Mechanisms of biocontrol against strawberry angular leaf spot disease by a constructed Paenibacillus polymyxa MY-J3 microbial consortium

Angular leaf spot (ALS) disease, caused by Xanthomonas fragariae, has emerged as a devastating bacterial pathogen, posing a serious threat to strawberry production. This study aimed to construct an effective synthetic microbial consortium using antagonistic bacteria and to elucidate its biocontrol mechanisms against ALS through an integrated approach including culturomics, real-time quantitative PCR (qPCR), and high-throughput sequencing of the phyllosphere microbiome. The main findings are as follows: Three synthetic microbial consortia were assembled following compatibility assessment. Among these, the combination of Paenibacillus polymyxa MY-J3 and Lysobacter antibioticus HY (designated M+H) demonstrated superior efficacy, achieving a relative control efficacy of 76.15% and 74.26% under greenhouse and field conditions, respectively. Using a tailored qPCR assay for X. fragariae detection, the M+H treatment reduced pathogen abundance by 99.99% compared to the control. The consortium M+H markedly up-regulated the expression of host defense-related genes while down-regulating key virulence genes of X. fragariae. The crude extract from the M+H consortium exhibited a minimum inhibitory concentration (MIC) of 80 mg/mL against X. fragariae and significantly disrupted bacterial biofilm formation, cell surface hydrophobicity, extracellular polysaccharide production, and reduced pathogenicity. Furthermore, treatment with the consortium notably altered the diversity and composition of the strawberry phyllosphere bacterial community. The microbial consortium M+H can suppress the occurrence of the ALS through multiple mechanisms, demonstrating promising application prospects.

1 Introduction

The strawberry (Fragaria × ananassa), a perennial herbaceous plant within the genus Fragaria (Rosaceae), is cultivated worldwide due to its high nutritional value. Strawberry angular leaf spot (ALS), caused by the bacterium Xanthomonas fragariae, represents a major bacterial disease affecting cultivated strawberry growing regions. The pathogen belongs to the family Xanthomonadaceae. This disease was first identified and described in Minnesota, USA, in 1960 (Kennedy and King, 1962a, 1962b). Since the 1970s, it has been progressively detected throughout Europe and is now present in strawberry production areas across the globe (Dye and Wilkie, 1973; Mazzucchi et al., 1973; Panagopoulos et al., 1978). ALS was classified as a quarantine pest by the European and Mediterranean Plant Protection Organization (EPPO) as early as 1986 (EPPO, 1986) and was subsequently listed in Annex II of the EU Council Directive 2000/29/EC (European Union, 2000). In 2007, it was also included in the Imported Plant Quarantine Pest List of the People’s Republic of China. The disease affects nearly all plant tissues, including leaves, calyces, corollas, fruit stalks, and stems (Gubler et al., 1999). The pathogen is capable of systemic movement through the plant’s vascular system, leading to infection of other leaf tissues, crown tissues, and developing daughter plants (Hildebrannd et al., 1967; Milholland et al., 1996).

Currently, chemical pesticides remain a primary measure for controlling ALS; however, their long-term use contributes to environmental pollution and may lead to the development of pesticide resistance. The development of resistant cultivars is another effective control strategy. Although breeding efforts are ongoing, no strawberry variety with complete resistance to ALS has been developed to date. Due to its environmental safety and reduced risk of inducing pathogen resistance, biological control has become a major research focus. Currently, research on the biocontrol of ALS is still in its early stages internationally. The (Daranas et al., 2019) reported that Lactobacillus strains TC92 and CC100, along with Leuconostoc mesenteroides strains CM160 and CM209, exhibited antagonistic effects against ALS. Bacillus subtilis QST713 has also shown some efficacy in controlling the disease. In recent years, research on phage-based biocontrol agents against plant pathogens has increased (Holtappels et al., 2019). McMahon et al (McMahon et al., 2020). isolated the xanthomonad phage RiverRider from naturally infected ‘Festival’ strawberry plants, which demonstrated infectivity against seven distinct strains of X. fragariae. Henry et al (Henry et al., 2016). identified several antagonistic bacteria, including Pseudomonas koreensis, Pseudomonas mandelii, and Rhizobium radiobacter, capable of inhibiting the growth of X. fragariae.

The use of mixed microbial consortia represents a common strategy for plant disease management. Such combinations can enhance plant defense mechanisms and improve efficacy against pathogens. For instance, Dunne et al (Dunne et al., 1998). demonstrated that co-inoculation of phloroglucinol-producing fluorescent pseudomonads with proteolytic Stenotrophomonas maltophilia significantly improved suppression of Pythium-induced damping-off in sugar beet. Mao et al (Mao et al., 2005). found that combining B. subtilis and Pseudomonas fluorescens in specific ratios enhanced inhibitory effects against Fusarium solani. Similarly, De Boer et al (De Boer et al., 1999). showed that a combination of resistance-inducing Pseudomonas putida RE8 and siderophore-producing P. putida WCS358 provided improved control of Fusarium wilt in radish.Ye (Ye, 2023) reported that a 3:1 mixture of two Bacillus velezensis strains acted synergistically against cucumber damping-off. Wang et al. (Wang, 2022) combined high-activity Bacillus safensis with broad-spectrum antifungal Lysobacter enzymogenes OH11 at a volumetric ratio of 1:3, achieving effective control against major bacterial and fungal diseases including kiwifruit canker and anthracnose.

Conventional biocontrol strategies predominantly relying on single-strain applications often face limitations due to inconsistent field performance and narrow modes of action. In contrast, synthetic microbial consortia utilize functional complementarity among constituent strains to establish multidimensional synergistic interactions with pathogens, thereby enhancing both the stability and efficacy of disease control. In this study, we constructed various synthetic consortia by combining the newly isolated antagonistic strain MY-J3 with established biocontrol strains. These consortia were systematically evaluated through in vitro and field experiments to assess their biocontrol activity, while preliminary investigations were conducted to elucidate the mechanisms underlying suppression of ALS. This research provides foundation for developing effective compound microbial inoculants and formulating sustainable eco-friendly strategies for ALS management.

2 Materials and methods

2.1 Identification of biocontrol strains and assessment of compatibility

2.1.1 Identification of biocontrol bacteria

In this study, a biocontrol strain MY-J3 with strong antagonistic activity against X. fragariae was isolated from healthy strawberry plants in Mayi Village, Daibu Town, Qujing City, Yunnan Province, and characterized through morphological observation, physiological and biochemical profiling, and molecular identification. Colony morphology was examined on Nutrient Agar (NA), and cellular features were assessed via Gram staining and spore staining under a light microscope. Physiological profiling was performed using the Biolog GEN III system. For molecular identification, a single colony was inoculated into Nutrient Broth (NB) and cultured for 48 hours. Genomic DNA was extracted and used as a template for PCR amplification of the 16S rRNA and gyrA genes. The amplicons were sequenced by Beijing Tsingke Biotech Co., Ltd. Kunming Branch. (http://Tsingke.com.cn), and resulting sequences were aligned against the NCBI GenBank database using BLAST. Phylogenetic analysis was conducted with MEGA 11 (Tamura et al., 2021).

