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. 2025 Oct 1;41(5):607–618. doi: 10.5423/PPJ.OA.01.2025.0007

Analysis of Bacterial Community Changes in Apple Trees Treated with Bacillus altitudinis KPB25, a Potential Biological Control Agent against Fire Blight

Na Ra Lim 1,†, Hyun Gi Kong 2,†, Eon Jin Jo 1, Min Kyu Kang 3, Duck Hwan Park 1,4,*
PMCID: PMC12488376  PMID: 41033673

Abstract

The balance of microbial communities in an ecosystem is the most important factor representing its healthy state, even when immigrant microorganisms, such as biological control agent, are introduced into agricultural fields. Thus, this study aimed to investigate the potential of the antagonistic bacterium KPB25 (Bacillus altitudinis) as a biological control agent against fire blight by analyzing the changes in the epiphytic and endophytic bacterial communities of apple tree leaves following treatment. The KPB25 treatment resulted in increased community richness and diversity in endophytic bacteria. Conversely, in epiphytic bacteria, community diversity decreased after treatment. Beta-diversity analysis revealed that the endophytic community formed distinct clusters following KPB25 treatment, indicating a shift in the community structure. Relative abundance analysis of the endophytic and epiphytic communities highlighted that some bacterial families, which increased in abundance following KPB25 treatment, oxidized sugars into organic acids or produced antibiotics, potentially creating an environment that makes it difficult for Erwinia amylovora to survive when attempting to infect its host. These findings suggest that KPB25 interacts with certain microbial taxa within apple trees, contributing to the regulation and alteration of the microbial community in a manner that promotes an environment unfavorable for E. amylovora. Overall, KPB25 may have enhanced certain microbial groups within the endophytic residual bacterial community of apple leaves that contribute to fire blight suppression, with minor structural changes but significant shifts in microbial diversity.

Keywords: Bacillus altitudinis, biological control, Erwinia amylovora, fire blight, microbial community

Fire blight, caused by Erwinia amylovora, is a severe bacterial disease that affects Rosaceae plants and significantly damages the global apple and pear industries. Fire blight was initially detected in apple (Myung et al., 2016) and pear trees (Park et al., 2016) in South Korea in 2015, subsequently cumulatively spreading to 2,323 orchards covering 1,227.6 ha in 34 cities/districts by 2023 (Ham et al., 2020, 2024). To control fire blight in Korea, the Rural Development Administration has registered 12 pesticides consisting of four antibiotics, including streptomycin, oxytetracycline, oxolinic acid, and validamycin, as well as eight copper-based agents, including copper hydroxide, copper sulfate basic, tribasic copper sulfate, and copper oxychloride, recommending their use during the blooming period (Choi et al., 2022; Park et al., 2017). However, excessive use of these chemical agents can lead to reduced crop yield and chlorophyll synthesis owing to altered photosynthesis and nutrient dynamics, as well as the emergence of antibiotic-resistant strains (Alengebawy et al., 2021). Consequently, there has been a global shift towards regulating the use of such chemicals. Alternatively, biological control using antagonistic microorganisms is considered safer than chemical methods (Elnahal et al., 2022). Thus, several strains of the Bacillus genus have been developed against E. amylovora as biological control agents for fire blight (Aldwinckle et al., 2002; Bahadou et al., 2018; Broggini et al., 2005; Chen et al., 2009; Shemshura et al., 2020).

However, biological control using antagonistic microorganisms remains challenging. Under laboratory conditions, observing the inhibitory effects of these antagonistic microorganisms on pathogens is easier when nutrients are abundant and conditions are optimized for microbial growth. However, under field conditions, factors such as nutrient deficiency, drought, UV radiation, and osmotic stress make it difficult for antagonistic microorganisms to successfully colonize the host, resulting in limited long-term control effects (Choi et al., 2022). In addition, plants have diverse microbial communities that interact with each other, making it challenging for the introduced antagonistic microorganisms to establish themselves within these complex communities. Previous studies on biological control have focused on the interactions between single strains of antagonistic microorganisms, pathogens, and host plants. However, to overcome these challenges, studying the interactions between antagonistic microorganisms and microbial communities is increasingly required (Sébastien et al., 2015). Plant microbial communities influence plant development and health as well as affect disease occurrence through interactions between the host plant, pathogens, and antagonistic microorganisms (Berg et al., 2014; Dicke, 2016; Hawkes and Connor, 2017; Sébastien et al., 2015). Despite the importance of these interactions, studies on their interactions remain limited. Furthermore, although apples are among the most consumed fruits globally, studies on apple microbial communities are limited. Most studies primarily focus on changes in microbial communities due to pathogen infections, apple cultivars, and post-harvest storage methods (Abdelfattah et al., 2020; Bösch et al., 2021; Cui et al., 2021).

