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Article

Comparative Effects of Sodium Metasilicate and Potassium Silicate in Enhancing Bacillus amyloliquefaciens PMB05 Plant Immune Responses and Control of Bacterial Soft Rot in Cabbage

1
Department of Plant Medicine, National Pingtung University of Science and Technology, Pingtung 912301, Taiwan
2
Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 912301, Taiwan
3
Department of Plant Pathology, National Chung-Hsing University, Taichung 40227, Taiwan
*
Author to whom correspondence should be addressed.
Agriculture 2025, 15(23), 2436; https://doi.org/10.3390/agriculture15232436
Submission received: 10 November 2025 / Revised: 24 November 2025 / Accepted: 24 November 2025 / Published: 26 November 2025
(This article belongs to the Section Crop Protection, Diseases, Pests and Weeds)

Abstract

Cabbage is an important vegetable crop worldwide. In Taiwan, during cabbage production, bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovorum often leads to significant yield losses. Aligning with the Sustainable Development Goals, there is a high demand for sustainable disease control strategies. Silicates are considered to be effective elicitors in activating plant defense responses and are reported to improve resistance to certain plant diseases. Bacillus amyloliquefaciens PMB05 fermentation liquid has been shown to enhance plant immunity and control many bacterial diseases. The supplementation of silicates to the PMB05 fermentation liquid may further improve its efficacy to control bacterial soft rot in cabbage. This study evaluated the effects of sodium metasilicate and potassium silicate on PMB05-mediated plant immune responses and disease control. Initial assays confirmed that treatment with B. amyloliquefaciens PMB05 suspension significantly increased HrpN-triggered reactive oxygen species (ROS) generation and callose deposition; moreover, PMB05 treatment alone reduced bacterial soft rot severity by 39.7%. When combined with B. amyloliquefaceins PMB05 fermentation liquid, sodium metasilicate at 2000 μM further enhanced ROS generation and callose deposition by 100% and 133%, respectively, compared to the treatment of PMB05 alone (p < 0.05). In contrast, potassium silicate exhibited inconsistent effects on ROS production, with both 500 and 1000 µM concentrations significantly reducing ROS generation by 26% and 38%, respectively, while none of the tested concentrations affected callose deposition (p < 0.05). Lastly, disease severity assessments in cabbage inoculated with P. carotovorum subsp. carotovorum PCCSB1 revealed that B. amyloliquefaciens PMB05 fermentation liquid was able to reduce bacterial soft rot symptoms by 60.3%. Supplementation with 1500 and 2000 µM sodium metasilicate further decreased disease severity by 77.9% and 76.4%, respectively (p < 0.05). Although the supplementation of potassium silicate also significantly reduced disease severity compared to P. carotovorum subsp. carotovorum PCCSB1 alone, it was less effective than PMB05 fermentation alone. Overall, these results demonstrate that sodium metasilicate enhances the biocontrol activity of B. amyloliquefaciens PMB05 by further intensifying plant immune responses. This approach may broaden the large-scale use of B. amyloliquefaciens PMB05 fermentation liquid for sustainable soft rot management in cabbage, although the stability and cost-effectiveness of sodium metasilicate under field conditions still require validation.