2.1.2 Compatibility assessment of strains

A compatibility assay was performed between P. polymyxa MY-J3 and three pre-selected antagonistic strains—Lysobacter antibioticus HY (isolated from konjac), Pantoea ananatis XP-1 (isolated from rice), and Pseudomonas mediterranea YX5-4 (isolated from strawberry)—all of which had demonstrated in vitro efficacy against X. fragariae YM2 and proven effective in controlling ALS under greenhouse conditions. The four strains were individually cultured in King’s B broth at 28 °C for 48 hours. Subsequently, 100 µL of each suspension was spread on a KB agar plate, while 200 µL aliquots from each of the other three strains were added into separate Oxford cups placed on the same plate. Following 48 hours of incubation at 28 °C, presence or absence of inhibition zones between strains was assessed.

2.2 Construction of synthetic consortia and evaluation of in vitro antagonistic activity

Strains without mutual inhibition were selected to construct synthetic consortia. Each strain was pre-cultured individually in KB broth to obtain seed cultures. The experiment comprised the following treatments:(1) Control: X. fragariae-inoculated plate without bacterial treatment; (2) Fermentation broth of P. polymyxa MY-J3 (M); (3) Fermentation broth of L. antibioticus HY (H); (4) Fermentation broth of P. ananatis XP-1 (X); (5) Fermentation broth of P. mediterranea YX5-4 (Y); (6) Co-culture: MY-J3 mixed equally with other seed cultures and inoculated (1% v/v) into fresh KB medium for 48 h; (7) Post-fermentation blend: individually fermented strains mixed equally after separate 48 h culture. Antagonistic activity was evaluated by spreading X. fragariae on WBN agar. Oxford cups were placed 2 cm from the bacterial lawn, and filled with 200 µL of each treatment broth. After 72 hours at 28 °C, inhibition zone diameters were measured. All treatments were performed in triplicate.

2.3 Validation of biocontrol efficacy under greenhouse and field conditions

2.3.1 Greenhouse experiment and control efficacy evaluation

The susceptible strawberry cultivar ‘Monterey’ was used in greenhouse trials. The pot experiment was conducted in a controlled greenhouse environment with regulated temperature and irrigation. Ambient conditions were maintained at 80–90% relative humidity and 20–30 °C. X. fragariae was inoculated into WBN medium and cultured at 28 °C with shaking at 160 rpm for 48 hours to obtain the bacterial suspension. The suspension concentration was adjusted to an OD₆₀₀ value of 0.5, followed by spray inoculation onto the abaxial surface of strawberry leaves until runoff occurred. Nine treatments were applied in triplicate, including a conventional chemical control (bromothalonil · bromonitrol, 1000× dilution). Each biocontrol bacterium was cultured separately in shake flasks at 28 °C and 160 rpm for 3 days to obtain fermentation broth. The concentration of all fermentation broths was uniformly adjusted to an OD₆₀₀ value of 2.0 prior to dilution. For single-strain treatments, the fermentation broth was diluted 20-fold. For synthetic consortium treatments, strains were first mixed at a 1:1 (v/v) ratio and then diluted 20-fold. Treatment details are provided in Table 1. All treatments were applied by spraying on the second day after pathogen inoculation, followed by subsequent applications at two-day intervals for a total of three sprays. The control group was sprayed with an equivalent amount of sterile water. ALS incidence was assessed 15 days after the final pathogen inoculation. Disease severity was graded according to Supplementary Table 1. The disease index (DI) and relative control effect (RCE) were calculated as follows:Disease Index (DI)= 100 × ∑ (Number of diseased leaves per grade × Grade value)/(Total leaves surveyed × Maximum grade value); Relative Control Effect (RCE, %) = (DI – control – DI – treatment)/DI – control × 100.

Table 1

Table 1. Greenhouse trial design for controlling strawberry angular leaf spot using three single-strain agents and four composite microbial agents.

2.3.2 Field validation of synthetic consortia efficacy

Field trials were conducted at Lujiajing, Daibu Town, Huize County, Qujing City, Yunnan Province, China (geographical coordinates: 26.223°N, 103.436°E, WGS84 datum). The trials were carried out in a greenhouse cultivation area under drip irrigation during the summer of 2024 (June to August). The experimental period was warm, humid, and rainy, with average temperatures ranging from 17 °C to 20 °C and relative humidity between 75% and 85%. The field trial was conducted using the strawberry cultivar ‘Monterey’ in plots with uniformly distributed incidence of ALS. Five treatments were evaluated, each with three replicates, totaling 15 plots. Each raised bed served as a replicate, containing 4 rows of strawberry plants, with each row comprising no fewer than 200 plants. Constructed bacterial consortia were applied as the main treatments, with a conventional chemical control (bromothalonil · bromonitrol, 1000× dilution) included for comparison (Table 2). The concentration of all fermentation broths was uniformly adjusted to an OD₆₀₀ value of 2.0 prior to dilution (28 °C, 160 rpm, 3 days). The compound microbial consortia were mixed in a 1:1 (v/v) ratio and diluted 50-fold. All solutions were sprayed onto the abaxial surfaces of strawberry leaves at three-day intervals for a total of three applications, with an equivalent amount of water sprayed as the control. Disease incidence and control efficacy were assessed according to the methods described in Section 2.3.1.

Table 2

Table 2. Field trial design for controlling strawberry angular leaf spot using three composite microbial agents in Daibu Town, Qujing City, Yunnan Province.

2.4 Quantification of pathogen colonization in planta using a tailored qPCR assay

2.4.1 Standard curve establishment

A standard curve for qPCR was constructed using ten-fold serial dilutions of genomic DNA from X fragariae YM2. The logarithm of DNA concentration (log₁₀) was plotted against Ct values to generate a regression equation for pathogen quantification. Method validity was evaluated based on the correlation coefficient (R²), slope, and amplification efficiency (E), calculated as: E = 10 ^(−1/slope) − 1.

2.4.2 Quantification of X. fragariae in greenhouse samples via qPCR

Leaf samples were collected 10 days post-treatment in the greenhouse. Total DNA was extracted using the SteadyPure Plant Genomic DNA Extraction Kit. Pathogen-specific primers XopAF-F/R were used for qPCR analysis, and bacterial abundance in leaf tissues was quantified by interpolating Ct values into the standard curve established in Section 2.4.1.