Therefore, this study aimed to examine the changes in the bacterial community of apple leaves following the reintroduction of B. altitudinis KPB25, a strain initially isolated from apple tree leaves and previously confirmed to inhibit E. amylovora (Choi et al., 2022).

Materials and Methods

Bacterial strain and culture conditions

In a previous study, the B. altitudinis KPB25 strain was identified, and its ability to inhibit the E. amylovora TS3128 strain was confirmed by inhibition zone and in planta assays using immature apple fruits and seedlings (Choi et al., 2022). In this study, KPB25 was used to determine its impact on the bacterial community in apple leaves (cv. Fuji) and evaluate the potential and safety of the residual bacterial communities as biological control agents. This strain was cultured in liquid or solid Luria-Bertani (LB) medium at 28°C for 24 h.

Field sampling and environmental condition

The sampling was conducted in an apple orchard located in Sinchon-ri, Dongnae-myeon, Chuncheon City, Gangwon province (37°51′16.2″N; 127°47′26.6″E), from May 24, to June 12, 2022. Three apple trees were randomly selected and KPB25 was applied to these trees at six-day intervals, with three treatments in total (Fig. 1A). The bacterial suspension used for the treatment was prepared at an optical density (OD) 600 nm of 0.1, and 500 mL of the suspension was sprayed onto each tree. The first and second treatments were applied on May 24 and 31, and the third on June 6, 2022. Because of the rain on May 30, the second treatment was postponed until May 31. Samples were collected before and after the application of the antagonistic bacteria KPB25. Pre-treatment samples were collected immediately before the first treatment on May 24, and post-treatment samples were collected five days after the final treatment on June 12. For each tree, 20–30 apple leaves were collected from one tree using pruning shears and the pruning shears were disinfected with 70% ethanol for 10 min to prevent cross-contamination when move to next tree during sampling. Collected leaves were kept in a zipper bag inside an ice box, transported to the laboratory, and stored at 4°C until sample processing the next day. Environmental conditions during the sampling period, including daily maximum and minimum temperatures, humidity, and precipitation were recorded using data from the Korea Meteorological Administration.

Fig. 1.

Fig. 1

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(A) Schematic diagram of the sampling procedure used to investigate changes in the residual microbiome of the apple leaves (cv. Fuji) episphere and endosphere following KPB25 treatment. (B) Relative abundance of the Class level of bacterial community in the episphere and endosphere of apple leaves before and after KPB25 treatment. BEp, epiphytic community before treatment (pre-treatment); AEp, epiphytic community after treatment (post-treatment); BEn, endophytic community before treatment; AEn, endophytic community after treatment.