1. Introduction

Cabbages (Brassica oleracea) are important vegetable crops that are cultivated on over 3.5 million hectares globally [1]. In Taiwan, cabbages are cultivated on more than 8 thousand hectares in planting area with cultivation concentrated in Taichung, Yunlin, and Changhua [2]. Bacterial soft rot disease is caused by the pathogen Pectobacterium carotovorum subsp. carotovorum and it is a major constraint in cabbage production [3,4]. Bacterial soft rot may occur during planting and harvesting activities, especially if the environment is high in temperature and humidity [5,6]. P. carotovorum subsp. carotovorum invades host tissues through wounds and natural entry sites including hydathodes and stomata [7]. Initial symptoms appear as small, water-soaked lesions along the leaf margins. If left untreated, these lesions rapidly progress to the other parts of the plant, leading to substantial yield losses. Traditionally, the application of chemicals such as copper-based fungicides are used to prevent and suppress the growth of P. carotovorum subsp. carotovorum [7]. However, the overuse of chemical pesticides contributes to resistant pathogen strains and environmental concerns. Under the issue of the Sustainable Development Goals, the use of microbial agents to replace the use of pesticides for disease control is crucial to ensure the safety of crop cultivation.
Biological control using beneficial microbes to manage diseases has been proven to be an efficient and safe alternative for sustainable disease management [2,7]. Among them, some Bacillus strains with antagonistic properties have shown inhibition against P. carotovorum subsp. carotovorum and effectively reduce bacterial soft rot [3,7]. In previous research, Bacillus amyloliquefaciens PMB05 has been shown to have the ability to reduce plant disease by intensifying plant immune responses of pathogen-associated molecular-pattern (PAMP)-triggered immunity (PTI) and promoting stomatal closure during PTI to limit pathogen entry, enhancing resistance to bacterial soft rot [2,8,9,10,11,12]. Due to their antagonistic and plant-growth-promoting characteristics in agriculture, Bacillus strains have been widely used in developing many commercialized products [13]. Microbial fermentation liquid provides more nutrients and results in the production and development of antibiotics, industrial enzymes, or other bioactive compounds, stabilizing the microbe cultures and making it feasible to use in fields [14,15]. The B. amyloliquefaciens PMB05 fermentation liquid has been shown to be effective in controlling diseases like lemon canker and cabbage black rot disease in the field [2,16]. Although microbial products have advanced considerably, their practical application in agriculture still faces several challenges. Because microbial pesticides are living organisms, their efficacy to control diseases in large-scale areas may be influenced by biological and non-biological factors [17]. Thus, further studies are required to strengthen the performance of biocontrol agents and broaden their applications.
Given the many benefits of silicon under both abiotic and biotic stresses, combining silicon with bacterial control agents may be a promising strategy to enhance their effectiveness. Silicon compounds are regarded to be safe compounds for humans and the environment [17,18,19,20,21]. Silicon helps to neutralize soil acidity, elevate pH, and enhance overall plant health [22]. They can also help to alleviate plant disease through various mechanisms, showing induced disease resistance activity. Silicon compounds promote several structural defenses, including a subcuticular silica layer, double cuticular layers, reinforced silicon–cellulose membranes, and papillae [17,22,23]. These physical barriers significantly restrict pathogen invasion. At the molecular level, silicon-induced resistance involves different biochemical events, including the activation of defense-related enzymes, metabolism of reactive oxygen species (ROS), stimulation of antimicrobial compound synthesis, and regulation of signaling molecules like salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) [18,20,23,24,25]. When silicon compounds were supplemented to beneficial microbial inoculants, plant growth and plant defense responses were significantly enhanced, reducing Fusarium wilt incidence in bananas [26]. Despite growing interest in silicon-mediated immunity, the interactions between silicate compounds and microbial-induced plant immunity remain poorly understood, particularly in the context of disease control.
An interesting concept is that silicate compounds may interact with B. amyloliquefaciens PMB05 by also participating in PTI. During pathogen infection, PTI activation relies heavily on ROS generation and mitogen-activated protein kinase (MAPK) signaling cascades, which subsequently activate SA, JA, and ET signaling pathways to cause plants to exhibit disease resistance [2,12,27]. It is also interesting to investigate whether differences in cation type or solubility of silicate compounds influence the nature of these immune responses. This mechanistic framework may provide a basis for exploring how silicate treatments interact with immunity-intensifying microbes like B. amyloliquefaciens PMB05 by intensifying early immune signaling and contributing to improved disease resistance. This warrants further investigation and may provide a new perspective on the integration of microbial inoculants with fertilizer management in field applications.
This study evaluated the effects of supplementing sodium metasilicate and potassium silicate to B. amyloliquefaciens PMB05 bacterial suspension and fermentation liquid on ROS generation, callose deposition, and the control of bacterial soft rot in cabbage.

2. Materials and Methods

2.1. Plant Growth Conditions

The cabbage Brassica oleracea L. var. capitate, sourced from Minghua Seed, New Taipei City, Taiwan, was used in this study. Seeds were surface-sterilized for 5 min by using 1% sodium hypochlorite solution, and then rinsed with sterile water over three times. After disinfection, seeds were air-dried (DHR-20TW, Cuisinart, Hong Kong, China) for 0.5 h. Cabbage seeds were considered clean and stored under 4 °C until further use. Stored seeds were replaced with fresh sterilized seeds every two months to prevent loss in viability. For cultivation, seeds were planted individually in pots with sterilized soil composed of peat moss, pearlite, and vermiculite at a 1:1:10 (v/v/v) ratio. Plants were maintained in a controlled-environment growth chamber (F-1200, Hipoint, Kaohsiung City, Taiwan) with a 16 h light/8 h dark photoperiod at 28 °C. Experimental plants were four weeks old and had developed four true leaves at the time of assessment.

2.2. Growth and Preparations of Microorganisms

The P. carotovorum subsp. carotovorum strain PCCSB1 was cultured on Nutrient Agar (NA, BD Difco™, Becton, Dickison & Co., Sparks, MD, USA) plates and incubated at 28 °C for 48 h. The PCCSB1 bacterial suspension was prepared by suspending colonies grown on the NA plates in sterile deionized water containing 0.1% of carboxymethylcellulose sodium (CMC; Sigma, St. Louis, MI, USA) obtaining a concentration of OD600 of 0.3 (3 × 108 CFU/mL). Bacillus amyloliquefaciens PMB05 is a PTI-intensifying microbe that can improve disease resistance in plants. B. amyloliquefaciens PMB05 was cultured under similar conditions on NA plates at 28 °C for 48 h. The PMB05 bacterial suspension was prepared by transferring a single colony into nutrient broth (5 mL) and kept in incubation at 28 °C with shaking (200 rpm) for 16–18 h. The resulting culture was centrifuged at a speed of 8000 rpm for 5 min under 4 °C, and the bacteria pellet was resuspended in sterile deionized water containing 0.1% of CMC, adjusted to OD600 of 0.3 (3 × 108 CFU/ mL).

2.3. Preparation of Bacillus amyloliquefaciens PMB05 Fermentation Liquid

The B. amyloliquefaciens PMB05 fermentation liquid was prepared from the standardized protocol, PMBFL-2A, from previous research [16]. Briefly, a 2% (v/v) overnight culture of PMB05 in Luria-Bertani (LB) broth was inoculated in fresh LB (400 mL) and kept under incubation at 37 °C for 6 h. This starter culture subsequently transferred into an automatic fermenter (BTF-B30L, 30 L, Biotop Process & Equipment Inc., Nantou County, Taiwan) that contained sterilized fermentation medium (20 L) composed of 1% yeast powder (Ensenmi Bio-technology Co., Ltd., Taichung City, Taiwan) and 3% granulated sugar. Fermentation was carried out at 37 °C with agitation at 120 rpm and aeration at 1.5 vvm until harvest, reaching a concentration of 1.6 × 109 CFU/mL and a pH of 5.0. Plate count was used to confirm the CFU. The fermentation liquid was collected and stored at 4 °C until further use. For experimental applications, the fermentation liquid was diluted 200-fold, resulting in a final bacterial concentration of approximately 7.9 × 106 CFU/mL in each assay.

