Antagonistic Mechanisms of Serratia plymuthica MM Against Phytophthora capsici and Its Growth-Promoting Traits

Abstract

Phytophthora blight, caused by Phytophthora capsici, an oomycete pathogen belonging to the phylum Oomycota, is a major soil-borne disease that limits the cultivation of pepper (Capsicum annuum). In this study, the bacterium Serratia plymuthica MM was evaluated for both its antagonistic ability and plant growth-promoting (PGP) potential. The sterile fermentation filtrate of S. plymuthica MM exhibited strong antifungal activity in vitro, inhibiting the mycelial growth of P. capsici by up to 88.32%. In pot experiments, Serratia plymuthica MM significantly reduced both disease incidence and disease severity of Phytophthora blight in pepper plants, achieving control efficacies of 88.33% (preventive application) and 55.56% (therapeutic application). Microscopic observations revealed severe hyphal abnormalities, including distortion, shrinkage, collapse, and fragmentation. Furthermore, propidium iodide (PI) and DAPI double staining provided cellular-level evidence of antifungal activity, demonstrating concentration-dependent disruption of membrane integrity and nuclear organization in P. capsici hyphae, which was supported by pronounced increases in ion leakage from pathogen cells. Further, S. plymuthica MM exhibited PGP traits, including nitrogen fixation, phosphate solubilization, siderophore production, and indole-3-acetic acid (IAA) synthesis. Pot experiments using the pepper cultivar ‘Longjiao’ (Capsicum annuum L. cv. Longjiao) confirmed significant growth promotion and enhanced activities of key defense-related enzymes (POD, PPO, PAL, and CAT). Stable colonization of pepper roots was verified by green fluorescent protein (GFP) labeling, demonstrating the strain’s persistence in the rhizosphere. Collectively, these results highlight the dual role of S. plymuthica MM in suppressing P. capsici and promoting pepper growth, supporting its potential as an eco-friendly biocontrol agent for sustainable pepper production.

1. Introduction

Pepper (genus Capsicum, family Solanaceae) is an economically important crop whose global cultivation area and yield have continually increased, with yearly production now exceeding 40 million metric tons annually from over 4 million hectares, led by China, Mexico, and Indonesia [1]. However, achieving high and stable yields of Capsicum species remains a pressing agricultural challenge because it is prone to phytopathogen invasions in cultivated areas. In particular, the Phytophthora blight caused by Phytophthora capsici is a major disease affecting many pepper crops [2]. In China, currently the world’s largest pepper producer, P. capsici poses a severe threat to production, especially in its warmer and wetter southern provinces, namely Yunnan, Fujian, Jiangxi, Zhejiang, and Guangdong [3].

Phytophthora capsici employs multiple infection strategies to enter its host tissues: either via the stomata or wound sites, or directly through the epidermis, leading to diffuse brown necrotic lesions forming on leaves. On the petioles and stems of infected hosts, the disease appears as dark brown lesions that girdle the stem, resulting in the wilting of upper plant parts, which translates into substantial yield reductions or even complete crop loss [4].

In addition to its aggressive infection behavior, P. capsici is well known for its ability to persist in soil for extended periods through the formation of long-lived survival structures, such as oospores. These propagules can remain viable in soil and plant debris for several years, serving as a persistent primary inoculum source and making eradication of the pathogen from infested fields extremely difficult. This long-term soil survival further limits the effectiveness of conventional chemical control strategies and underscores the need for sustainable soil-based disease management approaches [5].

Some research shows that certain fungicides, such as azoxystrobin and pyraclostrobin, can inhibit P. capsici. Yet Miao & Cai et al. [6] reported that, among 90 P. capsici isolates collected from southern China, a subgroup now exhibits resistance to azoxystrobin, accompanied by reduced sensitivity to pyraclostrobin. These observations indicate that P. capsici populations can rapidly adapt to repeated chemical selection pressure, resulting in reduced field efficacy and increased risk of cross-resistance. Such adaptive potential further challenges the long-term sustainability of fungicide-based control programs. Moreover, it turns out those isolates were resistant to famoxadone, which would suggest a stark decline in field efficacy [7]. As well, strong adaptability coupled with its dispersal capacity often renders conventional chemical control insufficient to effectively restrict the spread of P. capsici. Continued use of chemical pesticides also results in environmental pollution, pathogen resistance, and pesticide residues in agricultural products. Therefore, the development of sustainable, environmentally friendly, and effective biological control strategies has become a crucial focus in the integrated management of Phytophthora blight in cultivated pepper (Capsicum spp.).

Field studies have shown that either single or combined applications of antagonistic bacteria, particularly Pseudomonas fluorescens and Bacillus spp., are capable of reducing the P. capsici disease severity by 48–61% when applied as soil drenches (1 × 10⁹ CFU mL⁻¹) [8]. Supplementing these bacterial suspensions with olive-oil amendments raises their greenhouse efficacy to 57–81% [8]. Their further integration with low-dose chemical regimes (e.g., acibenzolar-S-methyl + mefenoxam) not only enhances disease suppression but also impedes the development of fungicide resistance in the phytopathogen [9]. Notably, endophytic Bacillus and Streptomyces isolates can inhibit both the spore germination and mycelial growth of P. capsici by producing lipopeptides and other bioactive metabolites [10,11]. Given its environmental compatibility, sustainability, and strong synergy with existing agronomic practices, biological control serves as an eco-friendly and essential strategy within integrated disease management for Phytophthora blight in Capsicum crops [12].

In a recent biocontrol study of Phytophthora blight in pepper, Syed-Ab-Rahman et al. reported that the dichloromethane crude extract of Bacillus velezensis UQ156 inhibited 61.8% of P. capsici’s zoospore growth on day 3 post-application. Meanwhile, gas chromatography–mass spectrometry (GC–MS) profiling revealed that this anti-oomycete activity is attributable to a suite of diketopiperazines (e.g., cyclo-Gly-Pro, cyclo-Ala-Val) and phenolic compounds rather than the canonical lipopeptide fengycin. Similarly, Bacillus velezensis UQ156 is able to suppress P. capsici by secreting these low-molecular-weight cyclic dipeptides, which likely interfere with pathogen membrane integrity and zoospore viability [13]. Yet these biocontrol strains face limitations in realistic field applications, largely in the form of their variable inhibitory efficacy and restricted environmental adaptability [14].

Although most biocontrol studies against P. capsici have focused on genera such as Bacillus and Pseudomonas, increasing evidence suggests that Serratia species also possess strong antagonistic potential against soil-borne pathogens. Several Serratia plymuthica strains have been reported to suppress fungal and oomycete diseases through the production of bioactive secondary metabolites, effective rhizosphere colonization, and plant growth–promoting traits. However, the biocontrol mechanisms of S. plymuthica against P. capsici, particularly in pepper systems, remain poorly understood, and strain-specific variation in efficacy and ecological adaptability is still largely unexplored.