Microbial DNA extraction and sequencing

Microbial DNA was extracted from both the episphere and endosphere before and after treatment with KPB25, according to a previously published method (Lee et al., 2023). Briefly, for the episphere, 5 g of apple leaves were placed in a 50 mL tube, followed by the addition of 40 mL of 1× phosphate buffered saline buffer (pH 7.4). The mixture was sonicated at 35 kHz for 45 s using a sonicator Q125 (Qsonica, Newtown, CT, USA), followed by vortexing for 30 s to obtain a suspension. The suspension was transferred to a new tube and centrifuged at 2,000 ×g for 20 min at 4°C. The resulting pellet was stored at −70°C until DNA extraction. For the endosphere, 10–13 apple leaves per sample were surface-sterilized in 70% ethanol for 30 s, followed by 30 s in 1% NaOCl, and then rinsed three times with sterile water. The samples were dried on a clean bench for 1 h. Once completely dry, the leaves were flash-frozen in liquid nitrogen and ground into a fine powder using a pestle. The ground samples were stored at −70°C until DNA extraction. Total microbial DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, Irvine, CA, USA), following the manufacturer’s instruction. A suspension (0.5 mL) was used for epiphytic microorganisms, and 0.3 g of ground leaf tissue was used for endophytic microorganisms. The extracted DNA was quantified using BioSpectrometer basic (Eppendorf, Hamburg, Germany), with concentrations between 6 and 20 ng/μL. Sequencing was performed using the Illumina MiSeq platform (Illumina, San Diego, CA, USA) by Macrogen (Seoul, Korea). The first round of PCR amplification was conducted using 2 ng of gDNA, 5× reaction buffer, 1 mM dNTP mix, 500 nM each of forward and reverse universal primers, 5 μM of each of the pPNA and mPNA oligo, and Herculase II Fusion DNA Polymerase (Agilent Technologies, Santa Clara, CA, USA). The universal primers 341F and 805R were used for amplification (Lee et al., 2023). The PCR cycling conditions were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, repeated for a total of 25 cycles, with a final extension at 72°C for 5 min. The first-round PCR products were purified using AMPure beads (Agencourt Bioscience, Beverly, MA, USA). For the final library preparation, a second round of PCR amplification was performed using 2 μL of purified PCR product and Nextera XT Indexed primers. The cycling conditions were the same as those used in the first PCR, except that the number of cycles was reduced from 25 to 10. Second-round PCR products were purified using AMPure beads. The purified products were quantified by qPCR following the KAPA Library Quantification protocol for Illumina sequencing platforms. The final libraries were sequenced on a MiSeq platform (Illumina) by paired-end sequencing.

Bacterial community analysis

Metagenomic analysis of the FASTQ files was conducted using QIIME2 (version 2024.10). Initially, the DADA2 plugin was employed to process the raw data, which involved chimera sequence removal, merging of forward and reverse reads, and filtering to create amplicon sequence variants (ASVs) (Supplementary Table 1). ASVs were clustered based on 97% sequence identity to generate operational taxonomic units (OTUs), and taxonomic classification was performed using the Silva 138 database (Yakubu et al., 2025). After taxonomic classification, sequences related to Archaea, mitochondria, cyanobacteria, and Rickettsia were removed to ensure an accurate community diversity analysis. The observed OTU, Chao1, evenness, and Shannon indices were used for the alpha diversity analysis. Beta-diversity was analyzed using the Bray-Curtis dissimilarity distance matrix.

In planta assay to determine density of KPB25

To determine the density of KPB25 in M9 apple tissue culture plant, an in planta assay was performed using a GFP-tagged strain of KPB25, where the plasmid pBAV1K-T5-gfp (KmR) was inserted. KPB25 (KmR) carrying the pBAV1K-T5-gfp was cultured overnight in LB medium containing kanamycin (50 μg/mL) at 28°C, and the cells were harvested and resuspended in 10 mM MgCl2 buffer to prepare a total of 150 mL of the suspension with an OD 600 nm of 0.1 (approximately 108 colony-forming unit [CFU]/mL). The KPB25 (KmR) bacterial suspension was sprayed onto the leaves 10 times per plant and the tissue culture plants were incubated at 28°C for 7 days. For isolation and enumeration of endophytic KPB25, tissue culture plants were surface-sterilized by immersing them in 70% ethanol for 30 s, followed by 1% NaOCl for 30 s, and rinsed three times with sterile water. The sterilized plant tissues were placed in extraction bags containing 3 mL of 10 mM MgCl2 and macerated to extract the internal microbial suspension. The extract was serially diluted from 100 to 10−7, and 10 μL of each dilution was spotted onto LB (Km) agar plates and plates were incubated at 28°C for 24 h, and CFU counts were recorded. In order to isolate and enumerate epiphytic KPB25, tissue culture plants were placed in 50 mL tubes containing 35 mL of 10 mM MgCl2 buffer and vortexed for 1 minute to dislodge epiphytic bacteria. The suspension was centrifuged at 4,000 rpm for 15 min, and the pellet was resuspended and serially diluted from 100 to 10−7. Each dilution (10 μL) was spotted onto LB (Km) agar plates and plates were incubated at 28°C for 24 h, and CFU counts were recorded. The experiment was repeated three times in different day with each KPB25 culture.