2.4. HrpN Protein Preparation

HrpN is a harpin protein secreted by P. carotovorum that acts as an elicitor to prime systemic acquired resistance and enhance plant immunity through ROS generation and defense gene activation. It was prepared according to previous studies [12]. Escherichia coli BL21 carrying pET-HrpN-Ecc3 was grown in LB broth containing 100 μg/mL ampicillin at 37 °C for 16 h. The culture was then inoculated into fresh LB medium and incubated for an additional 4 h before inducing protein expression with 1 mM isopropyl-β-D-thiogalactopyranoside. After a further 16 h of incubation, cells were harvested by centrifugation at 8000× g for 5 min at 4 °C. The pellet was resuspended in 25 mM Tris-HCl buffer (pH 7.0), sonicated, and heated at 100 °C for 10 min. The lysate was centrifuged at 10,000× g for 10 min, and the resulting supernatant was collected as the HrpN protein extract for further analyses. All experiments involving HrpN were conducted at a final HrpN concentration of 0.5 mg/mL.

2.5. Effects of Bacillus amyloliquefaciens PMB05 on HrpN-Triggered Plant Immune Responses

To evaluate whether B. amyloliquefaciens PMB05 can enhance plant immune responses such as ROS generation and callose deposition induced by HrpN in cabbage seedlings, assays were conducted according to established protocols [9]. Equal volumes of HrpN, PMB05 suspension, and Tris-HCl buffer (pH 7.0) were mixed to prepare the solutions used for infiltration. In treatments containing HrpN, the final concentration was adjusted to 0.5 mg/mL. Tris-HCl buffer (pH 7.0) was used as control. Leaves from four-week-old plants were infiltrated, excised into strips, and examined at 1 h post-infiltration. For ROS detection, leaf strips were stained with 20 µM of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes, Eugene, OR, USA) for 20 min in darkness, and fluorescence was visualized under a microscope (Leica DM2500, Wetzlar, Germany) with excitation and emission filters set at 465–495 nm and 515–555 nm, respectively. All images were cropped into 5 cm × 5 cm dimensions, and the fluorescence intensities were quantified under 8-bit mode and consistent threshold conditions using ImageJ software version 1.54 (https://imagej.net/ij/, accessed on 4 April 2025). For each treatment, 10 images were collected as technical repeats, and three independent biological experiments were performed.
For the callose deposition assay, leaves of four-week-old cabbage seedlings were infiltrated with the respective treatments, cut into strips, and sampled at 8 h post-infiltration. Leaf strips were stained with aniline blue (Sigma, USA) for 2 h in darkness and observed under a fluorescence microscope (Leica DM2500, Wetzlar, Germany) equipped with a 340–380 nm excitation filter and a 400–425 nm emission filter [11]. All images were cropped into 5 cm × 5 cm dimensions, and the fluorescence intensities were quantified under 8-bit mode and consistent threshold conditions using ImageJ software version 1.54 (https://imagej.net/ij/, accessed on 4 April 2025). For each treatment, 10 images were collected as technical repeats, and three independent biological experiments were performed.

2.6. Effects of Bacillus amyloliquefaciens PMB05 on the Biocontrol of Bacterial Soft Rot in Cabbage

The biocontrol effects of B. amyloliquefaciens PMB05 against bacterial soft rot in cabbage were evaluated using a spray inoculation method [12,28]. PMB05 and PCCSB1 bacterial suspensions were prepared in 0.1% CMC. Each bacterial suspension had a concentration of OD600 0.3 (3 × 108 CFU/mL). The bacterial suspension of PMB05 (25 mL) was sprayed evenly on 4-week-old cabbage leaves and left to dry naturally for 24 h. Subsequently, leaves were inoculated with PCCSB1 bacterial suspension (25 mL) and maintained in a humidified container at 22 °C for 48 h. Disease symptoms were scored using a four-level scale (0, no symptoms; 1, 1–10%; 2, 11–30%; 3, 31–50%; 4, >50% leaf area affected). A four-level disease severity scale (0–4) was adopted as it provides a practical and widely used method to classify symptom progression, allowing reliable comparison of treatment effects while reducing subjectivity in scoring. Disease severity was calculated using the following formula: disease severity (%) = [(0 × N0 + 1 × N1 + 2 × N2 + 3 × N3 + 4 × N4)/(4 × Ntotal)] × 100, where N0–N4 indicate the number of leaves corresponding to each disease severity scale (0–4), and Ntotal represents the total number of leaves assessed. For each treatment, 25 inoculated leaves were evaluated as technical repeats and three independent biological experiments were conducted.

2.7. Effects of Silicates on HrpN-Triggered Plant Immune Responses

To determine the effective concentrations of silicate alone, cabbage leaves were infiltrated with sodium metasilicate or potassium silicate at final concentrations of 0, 250, 500, 750, 1000, 1500, 2000, and 2500 μM. With some modifications, concentrations amongst this range were revealed to be optimal concentrations for both silicates in previous studies [19,20,29,30]. These concentrations did not show any phytotoxicity effects when applied to plants. To ensure there was no influence on bacterial activity, less than 5% of the silicate stock solution was added to the bacteria solution to minimize detrimental pH change. The ROS generation was assayed as previously described above. Based on these results, the concentrations that showed a statistically significant difference compared with the blank control were selected for the subsequent assays.