Hardly any studies have addressed the biological control of Phytophthora blight in pepper crops grown in Gansu Province, China, where chemical fungicides remain the key method for disease management in the field. This overreliance on chemical control not only elicits concerns about evolving pesticide resistance and environmental safety, but it also restricts the sustainability of pepper production. So, screening for novel biocontrol bacteria well adapted to Gansu’s local agroecological conditions that possess antagonistic activity against P. capsici and plant growth–promoting traits, along with elucidating the synergistic mechanisms between lipopeptide biosynthesis and rhizosphere colonization, holds great theoretical and practical value for devising a stable, efficient, and eco-friendly management system against Phytophthora blight in pepper [15].

This study aimed to identify biocontrol bacteria capable of effectively managing Phytophthora blight in Capsicum and to investigate their antagonistic mechanisms, as well as their benefits to plant growth and resistance. Our specific objectives were: (1) to confirm the pathogenicity of the obtained P. capsici strain; (2) to screen biocontrol bacterial strains with significant inhibitory activity against P. capsici and evaluate their antagonistic properties; (3) to elucidate the inhibitory mechanisms of selected biocontrol strains on P. capsici, including their respective effects on mycelial growth, cell membrane permeability, and hyphal morphology; (4) to assess the plant growth-promoting (PGP) traits of biocontrol bacteria, namely their siderophore production, nitrogen fixation, phosphate and potassium solubilization, indole-3-acetic acid (IAA) synthesis, and rhizosphere colonization ability; and (5) to comprehensively evaluate the biocontrol efficacy and PGP effects of selected strains in pot experiments, including their influence on defense-related enzyme activities in Capsicum plants. Therefore, screening locally adapted biocontrol bacteria from agroecosystems in Gansu Province, China, and systematically evaluating their antagonistic activity against P. capsici, plant growth–promoting traits, and rhizosphere colonization capacity is of both theoretical significance and practical importance.

2. Materials and Methods

2.1. Bacterial Strain, Culture Medium, and Pot Experiments

A collection of biocontrol bacterial strains preserved in the laboratory was cultured in Luria–Bertani (LB) medium, at 30 °C for 20 h, prior to their use. These strains consisted of (1) Bacillus tequilensis SY89 (NCBI accession number: MZ413285.1), isolated from Angelica sinensis rhizosphere soil [16]; (2) B. mucilaginosus (provided by the Microbiology Laboratory of Lanzhou Jiaotong University); (3) Paenibacillus polymyxa YF (NCBI accession number: MW205750), isolated from sheep-manure compost [17]; (4) B. amyloliquefaciens HT (NCBI accession number: MW776428), isolated from healthy vegetable rhizosphere soil [18]; (5) Bacillus velezensis BN (NCBI accession number: OR995192.1), isolated from Lilium brownii bulbs in Lintao County, Gansu [19]; (6) Serratia plymuthica MM (NCBI accession number: KY964326), isolated from pasture grass rhizosphere soil [20]; (7) Phytophthora capsici was provided by the Gansu Academy of Agricultural Sciences, China.

The plasmid pGFP4412 utilized in this study came from Fenghui Biotech. The culturing media used and their composition are described in the Supplemental Materials section. All pot experiments were conducted in a greenhouse facility at Lanzhou Jiaotong University, from May to August 2023, using only the pepper plant ‘Longjiao’ (Capsicum annuum L. cv. Longjiao), a commercial variety widely cultivated in China’s Gansu Province.

2.2. Pathogenicity Assay and Biocontrol Bacteria Screening

2.2.1. Pathogenicity Assay

To verify the pathogenicity of P. capsici, mycelial plugs of this oomycete were inoculated onto clarified V8 juice agar (composition provided in Supplementary Table S1) and incubated at 25 °C for 7–8 days. Zoospore production was induced following an established protocol [21]. Briefly, 5 mm-diameter mycelial plugs were excised from the actively growing margins of colonies, and 7–8 plugs were placed in 15 mL of sterile water. The cultures were incubated under fluorescent light for 72 h to promote sporangium formation, followed by incubation at 4 °C for 60 min to stimulate zoospore release. The resulting zoospore suspension was filtered through sterile cheesecloth, and the concentration was determined using a hemocytometer (Shanghai Qiujing Biochemical Reagent Instrument Co., Shanghai, China) and adjusted to 5.0 × 10⁴ zoospores mL⁻¹ with sterile water [22].

Pathogenicity assays were conducted following a modified Koch’s postulates workflow as described by Bhunjun et al. (2021), using P. capsici (Oomycota) to inoculate pepper plants (Capsicum annuum cv. Longjiao) [11,23]. For pathogen inoculation, the zoospore suspension was applied by soil drenching at a rate of 30 mL per kg of sterilized potting substrate to the root zone of healthy plants. Control plants received an equal volume of sterile water. Each treatment consisted of five replicate plants, and the entire experiment was repeated three times. All plants were maintained in a greenhouse at 25 °C for 14–20 days. Once typical disease symptoms appeared, lesion tissues were collected and the pathogen was re-isolated and purified on Martin’s selective medium, the composition of which is provided in Supplementary Table S1. Martin’s selective medium, supplemented with Rose Bengal and streptomycin (Sigma-Aldrich, St. Louis, MO, USA) to suppress bacterial growth, was used for the re-isolation of P. capsici. Pathogenicity was confirmed by comparing the morphological characteristics of the re-isolated pathogen with those of the original inoculated strain [24]. To further confirm the identity of the re-isolated pathogen at the molecular level, a two-step nested PCR assay targeting the YPT1 gene was performed [25]. The nested PCR protocol was adopted from a previously reported molecular detection method specific for Phytophthora capsici based on the YPT1 gene. The first-round PCR was used to confirm the affiliation of the pathogen to the genus Phytophthora, while the second-round PCR employed P. capsici–specific primers to verify species identity. PCR products obtained from both the original isolate and the re-isolated pathogen were sequenced, and the resulting YPT1 sequences were compared with reference sequences in the NCBI database using BLASTn (NCBI BLAST+ version 2.15.0; National Center for Biotechnology Information, Bethesda, MD, USA). The sequences showed high identity to P. capsici YPT1 reference sequences and were identical between the original and re-isolated isolates, confirming that the pathogen recovered after inoculation was P. capsica [26].