2.5 Gene expression profiling of host defense and bacterial virulence responses

2.5.1 Synthetic consortia effects on strawberry defense gene expression

Total RNA was extracted from liquid liquid nitrogen-snap-frozen leaves collected 10 days after consortia treatment and reverse-transcribed into cDNA. Using FaGAPDH2 as a reference, expression of defense-related genes (FaSnRK2, AOS, FaBG2-3, FaPR1, FaPAL, FaChi3, FaJAR1, FaEDS1) was quantified via qPCR (Xu, 2019). Primer sequences are listed in Supplementary Table 2. Reactions were performed on a Roche LightCycler^® 96 using a two-step protocol, with relative expression calculated by the 2^(–ΔΔCt) method.

2.5.2 Synthetic consortia effect on virulence gene expression in X. fragariae

Total RNA was extracted from liquid liquid nitrogen-snap-frozen leaves collected 10 days after consortia treatment and reverse-transcribed into cDNA. Expression of virulence genes (xopR, Hpa2, flgG, fliA, rpfE, gumG, rtxD, rtxE) was analyzed using the X. fragariae housekeeping gene gyrB as an internal control. Primer sequences are provided in Supplementary Table 3. Methods followed those described in Section 2.5.1.

2.6 Functional characterization of synthetic consortia

2.6.1 Biofilm formation assay

Biofilm formation was assessed using the crystal violet staining method (Jiu et al., 2020). Bacterial strains were cultured individually in KB medium for 48 h, then mixed in equal proportions according to the consortium treatment groups and adjusted to OD₆₀₀ ≈ 0.5. A 100 μL aliquot of each bacterial suspension was added to 96-well plates containing 100 μL of fresh KB medium per well. Uninoculated KB medium served as the blank control. All treatments were performed in triplicate and incubated at 28 °C for 48 hours.

2.6.2 Siderophore production assay

Siderophore production was evaluated with the chrome azurol S (CAS) assay (Sun et al., 2011). A 1% (v/v) inoculum of each bacterial consortium was cultured in MKB liquid medium for 72 hours. The supernatant was collected by centrifugation at 8000 ×g for 10 min and mixed with an equal volume of CAS detection solution. After 1 hours of reaction in the dark, the absorbance at 630 nm (A_s) was measured. The reference (A_r) was prepared using uninoculated MKB medium treated identically. Siderophore activity units (SU) were calculated as:SU = (A_r − A_s)/A_r × 100%.

2.6.3 Cellulose degradation assay

Cellulose decomposition ability was qualitatively determined using Congo red staining (Liao et al., 2014). After centrifugation and concentration, the bacterial consortium was inoculated onto carboxymethyl cellulose sodium (CMC-Na) plates and incubated at 28 °C for 48 hours. Plates were stained with 1 mg/mL Congo red for 10–15 min, then destained with 1 mol/L NaCl. The formation of a hydrolysis zone indicated cellulose degradation. The hydrolysis capacity (H) was evaluated as the ratio of the hydrolysis zone diameter (D) to the colony diameter (d): H = D/d.

2.7 Antibacterial and anti-virulence activity of M+H consortium crude metabolites

2.7.1 Preparation of crude metabolites

P. polymyxa MY-J3 (M) and L. antibioticus HY (H) were inoculated into their respective optimal media (KB medium for MY-J3 and YH medium for HY) and cultured under shaking conditions for 3 days. Using an equal volume of uninoculated medium as control, the cultures were mixed at a 1:1 volume ratio and extracted three times with an equal volume of ethyl acetate by vigorous shaking. The upper ethyl acetate phase was collected via a separatory funnel and concentrated using a rotary evaporator, yielding a brown oily crude extract. After weighing, the extract was dissolved in a defined volume of dimethyl sulfoxide (DMSO), filtered through a 0.22 μm membrane, and stored in amber vials for subsequent use. Since no extract was obtained from the uninoculated medium control, an equal volume of DMSO was used as the solvent control in subsequent bioassays to exclude any effects of the extraction solvent.

2.7.2 Effect of crude metabolites on X. fragariae growth

The crude metabolites (M+H) were added to 50 mL suspensions of X. fragariae YM2 to achieve final concentrations of 5, 10, 20, 40, 80, 120, and 160 μg/mL. A control was prepared using an equal volume of 1% DMSO. Bacterial growth was monitored by measuring OD₆₀₀ at 0, 12, 24, 36, 48, 60, and 72 h. The minimum inhibitory concentration (MIC) was determined as the lowest concentration showing no visible growth. All treatments were performed in triplicate.

2.7.3 Effect of crude metabolites on pathogenicity of X. fragariae

Pathogenicity was assessed by calculating the disease index following treatment with the crude metabolites. X. fragariae YM2 was cultured in WBN medium for 24 hours, then treated with crude metabolites at 2×MIC, MIC, and 1/2MIC concentrations. A control received 1% DMSO. After incubation (28 °C, 160 rpm, 24 h), cells were harvested, washed with PBS, and resuspended to OD₆₀₀ ≈ 0.5. Strawberry leaves were sprayed with the suspensions, and the disease index was recorded 10 days post-inoculation. Each treatment included three replicates with 10 leaves per replicate.

2.7.4 Effect of crude metabolites on biofilm formation and cell surface hydrophobicity

Biofilm biomass was quantified using crystal violet staining (Wang et al., 2021). X. fragariae YM2 suspensions were treated with crude metabolites at concentrations of 5–160 μg/mL (with 1% DMSO control) in 96-well plates and incubated at 28 °C for 48 hours. Cell surface hydrophobicity (CSH) was determined using the microbial adhesion to hydrocarbons (MATH) method (Danchik and Casadevall, 2020). Bacteria were treated with crude metabolites at 1/2MIC, MIC, and 2×MIC, incubated for 12–48 h, washed, and resuspended in PBS. A 5 mL aliquot was mixed with n-hexane, vortexed, and allowed to separate. The aqueous phase absorbance (OD₆₀₀) was measured. CSH was calculated as:CSH (%) = [1 − (OD₆₀₀ − treatment/OD₆₀₀ − control)] × 100.

2.7.5 Effect of crude metabolites on extracellular polysaccharide production

EPS production was measured using ethanol precipitation (Zhang, 2011). X. fragariae YM2 was treated with crude extract at 1/2MIC, MIC, and 2×MIC (1% DMSO control) and cultured for 5 days (28 °C, 160 rpm). A 10 mL sample was mixed with 20 mL absolute ethanol, stored at −20 °C overnight, and centrifuged at 4,000 ×g. The precipitate was dried and weighed to determine crude EPS yield.