2.8. Effect of Silicates with Bacillus amyloliquefaciens PMB05 on Regulation of Plant Immune Responses

The selected concentrations of each silicate were then tested for their effects on ROS generation and callose deposition supplemented with B. amyloliquefaciens PMB05 fermentation liquid upon treatment with HrpN. For these assays, PMB05 fermentation liquid was diluted 200-fold, and the final concentration of HrpN was 0.5 mg/mL. Stock solutions of sodium metasilicate were prepared to achieve final concentrations of 0, 500, 1500, 2000, and 2500 μM. Similarly, stock solutions of potassium silicate were prepared to achieve final concentrations of 0, 500, 1000, and 2500 μM. The ROS generation and callose deposition on cabbage leaves was assayed as described above, with samples evaluated at 1 h and 8 h post-infiltration, respectively.

2.9. Effects of Silicates with Bacillus amyloliquefaciens PMB05 Against the Biocontrol of Bacterial Soft Rot Disease in Cabbage

The biocontrol efficacy of B. amyloliquefaciens PMB05 against bacterial soft rot in cabbage was evaluated using a spray inoculation method. Bacterial suspensions of PCCSB1 were prepared in 0.1% CMC. PMB05 fermentation liquid was diluted 200-fold, and sodium metasilicate was added to reach final concentrations of 0, 500, 1500, 2000, and 2500 μM. Potassium silicate was applied at 0, 500, 1000, and 2500 μM concentrations. Then the B. amyloliquefaciens PMB05-silicate solutions (25 mL) were sprayed evenly on cabbage leaves and left to dry naturally for 24 h, followed by inoculation with PCCSB1. Inoculated plants were maintained in a humidified container at 22 °C for 48 h. Disease severity was calculated as previously described above. For each treatment, 25 inoculated leaves were evaluated as repeats and three independent biological experiments were conducted.

2.10. Data Analysis

SPSS Statistics version 25 (IBM Corp, Armonk, NY, USA) was used to conduct statistical analyses. Significant differences between multiple treatments were evaluated by an analysis of variance (one-way ANOVA) followed by Tukey’s HSD post hoc test with significance defined at p < 0.05. Significant differences between two treatments were evaluated by a t-test (p < 0.05)

3. Results

3.1. Effects of Bacillus amyloliquefaciens PMB05 on HrpN-Triggered Plant Immune Responses

To investigate whether B. amyloliquefaciens PMB05 induced PTI in cabbage, its effects on HrpN-triggered ROS generation and callose deposition were evaluated. The results revealed that the PMB05 treatment significantly enhanced HrpN-induced responses. It increased the fluorescent intensity by 173% and 208% in ROS generation and callose deposition, respectively, compared with HrpN alone (Figure 1A,B). PMB05 alone did not elicit ROS generation or callose deposition compared to the blank control.

3.2. Effects of Bacillus amyloliquefaciens PMB05 on the Biocontrol of Bacterial Soft Rot in Cabbage

To determine whether B. amyloliquefaciens PMB05 can effectively control bacterial soft rot in cabbage, the disease severity was evaluated. Based on a t-test, the results showed that PMB05 bacterial suspension significantly reduced disease severity by 39.7% (Figure 1C).

3.3. Effects of Silicates and HrpN on Reactive Oxygen Species Generation

To confirm if silicates can induce plant immune responses, concentrations of sodium metasilicate and potassium silicate were applied in the absence and presence of HrpN to assay their effects on ROS generation. The results revealed that in the absence of HrpN, sodium metasilicate was able to significantly increase ROS generation observed at concentrations ≥ 750 µM (Figure 2A,B). In the presence of HrpN, sodium metasilicate significantly increased ROS generation observed at ≥500 µM (Figure 2A,C). ROS levels at 2000 µM were significantly higher compared to other concentrations. In the absence of HrpN, all ROS generation produced by potassium silicate concentrations did not have any significant difference compared to that of the blank (Figure 3A,B). In the presence of HrpN, ROS generation produced by all potassium silicate concentrations was significantly lower compared to that of the blank (Figure 3A,C). Based on these results, sodium metasilicate at 0, 500, 1500, and 2500 μM concentrations and potassium silicate at 0, 500, 1000, and 2500 μM concentrations showed a statistically significant difference compared with the blank control. These concentrations were selected to be tested by the subsequent assays.