Statistical analysis

Data were analyzed using T-test in paired sample between before and after KPB25 treatments by Jamovi software (ver. 2.3.21.0), and the means were compared using Student’s t-test at a significance level of P < 0.05. For alpha diversity analysis was visualized using the ggplot2 and heatmap packages in R Studio (version 4.0.3, Posit, PBC, Boston, MA, USA). Differential abundance of endophytic microbial OTUs before and after KPB25 treatment was determined using the DESeq2 package (version 1.14.1) in R (R Foundation for Statistical Computing, Vienna, Austria).

Results

Environmental conditions in the tested orchard

For a comprehensive understanding of the effects of environmental conditions on microbial community changes, we investigated the temperature, humidity, and precipitation during the sampling period. Data were collected from May 24, 2022, before treatment with strain KPB25, to June 12, 2022, after treatment. The variability in minimum and maximum temperatures during this period was minimal; however, the highest temperature (May 24) and the lowest temperature (June 6) differed by approximately 11°C. The daily maximum humidity exceeded 80% for 17 of the 20 days of the sampling period. Rainfall was recorded for 10 of the 20 days.

Bacterial diversity in endosphere and episphere from apple leaves before and after KPB25 treatment

The microbiomes of the endosphere and episphere of apple leaves were analyzed before and after treatment with KPB25. At the class level, Gammaproteobacteria dominated the endosphere microbiome, accounting for more than 56.2% of the relative abundance, whereas Actinobacteria was the most abundant class in the episphere, comprising average of 49.6% of the community (Fig. 1B). Notably, the microbial composition of episphere samples showed greater variability across biological replicates, while the endosphere samples exhibited a more stable and consistent community structure. Following KPB25 treatment, Actinobacteria remained the most dominant class in the episphere, increasing approximately two-fold from 37.1% to 61.8%. Bacilli also increased in relative abundance, from 19.1% before treatment to 24.4% after treatment. In contrast, Gammaproteobacteria showed a marked decrease in the episphere, dropping from 38.1% to 8.1% (Fig. 1B). In the endosphere, the overall microbial community structure remained relatively stable before and after KPB25 treatment. However, one notable change was observed in the relative abundance of Alphaproteobacteria, which increased from 1.3% to 9.9% after treatment (Fig. 1B).

To evaluate the impact of KPB25 treatment on the endophytic and episphere bacterial communities, alpha diversity was assessed based on observed features, Chao1, evenness, and Shannon indices. In the episphere, the number of observed features representing species richness and the Chao1 index were significantly reduced following KPB25 treatment (Fig. 2A). Although the evenness and Shannon diversity indices did not show statistically significant differences, both exhibited decreasing trends, suggesting a reduction in both microbial species richness and evenness (Fig. 2A). This reduction may be attributed to the dominance of certain bacterial groups promoted by KPB25, which could have led to decreased evenness and overall diversity.

Fig. 2.

Fig. 2

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Alpha diversity of the bacterial community in the episphere (A) and endosphere (B) of apple leaves before and after KPB25 treatment. For variables, observed features, Chao 1, evenness, and Shannon were used. Asterisk indicates significant differences (*P < 0.05) before and after KPB25 treatments, whereas N.D. indicates non-significant differences between them by Student’s t-test. Principal coordinate analysis of representative sequences at the amplicon sequence variant level in the episphere (C) and endosphere (D) using the Bray-Curtis dissimilarity distance matrix. Statistical analysis was performed using pairwise two-way PERMANOVA. BEn, endophytic community before treatment; AEn, endophytic community after treatment.

