Inhibitory Effect of Bacillus velezensis dhm2 on Fusarium oxysporum f. sp. cucumerinum and Synergistic Activity of Crude Lipopeptide Extract with Chemical Fungicides

Abstract

Fusarium oxysporum f. sp. cucumerium, a resilient saprophytic fungus, poses a significant risk to cucumber crops. The research investigated the suppressive impact of Bacillus velezensis dhm2 on this pathogen and the synergistic performance of its crude lipopeptide extract with synthetic fungicides. Strain dhm2 inhibited the pathogen by 52.27% in confrontation culture. Its fermentation supernatant showed peak activity at 4 h bacterial age and 60 h fermentation duration, while the crude lipopeptide extract had an EC₅₀ of 9.99 g L⁻¹. Among the six chemical fungicides, prochloraz exhibited the highest toxicity, with an EC₅₀ value of 0.03 μg mL⁻¹. In all mixed combinations of the crude lipopeptide extract and chemical fungicides, there existed synergistic mixing ratios, particularly with difenoconazole (volume ratio 7:3, synergistic ratio 5.88) and propiconazole (7:3, 3.41), as confirmed by Wadley tests. Pot experiments revealed that the combined use of the crude lipopeptide extract and difenoconazole controlled cucumber Fusarium wilt by 80.95%. The mixture showed the highest SOD (315.76 U g⁻¹ FW min⁻¹), POD (281.63 U g⁻¹ FW min⁻¹), and CAT (23.39 U g⁻¹ FW min⁻¹), with increases over single treatments. This study provides an eco-friendly strategy for managing cucumber wilt, advocating reduced fungicide use via synergistic formulations.

1. Introduction

Cucumber Fusarium wilt, a systemic and polycyclic epidemic disease, presents a major obstacle in cucumber cultivation [1]. The causal agent, F. oxysporum f. sp. cucumerinum, is a soil-borne fungus that invades roots, quickly propagates through vascular bundles, obstructs xylem vessels, impedes water and nutrient transport, and secretes toxins such as fusaric acid, leading to rapid wilting and plant death [2]. Incidence rates of Fusarium wilt vary from 30% to 70% in both open-field and protected cultivation systems. The adoption of protected cultivation methods has facilitated year-round cucumber production, resulting in the buildup of pathogen inoculum, hastening disease dissemination, and potentially causing crop failure in severe instances, thereby imposing significant economic burdens [3]. Due to its typical soil-borne characteristics, managing Fusarium wilt in cucumbers remains challenging and poses a pressing industry dilemma. Hence, the development of effective control strategies is imperative to ensure the sustainable progress of the cucumber sector.

The main strategies for managing Fusarium wilt in cucumber cultivation encompass grafting, crop rotation, and the application of chemical pesticides. Grafting, although effective, is labor-intensive and expensive. Crop rotation, while advantageous for disease control, encounters limited adoption due to financial constraints. Chemical pesticides are currently the primary approach for managing Fusarium wilt in cucumbers [4]. Chemical pesticides, notably carbendazim, thiophanate-methyl, hymexazol, difenoconazole, propiconazole, tebuconazole, and fludioxonil, are commonly utilized to control Fusarium wilt in cucumbers. Fumigants like metam-sodium, chloropicrin, and metham-sodium are also employed [5]. However, the extensive use of these highly toxic fumigants can lead to the significant disruption of soil microecology, resulting in alterations to microbial community structures, decreased bacterial diversity in the cucumber rhizosphere, reduced populations of beneficial Bacillus species, and decreased cucumber resistance to Fusarium wilt [6]. Consequently, reducing reliance on chemical pesticides, eliminating highly toxic fumigants, and developing more effective control measures have become essential for managing cucumber Fusarium wilt. Current approaches to minimize pesticide use include biological control methods, the development of high-efficiency single- and mixed-pesticide formulations, and precision application techniques. Among these, biocontrol agents and their metabolites offer distinct advantages, including safety, diverse sources, and long-term disease suppression. These benefits make biocontrol increasingly important for reducing fungicide dependence, restoring ecological balance, and ensuring agricultural safety [7].

Effective and enduring antagonistic bacteria are pivotal for biologically managing plant diseases and utilizing their fermentation byproducts [8]. Various biocontrol strains have been isolated from different environments, showing diverse control efficacies against cucumber Fusarium wilt. Research indicates that biocontrol bacteria can impact the activity of defense enzymes in cucumber plants as well as soil enzymes. For example, the application of B. subtilis strain B579 via seed soaking and root irrigation can suppress cucumber Fusarium wilt effectively [9]. Likewise, B. subtilis strain 1JN2 impedes the pathogen growth causing cucumber Fusarium wilt, modulates rhizosphere fungal diversity in favor of cucumber plants, and boosts soil enzyme activity [10]. Additionally, B. subtilis JNF2, isolated from a Fusarium wilt-prone region, and B. subtilis YB-04, obtained from wheat straw, both demonstrate pathogen suppression capabilities and trigger the upregulation of multiple defense enzymes in cucumber plants [11,12]. Moreover, multiple strains of Paenibacillus polymyxa have demonstrated biocontrol potential against cucumber Fusarium wilt. Research indicates that strains such as HX-140 (isolated from rapeseed rhizosphere), endophytic strain Y-4, hg18, and PJH16 (obtained from healthy cucumber rhizospheres) inhibit pathogen invasion through the production of active metabolites or the enhancement of defense enzyme activity, thereby exhibiting biocontrol effects [13,14,15,16]. While numerous studies have explored the screening of biocontrol agents against cucumber Fusarium wilt and their influence on host physiological and biochemical traits, strains exhibiting strong efficacy in laboratory and pot experiments often underperform under field conditions. This discrepancy arises primarily from environmental variables, including soil physicochemical properties, crop variety and growth stage, and competition with indigenous microorganisms. Furthermore, the absence of suitable carriers or inadequate nutrient supply can significantly reduce the survival and proliferation of biocontrol agents in soil, ultimately compromising their efficacy.