2.2.2. Screening of Candidate Biocontrol Bacteria

An in vitro dual-culture bioassay was performed to evaluate the antagonistic activity of eight bacterial strains against P. capsici [27]. A mycelial plug of P. capsici (8 mm in diameter) was excised from a 4–6-day-old culture using a sterile cork borer and placed at the center of a PDA plate. Subsequently, 1 μL of each bacterial fermentation broth was spotted at three equidistant points (25 mm from the central pathogen plug) on the periphery of the plate. Control plates were prepared in the same manner, with sterile LB medium used instead of bacterial broth [28]. All plates were incubated at 25 °C for 5–7 days. until the mycelium in the control plates reached the edge of the Petri dish.

The antagonistic activity of each bacterial strain was evaluated based on the inhibition of radial growth of P. capsici. The radius of the pathogen colony in the treatment plates (Rt) was measured along the axis facing the bacterial inoculation point, defined as the distance from the center of the pathogen plug to the growth front of the colony on the side facing the bacterial strain. The radius of the pathogen colony in the control plates (Rc) was measured on the same day under identical conditions as the distance from the center of the plug to the colony margin [17].

The percentage inhibition of mycelial growth was calculated according to the following formula:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\mathrm{\%})={\displaystyle \frac{(\mathrm{R}\mathrm{c}-\mathrm{R}\mathrm{t})}{\mathrm{R}\mathrm{c}}}\times 100$$

The bacterial strain achieving the highest inhibition rate was selected for use in subsequent experiments.

2.3. Inhibitory Effect of Bacterial Fermentation Filtrate on the Mycelial Growth of P. capsici

Multiple bacterial isolates were obtained during the initial screening process, and the most promising strain was selected for further characterization. Based on their performance in the dual-culture bioassay, the most promising bacterial strain for biocontrol was chosen to evaluate the effect of its fermentation filtrate on the mycelial growth of Phytophthora capsici. An overnight culture was prepared by inoculating liquid LB medium with a single bacterial colony and incubating it at 30 °C, with shaking at 180 rpm, for 12 h. Cells were then harvested by centrifugation at 5000× g for 10 min. The ensuing supernatant was then aseptically filtered through a 0.22-μm membrane to obtain a cell-free, sterile filtrate for use in subsequent assays [29].

2.3.1. Effect of the Fermentation Filtrate of Serratia plymuthica on the Mycelial Growth of P. capsici

PDA plates containing a 1%, 10%, 20%, or 50% (v/v) concentration of the fermentation filtrate were prepared by mixing the sterile supernatant (V) with molten PDA medium (V_pad_a), in volume ratios of 1:99, 1:9, 2:8, or 5:5, respectively. Afterwards, a 5 mm-diameter mycelial plug of P. capsici was placed at the center of each prepared plate. Then all plates were incubated at 25 °C. The PDA plates without the fermentation filtrate served as the control [18]. When the mycelial growth in the control plates reached the plate margins, digital images were recorded. The colony radius of P. capsici was measured for both treatment (R_t) and control (R_c) plates, and the inhibition rate (%) of mycelial growth was calculated according to the formula described in Section 2.2.2.

2.3.2. Effect of the Bacterial Culture Supernatant on the Membrane Permeability of P. capsici

To assess the disruptive impact of the antagonistic bacterium on P. capsici cell membranes, changes in membrane permeability were evaluated by measuring extracellular conductivity. P. capsici mycelia were first cultured in PDB for 8–10 days, harvested by filtration through four layers of sterile gauze, and washed three times with 100 mM phosphate-buffered (PBS; Solarbio, Beijing, China) saline (PBS; pH 7.4). Approximately 0.5 g of the washed mycelia was then transferred into 50 mL of PBS supplemented with 10% (v/v) sterile bacterial culture filtrate (V_(filtrate):V_{(sterile water)} = 1:9) [30]. PBS without bacterial filtrate served as the control.

Extracellular conductivity of the same mycelial suspension was measured repeatedly at 0, 3, 6, 9, 12, 15, 18, 21, and 24 h using a conductivity meter (INESA DDS-307A, Shanghai, China). Relative membrane conductivity was calculated as described in Section 2.7.

2.3.3. Effect of the Bacterial Culture Supernatant on the Hyphal Morphology of P. capsici

Phytophthora capsici was cultured for 5–7 days and then inoculated on PDB medium supplemented with 10% sterile filtrate of the bacterial strain. The control group was cultured in standard PDB medium. All cultures were incubated at 18 °C, with shaking at 180 r/min, for 8–10 days. Mycelia were harvested by centrifugation (5000× g) for 10 min at 4 °C and washed thrice with 100 mM phosphate-buffered saline (PBS; pH 7.4) for their subsequent analysis [31,32].

For their use in scanning electron microscopy (SEM), samples were prepared using the method described by Zhao et al. [33]. Surface ultrastructural changes in P. capsici hyphae were observed under a ZEISS scanning electron microscope (Gemini 500, Oberkochen, Germany). All experiments were performed in triplicate.

2.3.4. PI and DAPI Double Staining Assay for Hyphal Membrane and Nuclear Damage

To visualize hyphal membrane disruption and nuclear damage, Phytophthora capsici mycelia were subjected to a Propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA) and DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA) double staining assay with minor modifications. Briefly, fresh mycelial plugs were inoculated into liquid medium and cultured at 25 °C until active hyphal growth was observed. The hyphae were then treated with the fermentation filtrate of Serratia plymuthica MM (at the concentration(s) specified in the corresponding experiment) for 24 h at 25 °C, while untreated hyphae served as the control. After treatment, mycelia were collected and washed twice with PBS (pH 7.2) [34].

For PI staining, the washed mycelia were incubated with propidium iodide (PI) at a final concentration of 50 µg/mL for 20 min in the dark, rinsed with PBS, and immediately examined under a fluorescence microscope (Olympus BX53, Tokyo, Japan). Subsequently, for DAPI staining, the same samples were stained with DAPI (1 µg/mL) for 30 min in the dark, washed with PBS, and observed under a fluorescence microscope. Representative fluorescence images were captured using identical exposure settings across treatments. The experiment was performed in triplicate [35].

2.4. Determination of Plant Growth-Promoting (PGP) Traits of the Putative Biocontrol Strain

To gauge the strain’s PGP potential, four key traits were assessed: indole-3-acetic acid (IAA) production, siderophore production, organic phosphate solubilization, and nitrogen fixation ability. For IAA production, the strain was cultured in LB liquid medium supplemented with L-tryptophan. After incubation, the culture was centrifuged, and the supernatant collected. The IAA concentration was determined according to the Salkowski colorimetric method, with absorbance measured at 530 nm [36]. Siderophore production was assessed using the Chrome Azurol S (CAS) agar plate assay, with positive production indicated by the formation of orange-yellow halos around the bacterial colonies [37]. The organic phosphate solubilization test was done by spot-inoculating the bacterial strain onto PKO inorganic-salt agar plates that contained lecithin as the sole source of phosphorus; the formation of a clear halo around a colony indicates positive phosphate-solubilizing activity [38]. To evaluate nitrogen fixation ability, the strain was streaked onto Ashby’s nitrogen-free agar plates and incubated at 28 °C for 2–3 days; nitrogen fixation was assessed by observing whether a clear halo zone formed around each colony [39].