2.8 High-throughput sequencing analysis of the strawberry phyllosphere bacterial microbiome

Strawberry leaves were sampled 21 days after the application of the bacterial consortium, immediately placed on dry ice and submitted to Guangzhou Gideo Biotechnology Co., Ltd. for analysis. Genomic DNA was extracted from strawberry leaves using the SteadyPure Plant Genomic DNA Extraction Kit (Precision Biotechnology (Hunan) Co., Ltd., China). DNA quality was assessed through PCR amplification and verified using a Nanodrop spectrophotometer to determine integrity, concentration, and purity.High-throughput sequencing of the bacterial 16S rRNA gene V3–V4 region was performed on the Illumina MiSeq platform using the primer pair 341F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Luo et al., 2023). Paired-end sequencing was conducted on PCR-amplified products. Raw sequencing data underwent quality filtering to generate high-quality Effective Reads for downstream bioinformatic analysis. Data processing and interpretation were performed following the methodology described by Wang et al (Wang et al., 2019).

2.9 Data statistics and analysis

Statistical analysis was performed using IBM SPSS Statistics. One-way analysis of variance (ANOVA) was employed to assess mean differences among treatments, and graphs were generated using GraphPad Prism software. For disease index and yield data from field trials, the measurements from each treatment plot (containing approximately 800 plants) were pooled as a single independent observation, resulting in a sample size of n=3 per treatment. Differences among treatments were compared using one-way analysis of variance (ANOVA).

3 Results and analysis

3.1 The M+H consortium enhanced biocontrol of ALS

Following the isolation and identification of the core antagonistic strain MY-J3, we subsequently evaluated its compatibility with other candidate strains and successfully constructed synthetic consortia. Among these, the combination of P. polymyxa MY-J3 and L. antibioticus HY (designated M+H) demonstrated superior synergistic antagonism in vitro.

3.1.1 The antagonistic strain MY-J3 as a core component for a synthetic consortium

Through systematic isolation and screening, a potent antagonistic strain MY-J3 was obtained from healthy strawberry tissues, demonstrating remarkable inhibitory activity against ALS pathogen X. fragariae YM2. The inhibition zone diameter produced by MY-J3 reached 2.70 cm, significantly surpassing other isolates. The strain was identified as a Gram-positive, spore-forming rod-shaped bacterium (Figures 1A–C). Based on 16S rRNA and gyrA gene sequence analysis, it was classified as P. polymyxa with a bootstrap value of 99% in the phylogenetic tree (Figures 1D, E). Physiological profiling using the Biolog GEN III system showed that MY-J3 could utilize 64 carbon sources (Supplementary Table 4) and exhibited strong metabolic reducing capacity. The 16S rRNA and gyrA gene sequences of strain MY-J3 have been submitted to GenBank with accession numbers PP732390.1 and PX676550, respectively.

Figure 1

Figure 1. Bacterial identification of Paenibacillus polymyxa MY-J3. (A) Colony morphology of the strain, (B) Gram staining, (C) Spore morphological characteristics, (D, E) Phylogenetic tree based on 16S rRNA gene and gyrA gene.

3.1.2 Bacterial consortia: In vitro synergy and strain compatibility

To ensure consortium stability, we systematically evaluated the compatibility between MY-J3 and three preserved antagonistic strains (L. antibioticus HY (H), P. mediterranea YX5-4 (Y), P. ananatis XP-1 (X)). Dual verification through cross-streaking and oxford cup assays demonstrated complete absence of inhibition zones in all strain combinations, with no inhibitory phenomena observed at colony junctions (Supplementary Figure 1), indicating excellent ecological niche compatibility. Comparison of two consortium construction strategies revealed that the “mix-then-ferment” approach (Figure 2A) consistently outperformed the “ferment-then-mix” method (Figure 2B). The M+H consortium (MY-J3 + HY) constructed using the “mix-then-ferment” strategy produced an inhibition zone of 3.53 cm, significantly larger than those of individual strains MY-J3 (2.47 cm) and HY (2.53 cm), demonstrating clear synergistic effects. Among other combinations, M+Y (5.20 cm) and M+H+Y+X (5.30 cm) showed prominent antibacterial activity, while the M+X combination exhibited relatively weaker effects. Although the M+Y and M+H+Y+X consortia produced larger inhibition zones in vitro, the M+H consortium was selected for subsequent mechanistic studies because it showed superior and more consistent disease control efficacy in field trials, coupled with the lowest pathogen load in planta as quantified by qPCR. These systematic in vitro evaluations provide experimental evidence for selecting the M+H consortium for further mechanistic investigation.

Figure 2

Figure 2. Two methods were used to construct composite microbial agents by combining P. polymyxa MY-J3 with Lysobacter antibioticus HY, Pseudomonas mediterranea YX5-4, and Pantoea ananatis XP-1, followed by evaluating their inhibitory activity against Xanthomonas fragariae YM2. (A) Mixing first and then fermentation, (B) Fermentation before mixing. M: Paenibacillus polymyxa MY-J3, H: Lysobacter antibioticus HY, Y: Pseudomonas mediterranean YX5-4, X: Pantoea ananatis XP-1. Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

3.2 The M+H consortium suppressed ALS and reduced pathogen load in greenhouse and field

Following the confirmation of its synergistic antagonism in vitro, we further evaluated the disease control potential of the M+H consortium under both greenhouse and field environments, and employed quantitative PCR to assess its capacity to reduce pathogen colonization in planta.

3.2.1 Effective disease control under greenhouse and field conditions

Following confirmation of in vitro synergy, we evaluated the practical efficacy of the M+H consortium against ALS under both greenhouse and field environments. Comparison of nine treatment schemes revealed that all treatments significantly reduced disease incidence (Supplementary Figure 2). Among them, the M+H consortium exhibited the optimal control efficacy in greenhouse trials, achieving a relative control effect of 76.15%, significantly higher than individual strain treatments M (58.33%) and H (60.00%) (Table 3) (P<0.05). The four-strain combination M+H+Y+X provided 77.69% control efficacy, showing no significant difference from M+H. Notably, the efficacy of M+H treatment significantly surpassed that of conventional chemical agent bromonitrol (60.66%). In field validation trials, the M+H treatment similarly exhibited excellent disease control performance, with a relative control efficacy of 74.26%. Under consistent conditions with biocontrol application as the only variable, the M+H consortium significantly outperformed the chemical agent bromonitrol NY, showing a higher yield increase (78.40% vs. 50.94%) and control efficacy (P < 0.05) (Table 4). It is therefore hypothesized that the yield improvement stems from the plant growth-promoting properties of the biocontrol agents, and that disease control further enhances strawberry quality. These results fully demonstrate that the M+H consortium possesses stable and reliable disease control capacity under practical application conditions.

Table 3

Table 3. Efficacy evaluation of three single-strain agents and four composite microbial formulations against strawberry angular leaf spot in the greenhouse.

Table 4

Table 4. Field efficacy evaluation of three composite microbial agents against strawberry angular leaf spot in Daibu Town, Qujing City, Yunnan Province.