In contrast to the episphere results, the endophytic bacterial community showed an increasing trend in richness following KPB25 treatment, although the differences were not statistically significant. The number of observed features increased (Fig. 2B), and the Chao1 index also showed a similar upward trend, suggesting that KPB25 may have contributed to an increase in the number of species within the endosphere (Fig. 2B). The evenness index remained stable before and after treatment, indicating that the relative distribution of species was maintained (Fig. 2B).

Bacterial community structure in the endosphere and episphere before and after KPB25 treatment

The similarity of endophytic and epiphytic bacterial communities before and after treatment was evaluated using principal coordinate analysis (PCoA) based on the Bray-Curtis dissimilarity distance matrix. Regarding the similarity between epiphytic microbial communities, the variance explained by the axes was 47.96% and 25.85% for the PCo1 and PCo2, respectively. The PCoA plot revealed that the microbial community was primarily dispersed along PCo1 before KPB25 treatment, whereas dispersion occurred along PCo2 after treatment (Fig. 2C). Although distinct clusters were not clearly formed before and after KPB25 treatment, the distribution patterns differed depending on each treatment condition (Fig. 2C). The lack of differentiation could be due to the influence of various environmental stresses affecting epiphytic bacteria, making it harder for KPB25 to significantly impact the community structure.

For the endophytic structure, according to the Bray-Curtis dissimilarity, the variance explained by the axes was 43.86% for the first principal coordinate (PCo1) and 21.86% for the second principal coordinate (PCo2). The PCoA plot showed a clear separation of groups along the PCo1 axis before and after KPB25 treatment (Fig. 2D). This suggests that KPB25 has a greater impact on the endogenous microbial community structure than on the epiphytic community structure.

Composition of the dominant bacterial community in the endosphere and episphere before and after KPB25 treatment

To identify OTUs that were uniquely present in the epiphytic bacterial community before and after KPB25 treatment, a Venn diagram was constructed. The analysis revealed that 25 OTUs were unique to the pre-treatment group, while 17 OTUs were exclusively observed after treatment (Fig. 3A). At the family level, community composition analysis showed that the relative abundance of Propionibacteriaceae increased from 17.52% before treatment to 42.54% after treatment. Conversely, Erwiniaceae decreased sharply from 28.88% to 1.99% (Fig. 3B), indicating that the majority of Enterobacterales—which were dominant before treatment—were primarily composed of Erwiniaceae. Families accounting for more than 5% of the total community included Propionibacteriaceae, Erwiniaceae, Streptococcaceae, Pseudomonadaceae, Corynebacteriaceae, Staphylococcaceae, and Streptomycetaceae. These seven families were grouped based on whether their relative abundance increased, decreased, or remained unchanged after KPB25 treatment.

Fig. 3.

Fig. 3

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Comparison of relative abundance at the family level and analysis of the top seven most abundant families in the episphere. (A) Venn diagram deduced from OTUs that were uniquely present in the epiphytic bacterial community before and after KPB25 treatment. (B) Seven families were grouped based on whether their relative abundance increased, decreased, or remained unchanged after KPB25 treatment. The families were identified based on a 5% cut-off of the total abundance, allowing for comparison of their relative abundance before and after treatment. BEp, epiphytic community before treatment (pre-treatment); AEp, epiphytic community after treatment (post-treatment); N.D., non-significant differences; OTU, operational taxonomic units.

Specifically, Propionibacteriaceae and Streptococcaceae showed more than a two-fold increase in abundance following treatment. In contrast, Erwiniaceae decreased approximately sevenfold (Fig. 3B). However, these changes were not statistically significant due to high variability among replicates. The abundances of Corynebacteriaceae and Streptomycetaceae remained unchanged (Fig. 3B). These findings suggest that KPB25 may exert selective positive or negative effects on specific bacterial families, such as Propionibacteriaceae and Erwiniaceae, thereby influencing episphere microbial community dynamics.