Biocontrol bacterial metabolites can serve as biogenic fungicides or be incorporated into mixed formulations with conventional fungicides to improve the stability of live biocontrol agents. Bacillus spp. produce bioactive lipopeptide compounds, which function as metabolites of plant disease-antagonistic bacteria. Extensive research has documented their inhibitory effects on plant pathogens. Noh et al. reported that component analysis of the lipopeptides produced by the isolated B. subtilis NM4 revealed that fengycin was the main active component. Its combination with chlorothalonil could significantly enhance antimicrobial efficacy and induce hyphal swelling and the formation of chlamydospores [17]. However, no studies have systematically evaluated combined formulations integrating lipopeptide antifungal substances with chemical fungicides for the control of cucumber Fusarium wilt. However, no research has systematically assessed combined formulations that merge lipopeptide antifungal compounds with chemical fungicides to inhibit the pathogen and manage cucumber Fusarium wilt [18]. Our hypothesis was that the crude lipopeptide extract from B. velezensis dhm2 could boost the effectiveness of chemical fungicides in combating cucumber Fusarium wilt, while also decreasing the reliance on fungicides. To test this hypothesis, the specific objectives of this study were as follows: (1) to assess antagonistic activity of strain dhm2 via plate confrontation assays, (2) to evaluate the antifungal activity of its fermentation supernatant under different conditions, (3) to determine the antimicrobial efficacy of the crude lipopeptide extract, (4) to screen for synergistic interactions between the crude lipopeptide extract and common chemical fungicides against the pathogen, and (5) to examine the effect of the most promising synergistic combination on defense-related enzyme activities (SOD, POD, CAT) in cucumber plants. The results aim to provide novel technical strategies for enhancing cucumber Fusarium wilt management through synergistic applications while reducing reliance on chemical fungicides.

2. Materials and Methods

This study was conducted at the Disease Prevention and Growth Promotion Microbial Fertilizer Laboratory, Anhui Science and Technology University (Geographical coordinates: 32°50′45″ N, 117°33′13″ E). Located in the transitional area between northern and southern Chinese climates. The experiments took place between March 2024 and March 2025.

2.1. Materials

2.1.1. Test Reagents

The technical-grade pesticides, carbendazim (98.0%), difenoconazole (95.0%), propiconazole (95.0%), azoxystrobin (95.0%), thiram (97.5%), and prochloraz (96.0%), were supplied by Jiangsu Fengdeng Crop Protection Co., Ltd. Stock solutions (5.0 × 10³ μg mL ⁻¹) were prepared by dissolving carbendazim in 1 mol L⁻¹ HCl and the other fungicides in acetone. TangShan QiuGua cucumber seeds were acquired from Cangzhou Jinkelifeng Seedling Co., Ltd. in Cangzhou city, China.

2.1.2. Test Strains

The pathogen of cucumber wilt (F. oxysporum f. sp. cucumerinum)was obtained from the Disease Prevention and Growth Promotion Microbial Fertilizer Laboratory at Anhui Science and Technology University. The biocontrol bacteria Bacillus velezensis dhm2 was isolated from soil samples taken at the Radar Hill Planting Base in Fucheng Town, Fengyang County, Anhui Province, China.

2.1.3. Test Media

Table 1. Components of the test media.

Test Media	Components
PDA	Potato 200.0 g, glucose 18.0 g, agar 15.0 g, distilled water 1000 mL.
NA	Tryptone 5.0 g, beef extract 3.0 g, yeast extract 1.0 g, glucose 10.0 g, 15.0 g agar, distilled water 1000 mL, pH 7.0–7.2. NB medium without agar.

presents the composition and preparation method of the culture media (PDA, NA and NB), the sterilization process was consistent with the method detailed by Meng et al. [19].