2.5. Host–Plant Colonization by the Putative Biocontrol Strain

To observe the colonization of Capsicum roots by the bacterial strain MM, a green fluorescent protein (GFP)-marked vector (pGFP4412) (Fenghui Biotechnology Co., Ltd., Changsha, China) was used to construct a GFP-labeled strain of Serratia plymuthica MM (GFP MM), following the methodology described by Xiong et al. [40]. Prior to GFP-based colonization experiments, preliminary laboratory tests were conducted to evaluate the growth characteristics and marker stability of the GFP-tagged strain. These tests indicated that the GFP-tagged Serratia plymuthica MM exhibited a growth rate comparable to that of the wild-type strain and maintained stable fluorescence during serial subculturing under laboratory conditions [41]. To further exclude potential effects of GFP labeling on antagonistic activity, the wild-type strain and the GFP-tagged strain were compared in dual-culture assays against Phytophthora capsici under identical conditions.

For colonization assays, the GFP-tagged strain was inoculated in 100 mL of LB broth supplemented with 100 µg·mL⁻¹ ampicillin (Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 30 °C. Five-week-old, vigorously growing Capsicum seedlings were surface sterilized at the roots and then transplanted into pots containing sterilized soil. Each seedling was root-drenched with 100 mL of the GFP MM suspension (10⁹ CFU·mL⁻¹), while the control group received 100 mL of sterile water. All treated plants were maintained at a constant temperature of 25 °C [42].

At 5, 10, 15, 20, and 25 days post-inoculation (dpi), stem and root samples were randomly collected. At each sampling time point, different plants were selected and destructively sampled. Colonization was observed under a laser confocal scanning microscope (Leica TCS SP8, Wetzlar, Germany) (excitation wavelength = 488 nm). For quantification, 0.1 g of each stem and root sample was ground in 0.9 mL of sterile water, serially diluted, and plated onto ampicillin-containing selective agar for their respective CFU enumeration [43], Prior to statistical analysis, CFU data were log10-transformed [log10(CFU + 1)], as bacterial population data typically do not follow a normal distribution.

2.6. Pot Experiments and Integrated Evaluation

2.6.1. Evaluation of Biocontrol Efficacy in Pot Trials

From a batch of uniformly grown Capsicum seedlings, healthy pepper seedlings with fully developed true leaves were randomly selected for cultivation. Seedlings were thoroughly washed and surface-sterilized prior to transplanting. Each seedling was individually planted in a sterile plastic pot containing autoclaved soil and grown under natural conditions for 5 weeks. Four experimental groups, each consisting of 10 pepper pots, were established following the methodology described by Lan et al. [17]. The pot experiment was conducted on three independent occasions.

For pathogen challenge, Phytophthora capsici was inoculated using a zoospore suspension prepared as described in Section 2.2.1 (Pathogenicity assay). The zoospore suspension (5.0 × 10⁴ zoospores mL⁻¹) was applied to the root zone by soil drenching at a rate of 30 mL per kg of sterilized soil. For biocontrol treatments, Serratia plymuthica MM was applied in the form of a fermentation-derived bacterial suspension, and the cell concentration was adjusted to 1 × 10⁹ CFU mL⁻¹ prior to application.

Four treatments were established as follows: (i) Blank Control, in which plants received an equal volume of sterile water; (ii) Negative Control, in which plants were inoculated with P. capsici only; (iii) Treatment Group, in which plants were first inoculated with P. capsici and subsequently treated with S. plymuthica MM 48 h later; and (iv) Prevention Group, in which plants were pretreated with S. plymuthica MM 48 h prior to inoculation with P. capsici. In addition, a biocontrol-only treatment (MM only, without pathogen inoculation) was included for plant growth and defense enzyme analyses; however, because no disease symptoms were observed in this group, it was excluded from disease incidence and severity evaluations.

At 14 days post-inoculation (dpi), disease incidence (DI) and disease severity index (DSI) of pepper blight were assessed to evaluate the biocontrol efficacy of the strain. The wilt severity grading criteria for pepper seedlings were evaluated according to the Chinese agricultural industry standard NY/T 2060.1–2011 (Rules for evaluation of pepper for resistance to diseases—Part 1: Rule for evaluation of pepper for resistance to Phytophthora blight) [44]. Disease severity was assessed using a 0–5 rating scale according to the Chinese agricultural industry standard NY/T 2060.1–2011, where 0 = no visible symptoms; 1 = slight wilting or small water-soaked lesions on the stem base; 2 = moderate wilting with lesions extending to less than one-third of the stem circumference; 3 = severe wilting with lesions covering one-third to one-half of the stem circumference; 4 = extensive stem necrosis or plant collapse; and 5 = plant death. Disease incidence and severity data were subsequently subjected to statistical analysis as described in Section 2.7.

2.6.2. Growth-Promoting Effects

A fermentation broth of Serratia plymuthica MM was prepared, with the bacterial suspension adjusted to a final concentration of 1 × 10⁹ CFU/mL. Healthy Capsicum seedlings of uniform size were selected and transplanted into pots containing sterilized soil. After 5 weeks of growth, each seedling was root-drenched with 50 mL of the bacterial suspension. Two weeks after the treatment, several growth parameters were measured (root length, plant height, fresh weight, and stem diameter). Each treatment consisted of 10 pots.

2.6.3. Determination of Defense-Related Enzyme Activities in Pepper (Capsicum) Plants

Key defense-related enzymes in plants, namely phenylalanine ammonia-lyase (PAL), peroxidase (POD), catalase (CAT), and polyphenol oxidase (PPO), were also investigated [45]. According to the treatment groups described above, four treatments (blank control, pathogen-only, therapeutic, and preventive groups) were used for disease-related comparisons, while an additional biocontrol-only group (MM only) was included specifically for the assessment of plant defense-related enzyme activities. Once the Capsicum plants had developed 7 or 8 true leaves, 0.5 g of their root tissue was randomly sampled per group for the determination of POD, PAL, CAT, and PPO activity levels. For each enzyme, its activity was quantified by following the given protocol of its commercial assay kit (AIDISHENG, Nanjing, China). Absorbance values were measured with a spectrophotometer (Thermo Scientific NanoDrop 2000, Waltham, MA, USA) and microplate reader (BioTek Synergy H1, Winooski, VT, USA), to calculate the enzyme activity levels [46].