3.2.2 Significant reduction of pathogen colonization in plant tissues

To elucidate the physiological basis of disease control, we quantified pathogen load in plant tissues using a specific qPCR assay. A standard curve was established with the regression equation Ct = -3.2193 log_[DNA] + 35.349 (R² = 0.9993) and amplification efficiency of 104%, ensuring accurate quantification (Supplementary Figure 3). Results showed that 10 days post-inoculation, X. fragariae YM2 load in control plants reached 1.58×10⁵ fg/μL. In contrast, the M+H treatment group exhibited only 9.12×10¹ fg/μL, representing a 99.99% reduction compared to the control and demonstrating the most significant suppression (Figure 3) (P<0.05). This molecular evidence confirms that the M+H consortium effectively inhibits pathogen colonization and spread in strawberry plants. The significant positive correlation between pathogen load and disease severity further demonstrates that the consortium controls disease through direct suppression of pathogen growth.

Figure 3

Figure 3. Quantification of X. fragariae YM2 in strawberry leaves after 10-day greenhouse treatment was performed by qPCR using strain-specific primers XopAF-F/R. M: P. polymyxa MY-J3, H: L. antibioticus HY, Y: P. mediterranean YX5-4, X: P. ananatis XP-1, NY: Bromothalonil·bronopol, CK: Clear water. Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

3.3 The M+H consortium primed strawberry defense via upregulation of defense genes

Based on the confirmed efficacy of the M+H consortium in suppressing pathogen spread, we further investigated its potential to enhance host immunity through activating plant systemic resistance. Systematic qPCR analysis of key defense pathway components and disease resistance-related genes in strawberry leaves revealed the molecular mechanisms underlying M+H-induced plant immunity.

3.3.1 Significant induction of pathogenesis-related gene expression

qPCR results demonstrated that compared to the control and individual strain treatments, the M+H treatment significantly upregulated the transcriptional levels of a series of core defense-related genes (Figure 4) (P<0.05). Specifically, the expression of FaBG2-3, a β-1,3-glucanase gene associated with cell wall reinforcement, was upregulated by 2.40-fold, the pathogenesis-related protein gene FaPR1 was upregulated by 2.32-fold, phenylalanine ammonia-lyase gene FaPAL by 2.31-fold, and chitinase gene FaChi by 3.63-fold, all significantly exceeding the levels observed in individual strain treatments. Notably, in the four-strain combination M+H+Y+X treatment, the upregulation of FaPR1, FaPAL, and FaChi3 expression reached maximum levels of 13.27-fold, 29.07-fold, and 10.41-fold compared to the control group, respectively, indicating that the multi-strain combination has a synergistic enhancing effect on inducing plant defense gene expression. The coordinated upregulation of these PR proteins demonstrates that the M+H consortium successfully triggers the plant’s basal immune response, establishing a systemic disease-resistant state.

Figure 4

Figure 4. The effect of composite microbial agents on the expression of strawberry resistance-related genes was analyzed by qPCR using specific primers after a 10-day greenhouse treatment. M: P. polymyxa MY-J3, H: L. antibioticus HY, Y: P. mediterranean YX5-4, X: P. ananatis XP-1, NY: bromothalonil·bromonitrol. The value > 1 indicates that the gene is up-regulated, and < 1 indicates that the gene is down-regulated. Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

3.3.2 Differential regulation of plant defense signaling pathways

To understand the initiation mechanisms of immune signaling, we systematically analyzed the expression patterns of marker genes from different hormone pathways (Figure 4) (P<0.05). The study found that AOS, a key gene in the jasmonic acid pathway, was significantly induced in all consortium treatments, with the M+H treatment group showing the induction amplitude of 5.08-fold compared to the control. Particularly noteworthy, in the M+H treatment, the key ABA signaling receptor gene FaSnRK2 showed specific upregulation, with expression levels 1.42-fold higher than the control, while in other treatment groups, the expression of this gene was lower than the control. In contrast, the expression pattern of FaEDS1, an important component of the salicylic acid pathway, varied among treatments: upregulated by 1.38-fold in the M treatment, 1.64-fold in the M+Y treatment, but downregulated in the M+H treatments. Another key jasmonic acid pathway gene, FaJAR1, showed significant down-regulation in the M+H treatment. Our results suggest that the M+H consortium fine-tunes the JA pathway, as indicated by the concurrent strong upregulation of the biosynthetic gene AOS and downregulation of the signaling gene FaJAR1.

3.4 The M+H consortium and its metabolites directly impair key virulence traits of X. fragariae

Beyond inducing plant resistance, we further investigated the direct inhibitory effects of the M+H consortium and its metabolites on the pathogen itself. This study provides multi-layered evidence from three aspects: consortium functional traits, metabolite activity, and virulence gene expression.

3.4.1 The M+H consortium enhanced colonization and competition potential

We first evaluated key functional traits of the consortium itself. The M + H consortium demonstrated the strongest biofilm production, with an OD₅₉₀ value of 0.7091, representing 2.18-fold and 1.41-fold increases over the H and M monocultures, respectively (Figure 5A) (P<0.05), indicating a significantly enhanced ability to colonize plant surfaces and form stable microenvironments. Meanwhile, Siderophore content in the fermentation broth was quantified using the CAS assay. The M+H consortium yielded significantly higher siderophore levels than the H or M monocultures, exceeding them by 3.66-fold and 1.47-fold, respectively (Figures 5B, D) (P<0.05), revealing a substantially improved capacity to compete with the pathogen for iron. Furthermore, assessment of cellulose degradation ability using Congo red staining revealed that the M+H consortium formed a hydrolysis zone-to-colony diameter ratio (H/d) of 3.29 on CMC-Na plates, significantly higher than those of the individual strains M (2.81) and H (2.77) (Figures 5C, E) (P<0.05), demonstrating enhanced substrate degradation capability and environmental adaptability. This cellulolytic activity was only detectable under in vitro conditions with highly concentrated cells. Crucially, no leaf damage was observed on strawberry plants treated with the 20-fold diluted fermentation broth, indicating minimal risk under practical application. These enhanced physiological traits collectively improve the consortium’s competitive survival on plant surfaces. This may directly or indirectly inhibit pathogen growth and colonization, and could also contribute to the activation of the plant’s immune system.