3.4. Effect of Silicates with Bacillus amyloliquefaciens PMB05 on Plant Immune-Triggered Response Assays

To determine whether silicates enhance immune responses induced by B. amyloliquefaciens PMB05 in cabbage, silicates were supplemented into both bacterial suspensions and fermentation liquid, and their effects on HrpN-induced ROS generation and callose deposition were investigated. Supplementation with sodium metasilicate further enhanced ROS generation, with fermentation liquid treatments showing stronger fluorescence than bacterial suspensions. In bacterial suspensions, ROS generation was significantly increased at 2000 and 2500 μM by 24% and 21%, respectively, compared to that of the blank (Figure 4A,B). In the fermentation liquid, concentrations at 500, 1500, 2000, and 2500 μM significantly increased ROS generation by 60%, 72%, 100%, and 90%, respectively, compared to the blank, with the highest increase observed at 2000 μM (Figure 4A,C). The increased effects with the fermentation liquid suggest that the bacterial metabolites secreted during growth interact synergistically with sodium metasilicate to intensify plant immune responses. In the bacterial suspension, concentrations at 500, 1500, 2000, and 2500 μM significantly increased callose deposition by 54%, 47%, 118%, and 78%, respectively, compared to that of the blank, with the highest increase observed at 2000 μM (Figure 5A,B). In the fermentation liquid, concentrations at 1500, 2000, and 2500 μM significantly increased callose deposition by 37%, 133%, and 84%, respectively, compared to that of the blank, with the highest increase observed at 2000 μM (Figure 5A,C).
In the bacterial suspension, all potassium silicate concentrations significantly decreased ROS generation. Concentrations at 500, 1000, and 2500 μM significantly decreased ROS generation by 57%, 54%, and 59%, respectively, compared to that of the blank (Figure 6A,B). In fermentation liquid, potassium silicate at 500 and 1000 μM significantly decreased ROS generation by 26% and 38% compared to that of the blank, while at 2500 μM, there were no significant differences with the blank (Figure 6A,C). In the bacterial suspension, potassium silicate at 1000 μM significantly increased callose deposition by 9%; at 2500 μM, callose deposition was significantly decreased by 29%, and at 500 μM, there were no significant differences observed compared to that of the blank (Figure 7A,B). In the fermentation liquid, no significant differences were observed in callose deposition among treatments compared to that of the blank (Figure 7A,C).

3.5. Effects of Silicates with Bacillus amyloliquefaciens PMB05 on the Biocontrol of Bacterial Soft Rot Disease in Cabbage

To determine whether silicate supplementation enhances the biocontrol efficacy of PMB05 fermentation liquid, the disease severity of bacterial soft rot was evaluated. The results revealed that PMB05 alone significantly reduced disease severity by 60.3% in fermentation liquid treatments (Figure 8). Supplementation with sodium metasilicate further enhanced disease control, with all concentrations showing significant disease reductions. Among them, the treatment containing 1500 and 2000 μM sodium metasilicate was more effective in reducing disease severity by 77.9% and 76.4%, respectively, with no significant difference between the two treatments (Figure 8A). Additionally, the supplementation of potassium silicate also significantly reduced disease severity across treatments; however, its addition reduced the disease control compared to that of PMB05 alone (Figure 8B).