2.7. Data Processing and Analysis

Statistical analyses were performed using GraphPad Prism (v10.1) and SPSS (v27.0). Unless otherwise stated, n represents the number of biological replicates within a single experiment. Continuous variables are presented as mean ± standard deviation (SD).

For pot experiments, disease incidence (DI) and disease severity index (DSI) were calculated within each independent experiment (10 plants per treatment), and the values reported represent the mean ± standard error of the mean (SEM) derived from three independent experiments (n = 3).

Continuous data were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test when the assumptions of normality and homogeneity of variance were met.

Disease severity scores (0–5), treated as ordinal data, were analyzed using a non-parametric marginal effects approach. This analysis reported mean disease ratings (MDR^x), mean ranks (Rij^y), relative treatment effects (Pij), and the corresponding 95% confidence intervals of Pij.

Disease incidence data (DI), expressed as proportions, were analyzed using Fisher’s exact test.

For time-series data (relative membrane conductivity), statistical analysis was conducted using a two-way repeated-measures analysis of variance (ANOVA) with treatment and time as fixed factors. When the assumption of sphericity was violated, Geisser–Greenhouse correction was applied.

A p value < 0.05 was considered statistically significant.

3. Results

3.1. Pathogen Pathogenicity Assay and Screening of Biocontrol Bacteria

3.1.1. Pathogenicity Assay of Phytophthora capsici

To fulfill Koch’s postulates, pathogen re-isolation from diseased tissues was performed. Briefly, lesion segments from a total of 10 infected pepper plants were surface-sterilized in 75% ethanol (analytical grade; Sinopharm Chemical Reagent Co., Shanghai, China) for 30 s, followed by 1% sodium hypochlorite solution (Sinopharm Chemical Reagent Co., Shanghai, China) for 3 min, and then rinsed three times with sterile water. These tissues were placed on Martin’s selective medium plates (three replicate plates per plant sample; composition shown in Supplementary Table S1) and incubated at 25 °C in the dark for 3–5 days. For each plate, the frequency of colony formation for the given re-isolated pathogen was recorded. The predominant pathogen emerging from the tissues was continually purified for its morphological characterization. The re-isolated pathogen formed white, petal-like colonies on Martin’s medium. The hyphae were aseptate, irregularly branched, and hyaline. Sporangia were mainly ovoid or elongated ellipsoidal.

To further confirm the identity of the re-isolated pathogen at the molecular level, a nested PCR assay targeting the YPT1 gene was performed. In the first round, YPT1 genus-specific primers were used to verify that both the original inoculated isolate and the re-isolated pathogen belonged to the genus Phytophthora. In the second round, P. capsici–specific primers were applied to discriminate P. capsici from other closely related Phytophthora species.

PCR amplification produced bands of the expected sizes for both primer sets in the original isolate and the re-isolated pathogen, whereas no amplification was detected in the negative controls (Supplementary Figure S1). The sequences of the amplified YPT1 fragments showed high identity to reference sequences of P. capsici in the NCBI GenBank database. Primer sequences and their corresponding references are listed in Supplementary Table S2. Together with morphological observations, these molecular results further confirmed that the pathogen re-isolated from diseased pepper plants was identical to the originally inoculated P. capsici strain.

3.1.2. Screening of Antagonistic Bacteria

Eight bacterial strains preserved in our laboratory were screened for their antagonistic activity against P. capsici (Figure 1). Among them, Serratia plymuthica MM showed the strongest inhibitory effect, with an inhibition rate of 79.05%, significantly exceeding that of other strains such as Bacillus polymyxa (p < 0.05;

Table 1. Inhibition effect of different bacterial strains on Phytophthora capsici’s mycelial growth.

Bacterial Strain	Treatment Group Radius (mm)	Control Radius (mm)	Inhibition Ratio (%)
Bacillus tequilensis	18.02 ± 1.51 bc	35.33 ± 0.15 ef	49.00 ± 1.73 e
Paenibacillus mucilaginosus	20.65 ± 1.30 a	34.10 ± 0.40 f	39.33 ± 0.58 f
Paenibacillus polymyxa	14.21 ± 1.48 e	39.23 ± 2.20 bc	64.00 ± 1.73 b
Bacillus amyloliquefaciens	19.22 ± 1.00 b	36.50 ± 0.10 de	47.00 ± 3.00 e
Bacillus velezensis	16.98 ± 1.94 cd	38.80 ± 0.90 bc	56.33 ± 1.53 cd
Serratia plymuthica	9.70 ± 0.64 f	46.31 ± 0.12 a	79.05 ± 0.58 a

Values are presented as mean ± SD (n = 3). Different lowercase letters within the same column indicate significant differences among treatments according to Tukey’s HSD test (p < 0.05).

). Due to its superior antagonistic performance, S. plymuthica MM was selected for further investigation and testing.

3.2. Inhibitory Effect of Serratia plymuthica MM on Phytophthora capsici

3.2.1. Effect of the S. plymuthica MM Culture Filtrate on the Mycelial Growth of P. capsici

The sterile fermentation filtrate strongly inhibited the mycelial growth of P. capsici. When applied at a 50% concentration, this inhibition rate reached 88.32%, significantly suppressing pathogen growth under the test conditions (Tukey’s HSD, p < 0.05) Prior to ANOVA, the data were checked for normality (Shapiro–Wilk) and homoscedasticity (Levene’s test) (Figure 2). These results were consistent with those obtained from the dual-culture assay, further confirming the biocontrol potential of S. plymuthica MM against P. capsici.

Microscopic observations revealed that hyphae of P. capsici treated with the sterile fermentation filtrate of S. plymuthica MM exhibited pronounced morphological abnormalities, including distortion, shrinkage, collapse, and fragmentation. In contrast, hyphae in the control group maintained a healthy morphology with smooth surfaces, uniform diameter, and intact structural integrity (Figure 3).

Furthermore, PI and DAPI double staining provided cellular-level evidence of hyphal damage induced by the fermentation filtrate. Hyphae exposed to increasing concentrations of the filtrate displayed progressively enhanced PI fluorescence, indicating concentration-dependent disruption of membrane integrity, whereas control hyphae showed negligible PI signals. Meanwhile, DAPI staining of treated hyphae became increasingly weakened and disorganized with increasing filtrate concentration, in contrast to the well-defined nuclear staining observed in the control group (Figure 4). Collectively, these observations demonstrate that the sterile culture filtrate of S. plymuthica MM exerts antifungal activity against P. capsici by inducing extensive structural and cellular damage to fungal hyphae.