In the endosphere, a Venn diagram comparing pre- and post-treatment OTUs revealed 8 OTUs unique to the pre-treatment group and 20 OTUs unique to the post-treatment group, indicating an increase in OTU diversity following KPB25 treatment (Fig. 4A). At the family level, Burkholderiaceae was the most dominant group, comprising approximately 45–50% of the total community (53% in untreated; 44.8% in treated samples) (Fig. 4B). To further assess the impact of KPB25, we applied a 5% abundance cutoff and selected the six most dominant families: Burkholderiaceae, Propionibacteriaceae, Pectobacteriaceae, Acetobacteraceae, Streptococcaceae, and Pseudomonadaceae. Relative abundance analysis showed that Burkholderiaceae, Propionibacteriaceae, Pectobacteriaceae, Pseudomonadaceae, and Acetobacteraceae increased after treatment, but these changes were not statistically significant (Fig. 4B). In addition, differential abundance analysis using DESeq2 revealed that Dickeya and Gluconobacter significantly increased in AEn following KPB25 treatment. Cutibacterium also exhibited an increasing trend, although the change was not statistically significant. In contrast, Prauserella and Ralstonia showed decreasing trends in AEn (Fig. 4C).

Fig. 4.

Fig. 4

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Comparison of relative abundance at the family level and comparison of the top six most abundant families in the endosphere. (A) Venn diagram deduced from OTUs that were uniquely present in the epiphytic bacterial community before and after KPB25 treatment. (B) The top six most abundant families were compared before and after KPB25 treatment. These top six families were selected using a 5% cut-off based on the total number of reads. Asterisks indicate significant differences. *P < 0.05, **P < 0.01, ***P < 0.001 before and after KPB25 treatments, whereas N.D. indicates non-significant differences between them by Student’s t-test. (C) Differential abundance analysis using DESeq2 identified five genera showing significant changes between before and after KPB25 treatment. BEn, endophytic community before treatment; AEn, endophytic community after treatment; OTU, operational taxonomic units.

Localization of KPB25

Bacillaceae, the family to which KPB25 belongs, was not detected before treatment but showed an increase in abundance after KPB25 application. However, this increase was not statistically significant (Fig. 5A). This suggests that while KPB25 may have contributed to the establishment or growth of Bacillaceae within the community, the effect was not statistically validated. Growth curve analysis and inhibition zone tests against E. amylovora confirmed that the plasmid (pBAV1K-T5-gfp) - carrying KPB25 strain exhibited growth and inhibitory effects similar to the wild-type KPB25 (data not shown). Thus, KPB25 (KmR) strain was considered morphologically and functionally equivalent to the wild type. After a 7-day incubation of M9 tissue culture plants inoculated with KPB25 (KmR), bacterial densities were quantified on the plant surface (3.5 × 107 CFU/mL) and within internal tissue (1.2 × 105 CFU/mL) (Fig. 5B). The higher density of KPB25 on the external surface compared to the internal tissues indicates that KPB25 predominantly colonizes the surface of the plant tissue while also being able to penetrate and persist internally. This dual colonization ability enhances the potential of KPB25 as a biological control agent by allowing it to target pathogens both externally and internally.

Fig. 5.

Fig. 5

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(A) The abundance of the Bacillaceae family, which includes KPB25, was compared before and after treatment. Although an increase in abundance was observed after KPB25 treatment, it was not statistically significant (N.D.) as determined by Student’s t-test (P < 0.05). (B) In planta assay to confirm the localization of KPB25 in apple tissue culture (M9). The graph illustrates the bacterial density (CFU/mL) of KPB25 (KmR) detected in the internal and external extracts of tissue culture plant, confirming the strain’s localization. An asterisk on the bar indicates a significant difference (*P < 0.05) between endosphere and episphere of localized KPB25 (KmR) determined by Student’s t-test. BEp, epiphytic community before treatment (pre-treatment); AEp, epiphytic community after treatment (post-treatment).