4. Discussion

Bacterial soft rot of cabbage caused by P. carotovorum subsp. carotovorum is a key limiting factor in global cabbage production [7,31]. While chemical pesticides are traditionally used for disease control, their overuse can lead to drug-resistant strains, encouraging the need for alternative strategies [2,32,33]. Among them, biological control is considered an environmentally friendly approach. Previous studies demonstrated that B. amyloliquefaciens PMB05 enhances PTI by intensifying ROS generation and callose deposition [8,9,11,16]. This allows B. amyloliquefaciens PMB05 to control multiple diseases, including bacterial soft rot, black rot, bacterial leaf spot, and bacterial wilt [2,4,8,12,14]. Thus, it is hypothesized that improving PMB05-induced defense efficiency could further enhance overall disease control. This study confirmed that B. amyloliquefaciens PMB05 can enhance PTI immune signals induced by P. carotovorum subsp. carotovorum elicitor HrpN in cabbage, as observed in Arabidopsis [12], speculating that its ability to control bacterial soft rot in cabbage is linked to intensified PTI responses.
In this study, the supplementation of sodium metasilicate was able to intensify the ROS generation and callose deposition produced by B. amyliquefaciens PMB05 in the presence of HrpN. Although our preliminary assays showed that silicate alone had no direct effect on P. carotovorum subsp. carotovorum PCCSB1, these results collectively suggest that sodium metasilicate may enhance the immune-priming effect of B. amyloliquefaciens PMB05 through intensified ROS generation, callose deposition, and other immune signaling pathways, increasing early PTI response (Figure 9) [34,35]. Similar mechanisms were observed in muskmelons, where sodium silicate stimulated superoxide anion and hydrogen peroxide accumulation during pathogen infection [18]. These enhanced responses may also be associated with PTI mechanisms involving phytohormone cross-talk [24]. Other biocontrol methods including Trichoderma species have demonstrated similar synergistic effects with its host plant, more specifically its involvement in the regulation of host-plant signal transduction pathways, where the cross-talk between ROS and hormones such as SA, JA, and ET is considerably important in plant disease resistance [36,37]. Application of silicates, such as sodium silicate and sodium metasilicate, has been reported to improve plant defense against pathogens and modulate soil microbial communities by supporting beneficial microbes while suppressing pathogens [38]. Specifically, sodium metasilicate has been reported to stimulate plant-growth-promoting and pathogen-inhibiting taxa, including Streptomyces, Cryptococcus, and Chaetomium [39]. Such microbial stimulation may contribute to improved biocontrol efficiency and strengthened plant resistance.
In contrast, the supplementation of potassium silicate to B. amyloliquefaciens PMB05 showed a suppressive or neutral effect on ROS generation and callose deposition, indicating that not all silicate forms provide the same benefit in microbial-mediated disease resistance. Silicon supplementation including potassium silicate can physically reinforce plant cell walls and improve resistance to stresses; however, the silicon effect on ROS generation and defense signaling varies with plant species and silicate form [22]. Previous studies have shown that potassium silicate enhances antioxidant enzyme activities, reduces oxidative damage markers such as ion leakage and malondialdehyde, and increases catalase activity, thereby maintaining redox homeostasis and mitigating oxidative stress [40,41]. This suggests that potassium silicate may lower ROS generation by enhancing antioxidant activity, potentially influencing ROS-mediated defense pathways. A study conducted on A. thaliana showed that potassium ion efflux was highly correlated with its induction of programmed cell death [29]. It is speculated that these contrasting effects between silicates may be attributed to the distinct physiological roles of Na+ and K+ ions in plant metabolism and signaling [40].
In plants, there are key ion transporters such as high-affinity K+ transporter (HKT), salt-overly-sensitive (SOS), and Arabidopsis K+ transporter (AKT) channels, which regulate the delicate balance of Na+ and K+ ions and membrane depolarization in plants [42,43,44]. HKT transporters control sodium uptake and exclusion, influencing membrane potential and interacting with potassium channels (AKT), while the SOS pathway helps remove excess Na+ to maintain ionic homeostasis. These ionic fluxes act upstream of downstream signaling cascades involving MAPK activation, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase-driven ROS bursts, and callose deposition, reinforcing PTI [45]. Thus, sodium metasilicate may enhance defense signaling by promoting stronger ionic currents that trigger these immune pathways. Additionally, although B. amyloliquefaciens PMB05 has not been confirmed to produce surfactin, iturin, or fengycin, these lipopeptides found in other Bacillus spp. are known to act as microbial elicitors and can stimulate host signaling networks [46]. The combination of microbial elicitors and silicate-induced modulation of ROS homeostasis may therefore intensify PTI signaling more than either treatment alone.
All treatments supplemented with sodium metasilicate were able to significantly reduce bacterial soft rot symptoms compared to P. carotovorum subsp. carotovorum treatment alone. Notably, the supplementation of 1500 and 2000 μM sodium metasilicate in the fermentation liquid reduced disease severity more effectively than plants treated with the fermentation liquid alone. In contrast, although all treatments supplemented with potassium silicate significantly reduced disease severity compared to the control, the extent of reduction was consistently lower than PMB05 fermentation liquid alone. The relationship between immune response markers and disease severity outcomes reinforces the functional role of ROS generation and callose deposition in cabbage resistance against P. carotovorum subsp. carotovorum. Biocontrol agents like Bacillus spp. are reported to suppress bacterial soft rot, but efficacy varies in the field [3,47]. Additionally, while the supplementation of potassium silicate can reduce disease severity in various plant types, including banana, strawberry, coffee, and onion [5,48,49,50], this study showed evidence that its activity to disrupt ROS/callose signaling can weaken plant immune activation leading to a limitation in full protection against diseases.
In this study, the supplementation of sodium metasilicate to B. amyloliquefaciens PMB05 fermentation liquid increases disease resistance to bacterial soft rot and can be a more suitable strategy to increase reliability and field applicability, though its performance may depend on soil pH, silicate stability, and environmental conditions. Practical implementation will require evaluation of cost-effectiveness and integration into existing cultivation systems. Further field trials will clarify whether the synergistic effects demonstrated in this study can be achieved at production scale. It is also important to highlight that the concentration-dependent effects further indicate that optimizing silicate dosage could maximize disease control.
Collectively, these results highlight the importance of the silicate source in modulating biocontrol efficacy. Sodium metasilicate synergizes with beneficial microbes to strengthen host resistance, whereas potassium silicate may attenuate microbe-induced defenses and compromise disease suppression.

5. Conclusions

This study shows that the supplementation of sodium metasilicate to B. amyloliquefaciens PMB05 fermentation liquid further enhances the bacterium’s ability to intensify plant immune responses and improve disease resistance. Supplementation of sodium metasilicate at 2000 μM enhanced ROS generation and callose deposition by 100% and 133%, respectively, compared to the treatment of PMB05 alone. It also decreased disease severity by 76.4%. Using this combined treatment could be especially valuable for crops like cabbage with limited resistant cultivars. Meanwhile, the supplementation of potassium silicate may reduce PMB05-induced defense responses, leading to reduced disease control efficacy. These findings highlight the importance of silicate source in modulating microbe-plant interactions. In response to the global need to reduce chemical use, sodium metasilicate could be supplemented to PMB05 fermentation liquid as a bio-enhancer, offering an effective and sustainable alternative for controlling bacterial soft rot in cabbage. Field application of this strategy warrants further investigation to evaluate its efficacy under diverse environmental conditions.

