wu H., Chen L., Yu X., Borriss R., Gao X.. 2015;Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial activity against Xanthomonas oryzae rice pathogens. Sci. Rep 5:12975.

Yang F., Jiang H., Ma K., Wang X., Liang S., Cai Y., Jing Y., Tian B., Shi X.. 2023;Genome sequencing and analysis of Bacillus velezensis VJH504 reveal biocontrol mechanism against cucumber Fusarium wilt. Front. Microbiol 14:1279695.

Yuan X., Eldred L. I., Sundin G. W.. 2022;Exopolysaccharides amylovoran and levan contribute to sliding motility in the fire blight pathogen Erwinia amylovora. Environ. Microbiol 24:4738–4754.

Zhang N., Wang Z., Shao J., Xu Z., Liu Y., Xun W., Miao Y., Shen Q., Zhang R.. 2023;Biocontrol mechanisms of Bacillus: improving the efficiency of green agriculture. Microb. Biotechnol 16:2250–2263.

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