Discussion

In South Korea, fire blight control primarily relies on chemical treatments involving antibiotics such as tetracycline and streptomycin, as well as copper-based agents. However, these chemical control methods present challenges, such as the emergence of antibiotic-resistant strains and negative effects on non-target microorganisms, which result in microbial community imbalance. Alternative methods, including the use of antagonistic microorganisms, essential oils, plant extracts, and bacteriophages, have been suggested (Abd El-Hack et al., 2022; Akhlaghi et al., 2020; Romero-Calle et al., 2019) in the agriculture and clinical fields to address these concerns. Among these, biological control using antagonistic microorganisms offers several advantages, such as colonization of host plants and production of secondary metabolites such as, antimicrobial peptides to inhibit pathogens or provide induced systemic resistance in plants to enhance defense against pathogens (Chen et al., 2009; García-Gutiérrez et al., 2013; Kim et al., 2020). The key to effective biological control using antagonistic microorganisms is their ability to successfully colonize host plants under field conditions characterized by environmental stress and nutrient scarcity. Bacillus spp. can form endospores that allow them to resist and survive under unfavorable conditions and nutrient limitations (Nicholson, 2002). These characteristics render them advantageous as antagonistic microorganisms. Successful colonization of host plants by antagonistic microorganisms requires a thorough understanding of the native microbial community of the host. Plants harbor diverse microbial communities that play significant roles in plant health and disease occurrence through mutual interactions (Sébastien et al., 2015). Thus, studies into the interactions between antagonistic microorganisms and the indigenous microbial communities of host plants are crucial; however, such studies remain limited. In this study, we investigated the microbial community changes in apple leaves after treatment with the antagonistic bacterium KPB25 (B. altitudinis), with known fire blight inhibitory effects (Choi et al., 2022).

The epiphytic community composition showed changes in the relative abundance at the class level following KPB25 treatment. The mean of abundance of Actinobacteria was the most dominant class (Fig. 1B). Analysis of community diversity and similarity indicated that the bacterial community diversity, richness, and evenness decreased in the epiphytic community after KPB25 treatment (Fig. 2A). This suggests that the increased abundance of Propionibacteriales following KPB25 treatment may have affected the community evenness and diversity. After KPB25 treatment, Propionibacteriaceae accounted for approximately 42% of the total community and was also observed in the endophytic community (Fig. 3B). The increased abundance of Propionibacteriaceae suggested that KPB25 interacted positively with this family. Additionally, Streptococcaceae showed increases after treatment (Fig. 3B). Streptococcaceae, belonging to Lactobacillales, includes pathogenic and non-pathogenic species. Streptococcaceae are abundantly present in the microbiomes of herbs (Lepidium sativum) and chicory (Cichorium endivia) and are also dominant in the endophytic microbiome of almond leaves, where some species produce H2O2 to inhibit pathogens (Guzmán et al., 2022; Patz et al., 2019). This suggests that Streptococcaceae can grow effectively in plants and potentially suppress plant pathogens. Beta-diversity analysis of the epiphytic community based on the Bray-Curtis similarity matrix showed that the intergroup patterns before and after KPB25 treatment were separated along different axes, with dispersion primarily along PCo1 and PCo2, respectively (Fig. 2C). In contrast, the endophytic community exhibited a clearer separation between treatment groups, indicating a more distinct impact of KPB25 compared to the epiphytic community (Fig. 2D). Given that the epiphytic surface is exposed to various environmental stresses (Dini-Andreote, 2020; Papp-Rupar et al., 2022), the PCoA results may suggest that the epiphytic community was more affected by external environmental factors other than KPB25.