Figure 5

Figure 5. Functional determination of composite microbial agents. (A) Determination of membrane generating capacity; (B, D) Determination of siderophile production capacity, (C, E) Determination of cellulose decomposition ability. M: P. polymyxa MY-J3, H: L. antibioticus HY, Y: P. mediterranean YX5-4, X: P. ananatis XP-1, CK: Clear water. Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

3.4.2 Crude metabolites inhibited pathogen growth and reduced pathogenicity

We further extracted secondary metabolites from the M+H fermentation broth. Growth curve assays demonstrated that the crude metabolites exhibited significant concentration-dependent inhibition against X. fragariae, with complete suppression of pathogen growth at 80 μg/mL, determined as the minimum inhibitory concentration (MIC) (Figure 6A). At concentrations of 120 μg/mL and 160 μg/mL, the pathogen failed to grow throughout the 72-hour incubation period. More importantly, in vivo pathogenicity assays revealed that when the pathogen was treated with MIC (80 μg/mL), and 2×MIC (160 μg/mL) concentrations of the crude metabolites, the resulting d X. fragariae isease indices after inoculation onto strawberry plants significantly decreased to 16.67 and 9.45, respectively (control: 43.89) (Figures 6B, C) (P<0.05), demonstrating that the crude metabolites effectively impair the pathogen’s pathogenic capability with a clear dose-response relationship.

Figure 6

Figure 6. Effects of Crude Extract from the Composite Microbial Agent on Antibacterial Activity, Disease index, Biofilm Formation, Hydrophobicity, and Extracellular Polysaccharide Production in X. fragariae YM2. (A) Effect of M + H metabolites on the growth of X. fragariae, (B, C) Effect of metabolites on the pathogenicity, (D) Effects of metabolites on biofilm, (E) Effects of metabolites on hydrophobicity; (F) Effect of metabolites on exopolysaccharides. M: P. polymyxa MY-J3, H: L. antibioticus HY, CK: DMSO. Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

3.4.3 Crude metabolites disrupted pathogen invasion by interfering with key virulence phenotypes

The crude metabolites markedly inhibited biofilm formation and altered cell surface hydrophobicity (CSH) of X. fragariae. Biofilm biomass decreased with increasing metabolite concentration, with an OD₅₉₀ of 0.70 at MIC compared to 1.89 in the control (Figure 6D) (P<0.05). CSH values were 50.0%, 16.7%, and 14.3% after treatment with 1/2MIC, MIC, and 2×MIC, respectively (Figure 6E) (P<0.05), indicating reduced surface hydrophobicity and potential impairment of host colonization. Furthermore, treatment with the crude metabolites significantly reduced the production of extracellular polysaccharides (EPS), EPS yields decreased to 75% and 68% of the control level at 1/2MIC and MIC, respectively. The 2×MIC treatment resulted in the most substantial reduction, confirming the dose-dependent inhibitory effect on EPS synthesis (Figure 6F) (P<0.05).

3.4.4 The M+H consortium attenuated pathogen virulence via downregulation of virulence genes

After 10 days of treatment, the expression of virulence-related genes in X. fragariae was analyzed. The results demonstrated that most consortia treatments downregulated virulence gene expression.In single-strain treatments, upregulation was observed only in fliA and flgG (flagellar motility genes) after YX5–4 treatment, and in rpfE (quorum sensing), gumG (exopolysaccharide synthesis), and Hpa2 (T3SS) after HY treatment, compared to the CK group. All other treatments, particularly the synthetic consortia, resulted in downregulation of these genes (Figure 7) (P<0.05).These findings suggest that biocontrol agents can impair motility, expansibility, and pathogenicity of X. fragariae in plant tissues. Overall, consortia treatments consistently suppressed the expression of pathogenicity-related genes, with single-agent treatments of XP-1 and MY-J3 also showing significant downregulatory effects.

Figure 7

Figure 7. The effect of composite microbial agents on the expression of X. fragariae virulence-related genes was analyzed by qPCR using specific primers after a 10-day greenhouse treatment. M: P. polymyxa MY-J3, H: L. antibioticus HY, Y: P. mediterranean YX5-4, X: P. ananatis XP-1, NY: bromothalonil·bromonitrol. The value > 1 indicates that the gene is up-regulated, and < 1 indicates that the gene is down-regulated. Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

It is noteworthy that these molecular-level changes are highly consistent with the phenotypic results we observed—the inhibition of virulence gene expression directly explains the reduced biofilm formation, decreased cell surface hydrophobicity, and diminished EPS production. This coordinated regulation of phenotypes and gene expression indicates that the M+H consortium comprehensively impairs the virulence of X. fragariae by simultaneously interfering with multiple pathways, including attachment capacity, motility, production of pathogenicity factors, and cell-cell communication. This multi-target mechanism of action may represent an important molecular basis for the exceptional disease control efficacy demonstrated by the M+H consortium in both greenhouse and field conditions.

3.5 The M+H consortium reshaped the phyllosphere bacterial community

Beyond direct antagonism and induced resistance, we investigated the ecological impact of the M+H consortium on the native strawberry phyllosphere bacteria at 21 days post-treatment, which is critical for assessing its role in achieving stable and sustainable biocontrol.

3.5.1 Quality assessment of high-throughput sequencing data

The 16S rRNA gene amplicon sequencing of nine samples from three treatment groups was performed on the Illumina MiSeq platform. After filtering raw reads and removing chimeras, the average proportion of high-quality Tags reached 92.24%, with low variability among triplicate samples per group, indicating that the sequencing data were suitable for downstream analysis (Table 5). Rarefaction curves were used to assess the adequacy of sequencing depth and indirectly reflect species richness across samples. The curves for all three treatment groups rose steeply initially and then plateaued, demonstrating sufficient sequencing coverage and confirming that the data met analytical requirements (Figure 8).

Table 5

Table 5. Statistical overview and quality control of preprocessing for Strawberry phyllosphere bacterial data from high−throughput Sequencing was performed on field−collected strawberry leaves sampled 21 days after treatment with two composite microbial agents.

Figure 8

Figure 8. Shannon diversity index rarefaction curve for high−throughput sequencing was performed on field−collected strawberry leaves sampled 21 days after treatment with two composite microbial agents. T1: P. polymyxa MY-J3 + L. antibioticus HY), T2: P. polymyxa MY-J3 + L. antibioticus HY + M. Pseudomonas YX5-4 + P. pineapple XP-1, CK: water control.

3.5.2 Significant enhancement of phyllosphere bacterial diversity and richness

High-throughput sequencing of the 16S rRNA gene V3–V4 region from field-grown strawberry leaves demonstrated that the M+H treatment significantly altered the structure of the phyllosphere bacterial community. Principal coordinates analysis (PCoA) revealed clear separation between the treatment and control groups (Figure 9A), indicating substantial structural shifts in community composition. Alpha diversity analysis further confirmed that the M+H-treated group exhibited significantly higher Chao1 (1250.43), Sobs (1146.00), and Shannon (1.82) indices, along with a significantly lower Simpson index, compared to the control (Figure 10) (P<0.05). These results demonstrate that the application of M+H significantly increased both species richness and evenness of the phyllosphere bacterial community. Venn diagram analysis showed that the M+H treatment group contained 1224 operational taxonomic units (OTUs), including 736 unique OTUs, while the control group had only 599 OTUs with 318 unique OTUs (Figure 9B), further supporting that the introduced consortium substantially enriched the indigenous bacteria microbial resources.