3.2.2. Effect of the S. plymuthica MM Culture Supernatant on the Membrane Permeability of P. capsici

As shown in Figure 5, relative membrane conductivity of P. capsici increased progressively over time in both the control and MM-treated groups. However, treatment with the bacterial culture supernatant resulted in a markedly higher increase in relative membrane conductivity compared with the control, indicating enhanced membrane permeability.

Statistical analysis using a two-way repeated-measures ANOVA revealed significant effects of treatment, time, and their interaction (p < 0.0001), demonstrating that MM induced a time-dependent disruption of membrane integrity in P. capsici.

3.3. Plant Growth-Promoting (PGP) Traits of the Antagonistic Strain

3.3.1. Evaluation of Siderophore Production, Nitrogen Fixation, Phosphate and Potassium Solubilization, and IAA Production

Serratia plymuthica MM formed distinct halos indicating organic phosphate and potassium solubilization, as well as yellow zones indicative of siderophore production on corresponding media. It also grew adequately on nitrogen-free plates, suggesting it harbors a nitrogen-fixation capability. Collectively, these results confirm that S. plymuthica MM can solubilize phosphate and potassium, fix atmospheric nitrogen, and produce siderophores. Furthermore, S. plymuthica MM synthesized IAA, with a yield of about 4 mg/L under standard conditions; however, when supplemented with L-tryptophan (Sigma-Aldrich, St. Louis, MO, USA), that IAA production increased more than quadrupled, to just over 17 mg/L (Figure 6), confirming its strong potential for IAA biosynthesis.

3.3.2. Colonization of Pepper Plants by the Biocontrol Strain

Root colonization by Serratia plymuthica MM increased during the early stages following inoculation, as reflected by CFU enumeration between 5 and 10 days post-inoculation (dpi). Bacterial population levels reached their highest values around 20 dpi and remained relatively stable thereafter, indicating successful establishment and sustained persistence of the strain in pepper root tissues.

Consistent with the quantitative CFU data, confocal microscopy observations revealed strong fluorescence signals in root tissues at 20 dpi, which remained detectable at 25 dpi, further confirming stable colonization by S. plymuthica MM (Figure 7 and Figure 8). In addition, comparative dual-culture assays showed no apparent differences in antagonistic activity between the wild-type and GFP-tagged strains (Figure S2).

3.4. Pot Experiment and Integrated Evaluation

3.4.1. Biocontrol Efficacy

Various plant growth states are shown in Figure 9. In the negative control group, plants inoculated with P. capsici alone exhibited severe stem blackening, constriction, extensive defoliation or wilting, and in some cases plant death. In contrast, plants in the preventive treatment group were largely symptom-free or showed only slight browning at the root collar, whereas those in the therapeutic treatment group displayed mild blackening at the root collar with leaves showing no wilting or only reversible wilting symptoms. Overall, these visual observations indicate that S. plymuthica MM effectively suppressed pepper blight, with preventive application showing stronger protective effects than therapeutic treatment.

Quantitative evaluation of disease incidence and severity is summarized in

Table 2. Evaluation of the control effect of Serratia plymuthica MM against Phytophthora blight in potted pepper (Capsicum) plants.

Treatment	Disease Incidence (%)	MDRx	$\overline{\mathit{R}}$ijy	$\widehat{\mathit{P}}$ij	Control Effect (%)	$95\%\mathbf{CI} \mathbf{for} \widehat{\mathit{P}}$ij
						Upper	Lower
Blank Control	0.00 e	0.00	7.00	0.23	-	0.23	0.23
Negative Control	100 a	3.60	23.00	0.85	-	0.78	0.91
Treatment Group	32.50 ± 0.48 c	1.80	17.80	0.65	55.56% ± 0.13 a	0.57	0.72
Prevention Group	14.60 ± 0.51 b	0.40	10.20	0.35	88.33% ± 0.06 a	0.14	0.57

Note: Values are presented as mean ± standard error of the mean (SEM) calculated from three independent experiments (n = 3). In each independent experiment, each treatment consisted of 10 individual plants. Disease incidence (DI) and disease severity index (DSI) were calculated at the experiment level and subsequently averaged across independent experiments. MDR^x, Rij^y, and Pij represent the mean disease rating, mean rank, and relative treatment effect, respectively, with 95% confidence intervals shown for Pij. Different lowercase letters within the same column indicate statistically significant differences among treatments according to Tukey’s HSD test (p < 0.05).

. The pathogen-inoculated group exhibited the highest disease incidence and severity, whereas both preventive and therapeutic treatments reduced disease development at 14 dpi. Consistent with the visual assessments, preventive treatment resulted in lower disease severity than therapeutic treatment, demonstrating superior biocontrol efficacy of S. plymuthica MM when applied prior to pathogen inoculation. The quantitative values shown in Table 2 represent the mean ± SEM calculated from three independent experiments.

3.4.2. Determination of the Plant Growth-Promoting (PGP) Traits of Serratia plymuthica MM

After 4 weeks, the morphological parameters of pepper (Capsicum) seedlings treated with S. plymuthica MM were evaluated. Compared with the control, the MM-treated seedlings increased significantly in plant height, root length, fresh weight, and dry weight, by 29.56%, 51.41%, 25.90%, and 20.00%, respectively (Tukey’s HSD, p < 0.05; Figure 10).

Similarly, in response to treatments combining P. capsici + MM or MM + P. capsici, the plant height, root length, stem diameter, and fresh weight were significantly greater vis-à-vis the group inoculated with P. capsici alone (Tukey’s HSD, p < 0.05; Figure 10). These results showed that S. plymuthica MM effectively promotes biomass accumulation in pepper plants.

3.4.3. Detection of Defense-Related Enzyme Activities in Pepper Roots

The activity levels of peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and catalase (CAT) in the roots of a pepper plant reflect its resistance capacity to pathogens. In comparison with the control, POD activity significantly increased 1.70-, 1.34-, and 1.55-fold in the S. plymuthica MM treatment, therapeutic, and preventive groups, respectively (Tukey’s HSD, p < 0.05; Figure 11A). Likewise, relative to the control, PPO activity was significantly 0.77- and 0.44-fold greater in the MM and preventive groups (Figure 11B), while PAL activity in the MM and therapeutic groups, respectively, 0.21- and 0.14-fold higher (Tukey’s HSD, p < 0.05) (Figure 11C). Standing out was CAT activity, which was markedly enhanced in the MM, pathogen-only (P.c), therapeutic, and preventive groups, with respective 6.91-, 4.94-, 5.22-, and 5.28-fold increases vis-à-vis the control (Tukey’s HSD test, p < 0.05) (Figure 11D). Enzyme activity data underwent Shapiro–Wilk and Levene’s tests before one-way ANOVA with Tukey’s HSD used to compare the means of groups (at p < 0.05).