The analysis of endophytic bacterial community changes owing to KPB25 treatment, revealed that Gammaproteobacteria was the most dominant order, accounting for more than 60% of the endophytic community before and after KPB25 treatment (Fig. 1B). At the family level, Burkholderiaceae was also the most abundant (Fig. 4B). This result is consistent with a previous study indicating that Burkholderiaceae is a major component of roots and fruits in healthy apple trees (Kim et al., 2021). Consequently, we inferred that Burkholderiaceae is important in interacting with other microbial community members within apple leaves. In the AEn, the relative abundance of Pseudomonadaceae, Propionibacteriales, Streptococcaceae, Acetobacterales, and Pectobacteriaceae increased (Fig. 4B). These results suggest that KPB25 treatment induced changes in the endophytic community. Additionally, we evaluated the changes in community richness, evenness, and diversity following KPB25 treatment. The endophytic community showed increases in richness and diversity after KPB25 treatment (Fig. 2B). Increased community diversity may enhance plant resistance to pathogen invasions. Cluster analysis using the Bray-Curtis matrix indicated that the endophytic communities formed distinct clusters before and after KPB25 treatment, suggesting that KPB25 influenced the endophytic community through interactions with other bacteria (Fig. 2D). Further analysis at the family level revealed that the abundance of Bacillaceae increased after KPB25 treatment, although this change was not statistically significant (Fig. 5A). In addition, in planta assay, localization of KPB25 was confirmed that primarily occupied on episphere and then could enter into endosphere of tissue culture plant (Fig. 5B). This implies that KPB25 might have infiltrated the plant’s endosphere. The top six most abundant families were selected, and their relative abundances were compared before and after KPB25 treatment (Fig. 4B). The families Burkholderiaceae, Pseudomonadaceae, Propionibacteriaceae, Streptococcaceae, Acetobacteraceae, and Pectobacteriaceae showed changes in abundance after KPB25 treatment. Burkholderiaceae inhabit a wide range of environments and include microorganisms that can metabolize and survive under conditions, ranging from facultative anaerobes to obligate anaerobes and aerobes (Coenye, 2014). The ability of Burkholderiaceae to adapt to various environments likely explains their consistently high abundance. Propionibacteriaceae are capable of thriving in diverse habitats, including dairy products, sludge, soil, water, and sewage treatment facilities, and consist of microorganisms that are either facultatively anaerobic or aerobic. The increase in Propionibacteriaceae abundance suggests that KPB25 treatment supports the growth of bacteria with versatile metabolic properties. Acetobacteraceae are obligate aerobes whose main characteristics are the oxidation of sugars and ethanol to organic acids via dehydrogenase enzymes (Komagata et al., 2014). They can survive in highly acidic environments, such as conditions with a pH of 3.0–3.5, and their optimal survival pH ranges from 5.0 to 6.5 (Reis and Teixeira, 2015). The increase in Acetobacteraceae abundance following KPB25 treatment suggests that it can alter the pH conditions within plant tissues, potentially creating a more acidic environment. Such an environment can be unfavorable for E. amylovora because it has an optimal survival pH of 7.5 and cannot survive in conditions with a pH below 5.0 (Shrestha et al., 2005). This suggests that an increase in Acetobacteraceae can contribute to the inhibition of E. amylovora invasion by creating conditions that hinder its survival.

This study aimed to investigate the interaction between an antagonistic bacterium, KPB25, and microbial communities in apple tree leaves. KPB25 grew within the plant as an endophyte and epiphyte as well as induced changes in the bacterial communities of apple leaves. In the endophytic community, diversity increased following KPB25 treatment, creating an unfavorable environment for E. amylovora invasion. In contrast, the diversity of the epiphytic community decreased, potentially because of exposure to various environmental stresses in addition to the effects of KPB25 treatment. However, a limitation of this study is the absence of control or mock-treated samples collected at the same time point as the KPB25 treatment group. Including such controls would have allowed us to more clearly distinguish whether the observed changes in endophytic and epiphytic microbial communities were due to KPB25 treatment or simply the result of temporal variation. Therefore, in our upcoming study examining the microbial community shifts in apple flowers before and after KPB25 treatment, we will ensure that control samples are collected at the same post-treatment time point. Nevertheless, KPB25 may interact with specific bacterial species within the leaf-associated community, leading to compositional changes that promote a community structure unfavorable to E. amylovora and potentially contribute to its suppression. Considering the important role of plant microbial communities in disease occurrence and plant health, studying the interactions between antagonistic microorganisms and native microbial communities is crucial for successful biological control. Therefore, this study provides foundational data for future research on the interactions between antagonistic microorganisms and apple tree microbial communities, contributing to a deeper understanding of these dynamics.

Footnotes

Conflicts of Interest

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

Acknowledgments

This work was carried out with the support of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (No. RS-2020-IP320041).

Electronic Supplementary Material

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

References

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Associated Data

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Supplementary Materials

PPJ-OA-01-2025-0007-Supplementary-Table-1.pdf (81.5KB, pdf)

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