Figure 9

Figure 9. PcoA clustering of species community composition and Venn diagram of common/endemic species abundance based on high−throughput sequencing was performed on field−collected strawberry leaves sampled 21 days after treatment with two composite microbial agents. (A) PcoA cluster diagram, (B) Venn diagram, T1: P. polymyxa MY-J3 + L. antibioticus HY, T2: P. polymyxa MY-J3 + L. antibioticus HY + M. Pseudomonas YX5-4 + P. pineapple XP-1, CK: water control.

Figure 10

Figure 10. Analysis of Alpha diversity indices from high−throughput sequencing was performed on field−collected strawberry leaves sampled 21 days after treatment with two composite microbial agents. T1: P. polymyxa MY-J3 + L. antibioticus HY, T2: P. polymyxa MY-J3 + L. antibioticus HY + M. Pseudomonas YX5-4 + P. pineapple XP-1, CK: water control). Different letters on the bars indicate significant differences according to Tukey’s HSD at P < 0.05.

3.5.3 Optimized community structure and specific pathogen suppression

After quality control, the obtained sequences met the requirements for downstream analysis. Comparative analysis of the top 12 most abundant bacterial phyla across samples revealed similar taxonomic profiles among treatments, though with variations in relative abundance.In treatments T1(M+H) and T2 (M+H+Y+X), Cyanobacteria (which may include chloroplast sequences from the host plant) and Proteobacteria were the dominant phyla, with relative abundances of 70.62% and 27.20% in T1, and 64.57% and 30.86% in T2, respectively. Compared to the CK group, T1 and T2 showed a significant increase in Cyanobacteria and a decrease in Proteobacteria. Minor phyla (<1% abundance) were slightly enriched in both treatments relative to CK (Figure 11A). The increase in Cyanobacteria relative abundance in T1 and T2 suggests an enrichment of potentially beneficial taxa, potentially enhancing plant growth and disease resistance. At the genus level, Methylobacterium, Prevotella, Cossackia, and Halomonas showed increased abundance. The consortium treatments may have enriched genera such as Sphingomonas, Cossackia, and Halomonas, which are associated with toxin degradation, pathogen cell wall lysis, and nitrogen fixation, thereby improving plant stress resistance. Notably, the relative abundance of Xanthomonas was significantly reduced in T1 and T2, accounting for only 3.20% and 0.28% of that in the CK group, respectively (Figure 11B). These findings indicate that the M+H consortium did not disrupt the native ecology but instead steered the phyllosphere microbiome toward a healthier, more stable, and pathogen-suppressive state.

Figure 11

Figure 11. Species composition of the top 12 taxa at phylum and genus levels based on high−throughput sequencing was performed on field−collected strawberry leaves sampled 21 days after treatment with two composite microbial agents. (A) phylum level species composition, (B) genus-level species composition. T1: P. polymyxa MY-J3 + L. antibioticus HY, T2: P. polymyxa MY-J3 + L. antibioticus HY + M. Pseudomonas YX5-4 + P. pineapple XP-1, CK: water control.

Notably, compared to the control group (CK), the overall abundance of Proteobacteria in the treatment groups (T1, T2) showed a decreasing trend, and the exogenously applied Firmicutes biocontrol agent was also not significantly detected at the phylum level. This pattern reflects the distinct ecological dynamics at the 21-day sampling point. In the control, explosive growth of the pathogen X. fragariae (Proteobacteria) dominated the community, leading to high Proteobacteria abundance (Figure 11A). In the treatment groups, after achieving early pathogen suppression, the populations of the exogenous biocontrol agents (including MY-J3 from Firmicutes and HY from Proteobacteria) naturally declined to low levels due to ecological competition. Simultaneously, through ecological regulation, they reshaped a microbial network centered on indigenous beneficial microbiota. This network continuously suppressed pathogen resurgence, resulting in a reduction in pathogen biomass that far exceeded the transient colonization contribution of the exogenous agents. Consequently, this led to a net decrease in Proteobacteria abundance and an overall optimization of the community structure in the treatment groups. These findings reveal the ecological principle of biocontrol treatment achieving sustained disease suppression through a “priming-remodeling” mechanism.

4 Discussion

4.1 Construction and synergistic effects of synthetic consortia

Research on individual biocontrol agents in plant disease management has advanced significantly. Studies demonstrate that specific beneficial strains can effectively suppress pathogens by producing antimicrobial compounds, competing for resources, and inducing plant systemic resistance (Meng et al., 2024). The secondary metabolites of P. polymyxa MEZ6 suppress methicillin-resistant Staphylococcus aureus (MRSA) by inhibiting growth, preventing biofilm formation, downregulating virulence genes (e.g., agrA, spa), and disrupting membrane integrity, leading to ROS accumulation (Na et al., 2025). Similarly,Myxin from L. antibioticus targets X. fragariae by increasing membrane permeability, inducing ROS, inhibiting biofilm and EPS synthesis, and modulating gene expression, thereby achieving multi-target control of pathogen proliferation (Deng et al., 2025). Most commercial microbial agents rely on single exogenous strains, which often underperform under field conditions. In contrast, synthetic microbial consortia demonstrate enhanced practical value through greater functional diversity and ecological adaptability (Zhao et al., 2020). Based on strain compatibility and plate antagonism assays, multiple microbial consortia including M+H were successfully constructed using strains such as P. polymyxa MY-J3 (M) and L. antibioticus HY (H). This approach aligns with current trends in microbial community research, as exemplified by Hu et al.’s Pseudomonas consortium that improved colonization and reduced tomato bacterial wilt incidence (Hu et al., 2021), Tan et al.’s multi-strain combination that enhanced antibacterial efficacy against tomato wilt (Tan et al., 2025), and Zhang et al.’s B. subtilis consortium that suppressed cabbage clubroot by modifying soil acidity and rhizosphere microbiota (Zhang et al., 2022). The M+H consortium significantly outperformed individual strains in biofilm formation, siderophore production, and cellulose degradation. Biofilms provide a stable microenvironment (Jiu et al., 2020), while siderophores mediate iron competition with pathogens (Cao et al., 2011). These synergistic functional traits collectively enhance the ecological competitiveness of the consortium.