4. Discussion

Phytophthora blight caused by Phytophthora capsici is among the most destructive soil-borne diseases impacting pepper cultivation, resulting in devastating losses in yield and severe declines in its quality [47]. This pathogen has a broad host range, infecting more than 200 plant species, including solanaceous and cucurbit crops, rendering it particularly difficult to eradicate from infested fields [2,5]. Current management approaches still rely heavily on synthetic fungicides, such as metalaxyl, oxathiapiprolin, and strobilurins; however, their repeated application is accelerating the emergence of resistant populations of P. capsici, while also exacerbating environmental contamination and the accumulation of pesticide residues in soil [48,49]. Evolved metalaxyl resistance in greenhouse pepper production in southern Europe underscore the limitations of this dependence on chemical control [50]. These issues emphasize the urgent need for environmentally friendly and sustainable biological control strategies.

In the present study, Serratia plymuthica MM demonstrated remarkable antagonistic activity against P. capsici. Dual culture and cell-free filtrate assays confirmed that this bacterial strain strongly decreases mycelial growth, with inhibition rates reaching 88.32% at the 50% filtrate concentration. These findings are consistent with earlier reports that Serratia species produce diverse bioactive secondary metabolites, including pyrrolo[1,2-a] pyrazine derivatives and lipopeptides, which disrupt the physiology of certain pathogens [20,31]. The high inhibition rate observed in our assays suggests S. plymuthica MM secretes potent antifungal compounds capable of impairing pathogen growth considerably.

A key contribution of the present study is the identification of membrane-associated cellular damage as a major feature underlying the antifungal activity of S. plymuthica MM against P. capsici. Treatment with the sterile filtrate of S. plymuthica MM induces a rapid and substantial increase in extracellular electrolyte leakage, indicative of weakened or lost membrane integrity and function. This finding was corroborated by SEM observations, which uncovered severe ultrastructural aberrations in the pathogen’s hyphal morphology, including widespread plasmolysis, distortion, collapse, and fragmentation.

Importantly, the membrane-damage hypothesis was further substantiated at the cellular and subcellular levels by propidium iodide (PI) and DAPI double staining. PI is a membrane-impermeable nucleic acid dye that selectively penetrates cells with compromised plasma membranes, whereas DAPI preferentially binds to intact nuclear DNA. In the present study, hyphae of P. capsici exposed to increasing concentrations of the sterile fermentation filtrate of S. plymuthica MM exhibited progressively intensified PI fluorescence signals, indicating concentration-dependent loss of membrane integrity. Concurrently, DAPI staining patterns in treated hyphae became weakened, fragmented, and disorganized, suggesting pronounced alterations in nuclear organization and integrity. In contrast, control hyphae displayed minimal PI uptake and well-defined DAPI-stained nuclei, reflecting intact cellular organization.

The combined PI/DAPI staining results provide cellular-level support for the involvement of membrane and intracellular damage in the antifungal activity of S. plymuthica MM is associated not only with surface ultrastructural disruption, as revealed by SEM, but also with profound intracellular damage. Such simultaneous impairment of membrane integrity and nuclear organization likely accelerates irreversible cellular dysfunction and cell death, thereby explaining the strong inhibitory effects observed in both plate-based assays and pot experiments. Similar PI/DAPI-based evidence of membrane- and nucleus-targeting antifungal mechanisms has been reported for bioactive metabolites produced by antagonistic bacteria and plant-derived compounds, further supporting the biological relevance of this mode of action [34].

Other studies have shown that reactive oxygen species (ROS)-induced lipid peroxidation plays a prominent role in membrane destabilization [40]. More precisely, ROS can oxidatively break down unsaturated fatty acids with phospholipid bilayers, leading to the accumulation of malondialdehyde (MDA), greater membrane permeability, and eventually cell lysis [51]. In plant physiology and biochemistry, similar processes have been reported in pathogen–antagonist ecological interactions: oxidative bursts on the pathogen’s cell surface trigger the peroxidation of membrane lipids, thereby compromising its structural and functional integrity [32,36]. In the present study, the observed electrolyte leakage and hyphal deformation in P. capsici treated with S. plymuthica MM filtrates are consistent with these previously described oxidative-damage patterns. It should be noted that oxidative stress-related parameters, including ROS accumulation and lipid peroxidation products such as MDA, were not directly quantified in this study. Although ROS or MDA levels were not directly measured, the morphological alterations observed provide supportive evidence that oxidative stress-associated membrane disruption may contribute to the antifungal effects of S. plymuthica MM. Such a mode of action, if confirmed, would be advantageous because membrane damage is largely irreversible, limiting pathogen recovery. This proposed “filtrate → membrane permeability → hyphal malformation” process therefore offers a plausible explanation for the strong inhibitory effects of S. plymuthica MM against P. capsici [52]. To the best of our knowledge, no previous study has described a comparable membrane-damage-related mechanism induced by S. plymuthica against P. capsici in pepper, underscoring the novelty of this observation.

Apart from its direct inhibition of the pathogen, this bacterial strain also harbors several plant growth-promoting (PGP) attributes, including phosphate solubilization, nitrogen fixation, siderophore production, and indole-3-acetic acid (IAA) biosynthesis. These functions are crucial for improving nutrient acquisition by plants and stimulating their root development. In our pot experiments, pepper plants treated with S. plymuthica MM improved significantly in terms of their height, root length, fresh weight, and dry weight when compared with the untreated controls. This enhanced biomass accumulation suggests that this bacterial strain not only mitigates disease pressure but also improves the overall growth performance of pepper plants, with potential applicability to other crops.

No less important is the ability of S. plymuthica MM to activate host–plant defense systems. The levels of peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), and catalase (CAT) were all markedly elevated in MM-treated plants. These enzymes are known to assist in various processes, such as lignification, phenolic metabolism, and ROS scavenging, thereby fortifying cell walls and activating systemic resistance in plants [53,54]. For example, POD and PPO catalyze the oxidation of phenolics to quinones, which are polymerized into lignin-like substances, strengthening plant tissues against pathogen penetration. PAL functions as a key enzyme in the phenylpropanoid pathway, leading to the synthesis of phytoalexins and lignin precursors, and CAT detoxifies hydrogen peroxide, reducing oxidative stress while maintaining the ROS signaling required for defense activation. Hence, the concurrent upregulation of both enzymes in MM-treated pepper plants suggests this distinguished biocontrol bacterium does more than act externally on the targeted pathogen: it also primes the internal defense responses of its host, resulting in integrated disease protection response [55].