Author Contributions

Y.-H.L.: conceptualization, methodology, data curation, writing—review and editing, supervision, and funding acquisition; S.D.B.: methodology, software, formal analysis, investigation, writing—original draft preparation, and visualization; J.-R.L. and J.-C.Y.: software and formal analysis; T.-B.Y. and T.-P.H.: review and supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Science and Technology Council (NSTC), Taiwan, through grants (NSTC-113-2313-B-020-011) to Yi-Hsien Lin.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effect of B. amyloliquefaciens PMB05 bacterial suspension on HrpN-induced plant immune responses and disease control of bacterial soft rot in cabbage. Panel (A) reveals the fluorescent intensity of ROS under treatments with the inclusion/exclusion of HrpN. Panel (B) reveals the fluorescent intensity of callose deposition under treatments with the inclusion/exclusion of HrpN. Panel (C) reveals the effect of B. amyloliquefaciens PMB05 bacterial suspension on the control of bacterial soft rot disease caused by PCCSB1. To observe ROS generation, the infiltrated leaves were stained with H2DCFDA after 1 h post-infiltration. In the callose deposition assay, the infiltrated leaves were stained with aniline blue at 8 h post-infiltration. The disease severity was calculated at 48 h post-inoculation in 4-week-old cabbage seedlings after being treated with the bacterial suspension of PCCSB1 at 3 × 108 CFU/mL. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them (p < 0.05). The * indicates a significant difference compared with the blank treatment, as assessed using a t-test (p < 0.05). The yellow bar measures 20 μm in length.
Figure 1. Effect of B. amyloliquefaciens PMB05 bacterial suspension on HrpN-induced plant immune responses and disease control of bacterial soft rot in cabbage. Panel (A) reveals the fluorescent intensity of ROS under treatments with the inclusion/exclusion of HrpN. Panel (B) reveals the fluorescent intensity of callose deposition under treatments with the inclusion/exclusion of HrpN. Panel (C) reveals the effect of B. amyloliquefaciens PMB05 bacterial suspension on the control of bacterial soft rot disease caused by PCCSB1. To observe ROS generation, the infiltrated leaves were stained with H2DCFDA after 1 h post-infiltration. In the callose deposition assay, the infiltrated leaves were stained with aniline blue at 8 h post-infiltration. The disease severity was calculated at 48 h post-inoculation in 4-week-old cabbage seedlings after being treated with the bacterial suspension of PCCSB1 at 3 × 108 CFU/mL. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them (p < 0.05). The * indicates a significant difference compared with the blank treatment, as assessed using a t-test (p < 0.05). The yellow bar measures 20 μm in length.
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Figure 2. Effect of sodium metasilicate on ROS generation when treated with HrpN in cabbage. The assay was performed with the application of sodium silicate at 0, 250, 500, 750, 1000, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) is different concentrations of sodium metasilicate without HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). Panel (C) is different concentrations of sodium metasilicate with HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d, e) above them. The yellow bar measures 20 μm in length.
Figure 2. Effect of sodium metasilicate on ROS generation when treated with HrpN in cabbage. The assay was performed with the application of sodium silicate at 0, 250, 500, 750, 1000, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) is different concentrations of sodium metasilicate without HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). Panel (C) is different concentrations of sodium metasilicate with HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d, e) above them. The yellow bar measures 20 μm in length.
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Figure 3. Effect of potassium silicate on ROS generation when treated with HrpN in cabbage. The assay was performed with the application of potassium silicate at 0, 250, 500, 750, 1000, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) is different concentrations of potassium silicate without HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). Panel (C) is different concentrations of potassium silicate with HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d) above them. The yellow bar measures 20 μm in length.
Figure 3. Effect of potassium silicate on ROS generation when treated with HrpN in cabbage. The assay was performed with the application of potassium silicate at 0, 250, 500, 750, 1000, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) is different concentrations of potassium silicate without HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). Panel (C) is different concentrations of potassium silicate with HrpN and reveals the fluorescent intensity of ROS calculated by ImageJ (p < 0.05). The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d) above them. The yellow bar measures 20 μm in length.
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Figure 4. Effect of sodium metasilicate on B. amyloliquefaciens PMB05-intensified ROS generation upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of sodium metasilicate at 0, 500, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) reveals the fluorescent intensity of ROS by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of ROS by fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d) above them. The yellow bar measures 20 μm in length.
Figure 4. Effect of sodium metasilicate on B. amyloliquefaciens PMB05-intensified ROS generation upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of sodium metasilicate at 0, 500, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) reveals the fluorescent intensity of ROS by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of ROS by fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d) above them. The yellow bar measures 20 μm in length.
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Figure 5. Effect of sodium metasilicate on B. amyloliquefaciens PMB05-intensified callose deposition upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of sodium metasilicate at 0, 500, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of callose staining taken at 8 h post-infiltration. Panel (B) reveals the fluorescent intensity of callose deposition by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of callose deposition by fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d) above them. The yellow bar measures 20 μm in length.
Figure 5. Effect of sodium metasilicate on B. amyloliquefaciens PMB05-intensified callose deposition upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of sodium metasilicate at 0, 500, 1500, 2000, and 2500 μM of the final concentration. Panel (A) indicates the image of callose staining taken at 8 h post-infiltration. Panel (B) reveals the fluorescent intensity of callose deposition by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of callose deposition by fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c, d) above them. The yellow bar measures 20 μm in length.
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Figure 6. Effect of potassium silicate on B. amyloliquefaciens PMB05-intensified ROS generation upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of potassium silicate at 0, 500, 1000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) reveals the fluorescent intensity of ROS generation by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of ROS generation by the fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them. The yellow bar measures 20 μm in length.
Figure 6. Effect of potassium silicate on B. amyloliquefaciens PMB05-intensified ROS generation upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of potassium silicate at 0, 500, 1000, and 2500 μM of the final concentration. Panel (A) indicates the image of ROS staining taken at 1 h post-infiltration. Panel (B) reveals the fluorescent intensity of ROS generation by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of ROS generation by the fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them. The yellow bar measures 20 μm in length.
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Figure 7. Effect of potassium silicate on B. amyloliquefaciens PMB05-intensified callose deposition upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of potassium silicate at 0, 500, 1000, and 2500 μM of the final concentration. Panel (A) indicates the image of callose staining taken at 8 h post-infiltration. Panel (B) reveals the fluorescent intensity of callose deposition by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of callose deposition by fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them. The yellow bar measures 20 μm in length.
Figure 7. Effect of potassium silicate on B. amyloliquefaciens PMB05-intensified callose deposition upon HrpN treatment in cabbage. The concentration in the B. amyloliquefaciens PMB05 bacterial suspension is 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. The assay was performed with the application of potassium silicate at 0, 500, 1000, and 2500 μM of the final concentration. Panel (A) indicates the image of callose staining taken at 8 h post-infiltration. Panel (B) reveals the fluorescent intensity of callose deposition by bacterial suspension (p < 0.05) calculated by ImageJ. Panel (C) reveals the fluorescent intensity of callose deposition by fermentation liquid (p < 0.05) calculated by ImageJ. The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them. The yellow bar measures 20 μm in length.
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Figure 8. Effects of the supplementation of sodium metasilicate and potassium silicate to B. amyloliquefaciens PMB05 fermentation liquid on the biocontrol of bacterial soft rot disease in cabbage. The disease severity was calculated at 48 h post-inoculation in 4-week-old cabbage seedlings after being treated with the bacterial suspension of PCCSB1 at 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. Panel (A) shows the disease severity after the supplementation of sodium metasilicate at 0, 500, 1500, 2000, and 2500 μM of the final concentration in the fermentation liquid (p < 0.05). Panel (B) shows the disease severity after the supplementation of potassium silicate at 0, 500, 1000, and 2500 μM of the final concentration in the fermentation liquid (p < 0.05). The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them. The yellow bar measures 20 μm in length.
Figure 8. Effects of the supplementation of sodium metasilicate and potassium silicate to B. amyloliquefaciens PMB05 fermentation liquid on the biocontrol of bacterial soft rot disease in cabbage. The disease severity was calculated at 48 h post-inoculation in 4-week-old cabbage seedlings after being treated with the bacterial suspension of PCCSB1 at 3 × 108 CFU/mL. The concentration in the B. amyloliquefaciens PMB05 200-fold-dilution fermentation liquid is 106 CFU/mL. Panel (A) shows the disease severity after the supplementation of sodium metasilicate at 0, 500, 1500, 2000, and 2500 μM of the final concentration in the fermentation liquid (p < 0.05). Panel (B) shows the disease severity after the supplementation of potassium silicate at 0, 500, 1000, and 2500 μM of the final concentration in the fermentation liquid (p < 0.05). The calculations of the significant differences between the results were performed by using Tukey’s HSD test, which are indicated by the different letters (a, b, c) above them. The yellow bar measures 20 μm in length.
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Figure 9. An overview of the proposed synergistic mechanism between sodium metasilicate and B. amyloliquefaciens PMB05 in enhancing PTI. Upon PAMP recognition by protein receptors, early immune signaling pathways (MAPKs, NADPH, and RBOHD-mediated ROS generation) are rapidly activated [8,12]. This synergistic effect is proposed to upstream recognition and signaling events, thereby intensifying early PTI response. This study indicates that the improved plant resistance to bacterial soft rot resulting from the combined treatment is primarily associated with enhanced MAPK and ROS signaling, and callose deposition.
Figure 9. An overview of the proposed synergistic mechanism between sodium metasilicate and B. amyloliquefaciens PMB05 in enhancing PTI. Upon PAMP recognition by protein receptors, early immune signaling pathways (MAPKs, NADPH, and RBOHD-mediated ROS generation) are rapidly activated [8,12]. This synergistic effect is proposed to upstream recognition and signaling events, thereby intensifying early PTI response. This study indicates that the improved plant resistance to bacterial soft rot resulting from the combined treatment is primarily associated with enhanced MAPK and ROS signaling, and callose deposition.
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Blanco, S.D.; Li, J.-R.; Yan, J.-C.; Yen, T.-B.; Huang, T.-P.; Lin, Y.-H. Comparative Effects of Sodium Metasilicate and Potassium Silicate in Enhancing Bacillus amyloliquefaciens PMB05 Plant Immune Responses and Control of Bacterial Soft Rot in Cabbage. Agriculture 2025, 15, 2436. https://doi.org/10.3390/agriculture15232436