4.2 Plant immune activation and defense mechanisms

Microbial consortia can enhance plant health by inducing systemic resistance, often through the regulation of secondary metabolic pathways and the enhancement of defense-related gene expression and enzyme activity (Mei et al., 2019). Supporting this, Li et al. demonstrated that a synthetic community activated plant immunity and suppressed pathogen growth (Li et al., 2021). In line with these findings, our study revealed that the M+H consortium activated the strawberry immune system via multi-level, multi-pathway regulation. It significantly upregulated core defense-related genes (e.g., FaBG2-3, FaPR1, FaPAL, and FaChi3) and differentially modulated key hormone signaling pathways involving JA, ABA, and SA. The observed expression pattern—strong upregulation of the JA biosynthetic gene AOS concurrent with the downregulation of the JA signaling gene FaJAR1 and the SA pathway marker FaEDS1—leads us to propose a potential “rapid induction, timely attenuation” strategy. This mechanism would enable an effective initial defense response while potentially mitigating fitness costs by fine-tuning the JA pathway and strategically reallocating resources away from the SA pathway, thereby aligning with the plant’s growth-defense balance. This complex network regulation and cross-talk likely underpin the efficient induction of systemic resistance by the M+H consortium. Concurrently, our results demonstrate that the M+H consortium and its metabolites exerted multi-target inhibition against X. fragariae by disrupting biofilm formation, reducing cell surface hydrophobicity, and suppressing extracellular polysaccharide (EPS) production. These phenotypes are critically linked to pathogenicity, as EPS is a key virulence factor in Xanthomonas, facilitating attachment, colonization, and protection (Kumari et al., 2025), while cell surface hydrophobicity directly influences pathogen adhesion and aggregation on host surfaces (Wang et al., 2023). This study provides the first evidence that co-inoculation with B. thuringiensis CAPE95 and P. polymyxa CAPE238 functions as an effective biofertilizer for Tropaeolum majus, significantly enhancing plant growth. Genomic analysis identified key pathways involved in nutrient solubilization (nitrogen fixation, sulfur assimilation), phytohormone synthesis (IAA precursors), and antimicrobial production (bacilysin, paenibacillin) (Dal’Rio et al., 2024). This aligns with studies showing that microbial consortia can promote plant growth through mechanisms such as nutrient solubilization and phytohormone synthesis, which may partly explain the observed yield increase in our study.

4.3 Pathogen inhibition and microecological remodeling

At the molecular level, M+H treatment significantly downregulated the expression of key virulence genes in X. fragariae, including flagellar motility genes (fliA, flgG), EPS-related gumG, T3SS-related Hpa2, quorum sensing-related rpfE, effector protein gene XopR, and specific virulence factors rtxD and rtxE —all of which are part of major pathogenicity clusters (gum, hrp, rpf, xps) in the X. fragariae YM2 genome (Qiu et al., 2023). The consistency between gene suppression and phenotypic inhibition indicates that the consortium comprehensively suppresses the pathogen through transcriptional reprogramming, reducing the risk of resistance development. Real-time quantitative PCR analysis confirmed that the M+H consortium significantly reduced the density of X. fragariae in strawberry leaves, thereby effectively controlling pathogen spread.Recent studies highlight microbial community modulation as a promising disease control strategy. Phyllosphere and rhizosphere microbes influence plant health through direct and indirect mechanisms, including pathogen inhibition, mutualistic symbiosis, and regulation of plant metabolism and gene expression (Liu et al., 2020). The bacteria in the synthetic consortium not only enhance nutrient availability by secreting phytohormones and siderophores but also modulate the microbial community, leading to an increase in the abundance of beneficial genera such as Acinetobacter and Anaeromyxobacter (Sarver et al., 2025). For instance, Fan et al. reported that biocontrol agents increased bacterial abundance and reduced fungal load in the soybean rhizosphere, suppressing Fusarium and Rhizoctonia while promoting beneficial microbes such as rhizobia, Trichoderma, and Pseudomonas (Fan et al., 2012). Zhang et al. found that specific consortia enriched beneficial bacteria in maize rhizosphere soil, improved microbial composition, and upregulated growth-promoting functional genes (Zhang et al., 2024). This study revealed that the M+H consortium significantly enhanced phyllosphere microbial diversity and richness, enriching beneficial genera such as Sphingomonas and Methylobacterium. Strawberry plants naturally host diverse microbial communities (Olimi et al., 2022), and beneficial bacteria like Pseudomona and Sphingomonas not only directly inhibit pathogens but also promote plant growth and stress tolerance (Zhu et al., 2023). Yuan et al. demonstrated Pseudomonas reshaped the phyllosphere microbiome by both direct antagonism and indirect virulence disruption, thereby suppressing disease and increasing yield (Yuan et al., 2024). Notably, Cyanobacteria as plant growth-promoting agents (El-Bestawy et al., 2007; Ramakrishnan et al., 2023) and the Type VI Secretion System (T6SS) in microbial interactions (Lin et al., 2025) play important roles in microecological regulation and warrant further investigation.

4.4 Conclusion and future directions

In this study, a synthetic microbial consortium designated M+H, with P. polymyxa MY-J3 as the core strain, was successfully constructed. It significantly enhanced the control efficacy against ALS through multiple mechanisms, including synergistic biocontrol activities, activation of plant systemic resistance, suppression of pathogen virulence, antibacterial action of crude extracts, and remodeling of the phyllosphere microbiome. This consortium provides an integrated solution that overcomes the limitations of single-strain approaches and establishes a theoretical foundation for developing next-generation compound microbial pesticides. Future research should focus on (1) optimizing consortium ratios to elucidate inter-strain interactions; (2) characterizing metabolomic profiles to identify key active compounds; (3) evaluating the consortium’s stability and efficacy under diverse environmental conditions; (4) elucidating the mechanisms underlying the observed yield increase, specifically by quantifying plant growth promotion traits (e.g., phytohormone production, nutrient solubilization) of the M+H consortium and their relative contribution to yield under both diseased and non-diseased conditions; and (5) investigating the molecular mechanisms of plant-consortium interactions.

Data availability statement

The original datasets presented in this study are openly available in Mendeley Data at doi: 10.17632/fg5hxsj5kt.1.

Author contributions

MM: Data curation, Formal Analysis, Resources, Software, Supervision, Validation, Visualization, Writing – review & editing. HH: Writing – original draft. YL: Supervision, Validation, Writing – review & editing. CD: Supervision, Validation, Writing – review & editing. JZ: Writing – review & editing. RL: Writing – review & editing. HML: Writing – review & editing. HL: Writing – review & editing. KO: Writing – review & editing. GJ: Writing – review & editing, Supervision, Validation.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This research is supported by the fund project of Yunnan Provincial Department of Science and Technology Basic Research Special-Key Project(202201AS070313), China. This work was supported by a key project fund(202201AS070313).

Acknowledgments

This article draws upon data from the master’s thesis of WH (Class of 2025), which have undergone subsequent reorganization and synthesis. We gratefully acknowledge the experimental data provided by WH from her master’s thesis. We also extend our sincere thanks to GJ for his guidance on this article. All authors have read and approved the final version of this manuscript for submission.

Conflict of interest

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fagro.2025.1732161/full#supplementary-material

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