In the present study, GFP labeling was used solely as a tracking tool for colonization assays, and comparative tests confirmed that the GFP-tagged strain retained antagonistic activity comparable to that of the wild-type strain. Long-term colonization of the rhizosphere is a prerequisite for reliable field performance of any biocontrol agent. The GFP-labeling experiments proved that S. plymuthica MM successfully colonizes pepper’s roots, with strong fluorescence signals detected up to 25 dpi (days post-inoculation). Colony counts confirmed the stable persistence of the strain in root tissues and their surrounding soil. These findings indicate S. plymuthica MM can quickly adapt to the rhizosphere’s environment, establishing itself as a resident member there of its microbial community. Stable colonization ensures the continuous production of antifungal metabolites and the sustained induction of plant resistance. Similar studies with Pseudomonas and Bacillus spp. have emphasized the importance of rhizosphere competence for consistent biocontrol efficacy [56]. Although GFP-based microscopy provided direct visualization of root colonization in this study, molecular approaches such as quantitative PCR (qPCR) may offer complementary quantitative insights into bacterial abundance and persistence, and should be considered in future investigations. Thus, the ecological adaptability of MM provides a sound biological basis for its further development as a commercial inoculant for use in the production of pepper and other crops.

Although some members of the genus Serratia have been reported as opportunistic human pathogens, biosafety assessment should be conducted at the strain level rather than at the genus level. Serratia plymuthica MM used in this study was isolated from the plant rhizosphere and has previously been reported as an effective biocontrol agent in agricultural systems. In earlier work, this strain was applied in long-term pot experiments and demonstrated stable root-surface and endophytic colonization without causing visible phytotoxic effects or abnormal plant phenotypes [20].

Consistent with these previous observations, repeated pot experiments in the present study did not reveal any adverse effects of S. plymuthica MM on pepper growth or development. Instead, MM-treated plants exhibited improved growth performance and enhanced disease resistance. Together, these findings support the suitability of S. plymuthica MM for agricultural biocontrol research under controlled conditions.

Nevertheless, a comprehensive biosafety evaluation, including genomic screening for virulence- and antibiotic resistance–associated markers and environmental risk assessment, will be required prior to any large-scale or field application. Such analyses will be essential to fully assess the long-term ecological safety of this strain.

Despite the promising biocontrol performance of Serratia plymuthica MM observed under controlled pot conditions, several challenges must be considered before its practical application in open-field production systems. Field environments are inherently more complex and variable than greenhouse conditions, and factors such as soil physicochemical properties, native microbial communities, climate fluctuations, and agricultural practices may substantially influence the survival, colonization efficiency, and biocontrol consistency of introduced bacterial agents.

One major challenge for field application lies in ensuring stable establishment and persistence of the biocontrol strain in competitive soil microbiomes. Although GFP-labeling experiments in the present study demonstrated effective root colonization under controlled conditions, long-term persistence and functional expression of antagonistic traits in diverse field soils remain to be verified. In addition, formulation development, storage stability, and delivery methods (e.g., seed coating, root drenching, or soil amendment) will be critical determinants of field performance.

Currently commercialized bacterial biocontrol agents, such as Bacillus-based products (e.g., Bacillus subtilis and Bacillus amyloliquefaciens formulations), provide useful benchmarks for evaluating the translational potential of new candidates. These products have demonstrated that consistent field efficacy often requires optimized formulations, repeated applications, and integration with other disease management strategies rather than reliance on a single control measure.

Therefore, while the present findings establish a solid experimental foundation for the use of S. plymuthica MM as a biocontrol candidate against Phytophthora blight, further studies under field conditions, together with formulation optimization and ecological risk assessment, will be essential to determine its practical applicability in sustainable pepper production systems.

While greenhouse and pot experiments can provide valuable mechanistic insights, validation under field conditions is essential for the commercialization of potential biocontrol agents. Fortunately, several studies have demonstrated that microbial inoculants can achieve meaningful disease control and yield enhancement in diverse horticultural systems. For example, work by Xu et al. [45] showed that Bacillus subtilis YB-04 reduced the incidence of Fusarium wilt and promoted cucumber growth in field trials. Later, Li et al. [20] reported that S. plymuthica MM decreased Fusarium wilt in watermelon under open-field conditions, highlighting its broad-spectrum biocontrol potential. More recently, Wang et al. [54] showed that S. plymuthica HK9-3 strengthened tomato resistance against P. capsici by modulating antioxidant defenses and improving rhizosphere micro-ecology. Finally, pre-harvest application of B. subtilis extended the shelf life of tomato fruits and enhanced postharvest quality, further supporting the feasibility of bacterial inoculants in open-field production systems [57]. Taken together, these studies underscore the feasibility of deploying bacterial biocontrol agents in sustainable horticultural systems.

Our findings are timely because they provide the necessary experimental foundation for advancing the evaluation of S. plymuthica MM in actual field settings. The proven ability of this bacterial strain to directly inhibit P. capsici, promote the growth of pepper plants, and induce their systemic resistance suggests it could minimize blight infestations while also enhancing their yield stability. We propose the prudent integration of S. plymuthica MM into current production systems could also greatly reduce the present reliance on synthetic fungicides, mitigating risks of resistance development and environmental contamination. Moreover, combining S. plymuthica MM with other beneficial management practices—like using resistant cultivars, crop rotation, and optimized irrigation—could form a holistic integrated pest management (IPM) framework for pepper blight.

Collectively, these results deepen our understanding of P. capsici’s pathogenic mechanisms vis-à-vis the antagonistic responses of biocontrol bacteria, while also providing scientific evidence and technical support for the biological control of Phytophthora blight in pepper. Furthermore, this work holds significant theoretical and practical value for advancing green agriculture and reducing dependence on chemical pesticides. Through the screening and application of efficient biocontrol strains, the sustainable management of Phytophthora blight in pepper production could be achieved, thereby maintaining high and stable yields, minimizing environmental pollution, and promoting the development of ecological agriculture.

5. Conclusions

Serratia plymuthica MM exhibited strong antagonistic activity against the pepper pathogen Phytophthora capsici, which was closely associated with membrane-related cellular damage, as reflected by increased membrane permeability, electrolyte leakage, and severe hyphal deformation. In addition to its direct inhibitory effects on the pathogen, this strain displayed multiple plant growth–promoting traits and effectively enhanced host defense responses in pepper plants, as evidenced by the elevated activities of POD, PPO, PAL, and CAT enzymes. Furthermore, GFP labeling confirmed stable root colonization by S. plymuthica MM, indicating its ecological adaptability and persistence in the rhizosphere. Collectively, these results highlight the integrated biocontrol potential of S. plymuthica MM under controlled conditions, while further biosafety evaluation and field-scale validation will be required prior to agricultural application.