AMA Style

Blanco SD, Li J-R, Yan J-C, Yen T-B, Huang T-P, Lin Y-H. Comparative Effects of Sodium Metasilicate and Potassium Silicate in Enhancing Bacillus amyloliquefaciens PMB05 Plant Immune Responses and Control of Bacterial Soft Rot in Cabbage. Agriculture. 2025; 15(23):2436. https://doi.org/10.3390/agriculture15232436

Chicago/Turabian Style

Blanco, Sabrina Diana, Jia-Rong Li, Jo-Ching Yan, Tsair-Bor Yen, Tzu-Pi Huang, and Yi-Hsien Lin. 2025. "Comparative Effects of Sodium Metasilicate and Potassium Silicate in Enhancing Bacillus amyloliquefaciens PMB05 Plant Immune Responses and Control of Bacterial Soft Rot in Cabbage" Agriculture 15, no. 23: 2436. https://doi.org/10.3390/agriculture15232436

APA Style

Blanco, S. D., Li, J.-R., Yan, J.-C., Yen, T.-B., Huang, T.-P., & Lin, Y.-H. (2025). Comparative Effects of Sodium Metasilicate and Potassium Silicate in Enhancing Bacillus amyloliquefaciens PMB05 Plant Immune Responses and Control of Bacterial Soft Rot in Cabbage. Agriculture, 15(23), 2436. https://doi.org/10.3390/agriculture15232436

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