2.2. Methods

2.2.1. Confrontation Culture Assay Between B. velezensis dhm2 and the Pathogen

B. velezensis strain dhm2 was streaked onto NA medium and incubated at 26 °C for 48 h. A single colony was subsequently transferred to the center of a PDA plate and incubated at aforementioned temperature for a further 24 h. Thereafter, four 7 mm diameter fungal disks of the cucumber Fusarium wilt pathogen, pre-incubated at 26 °C for 96 h, were inoculated at right angles in a crosswise arrangement, each positioned 2.0 cm from the plate center. Parallel control plates lacking central inoculation were also established. The antifungal inhibition rate was determined once the control colonies had fully overgrown the plate, using Formula (1) [20].

$$\mathrm{Antifungal} \mathrm{rate} \left(\%\right)={\displaystyle \frac{\mathrm{Control} \mathrm{colony} \mathrm{diameter}–\mathrm{Treatment} \mathrm{colony} \mathrm{diameter}}{\mathrm{Control} \mathrm{colony} \mathrm{diameter}–7 \mathrm{mm}}}\times 100$$

(1)

2.2.2. Assessment of Supernatant Activity from Seed Inoculum of B. velezensis dhm2 with Different Bacterial Ages Against F. oxysporum f. sp. cucumerinum

(1) Seed inoculum preparation: A single colony was transferred into a 10 mL NB medium tube (18 × 180 mm) and subcultivated at 33 °C with shaking at 140 rpm for 12 h to establish the primary seed inoculum. Thereafter, the primary seed culture was inoculated at a 10% volume ratio into fresh 10 mL NB medium tubes (18 × 180 mm) and incubated for 4, 6, 8, 10, 12, and 14 h under identical conditions. Post-incubation, bacterial cells were harvested by centrifugation at 10,000 rpm and 4 °C for 20 min. The resultant cell suspension was resuspended in sterile water to adjust the optical density at 600 nm to 1.0, generating seed cultures of distinct bacterial ages.

(2) Fermentation supernatant preparation: The Seed inoculum was inoculated at a 0.5% volume ratio into 250 mL Erlenmeyer flasks containing 100 mL of NB medium and shaken for 72 h (consistent with seed inoculum culture conditions). After fermentation, the culture was centrifuged at 10,000 rpm for 20 min at 4 °C to acquire the supernatant. The supernatant was then filtered through a 0.22 μm membrane and stored at 4 °C for subsequent use.

(3) Inhibition rate determination: Mycelial growth rate method was employed [21]. 5 mL aliquot of fermentation supernatant from cultures of different bacterial ages was mixed with 45 mL of molten PDA medium to prepare agar plates. After incubating at 26 °C for 120 h, colony diameters were measured via the cross method. The inhibition rate was calculated using the formula in Section 2.2.1. Each bacterial age treatment for the seed inoculum was replicated in triplicate.

2.2.3. Determination of the Inhibitory Activity of Fermentation Supernatants from B. velezensis dhm2 at Different Fermentation Duration Against the Pathogen

The flasks were incubated with agitation as described 2.2.2 for 24, 48, 60, 72, 84, and 96 h. Following incubation, the cultures were centrifuged and filtered to collect fermentation supernatant. Their inhibitory activity was assessed against the pathogen by determining the inhibition rates using the formula provided in Section 2.2.1. Each fermentation duration was repeated in triplicate.

2.2.4. Determination of the Inhibitory Activity of Fermentation Supernatant at Different Concentration Against the Pathogen

The fermentation supernatant was diluted to the required concentrations using sterile water and the final concentrations of 200.0, 100.0, 66.7, 50.0, 25.0 and 16.7 mL L⁻¹. The following was performed in accordance with the method outlined in Section 2.2.2. Three replicates were set up for each concentration of the fermentation supernatant. The EC₅₀ value of the fermentation supernatant against the pathogen was calculated using SPSS 26.0 software.

2.2.5. Preparation and Inhibitory Activity Assay of Crude Lipopeptide Extract

Following the protocol of Wen et al. [22], the concentration of the crude lipopeptide extract was quantified as 8.65 × 10⁵ μg mL⁻¹. For bioactivity assessment, the final concentrations of crude lipopeptide extract were 0.70, 1.10, 2.20, 4.30, 8.70, and 17.40 g L⁻¹. The following method was described in Section 2.2.2. Each concentration of the fermentation supernatant was tested in triplicate to evaluate the inhibitory effects on the pathogen, and the EC₅₀ value was determined using SPSS 26.0 software. Additionally, mycelia were separately picked from the edge of the pathogen colonies in the control group and those treated withcrude lipopeptide extract(17.4 g L⁻¹) for 120 h. Microscopic photography was performed using an Olympus biological microscope (Model: BX53F; Olympus Corporation, Hachioji-shi, Tokyo, Japan) to compare the differences in mycelial morphology between the control and crude lipopeptide extract-treated groups.

2.2.6. In Vitro Toxicity Determination of Six Chemical Fungicides Against the Pathogen

Inhibitory effects of six fungicides (prochloraz, propiconazole, difenoconazole, azoxystrobin, carbendazim, and thiram) on Fusarium wilt pathogens in cucumber were evaluated using the methodology described in Section 2.2.2. Serial concentrations of each fungicide were tested to determine their inhibitory activities, and inhibition rates were calculated (

Table 2. Concentration gradients of six chemical fungicides.

Fungicide	Concentration (μg mL−1)
Prochloraz	0.02	0.04	0.08	0.16	0.32	0.48
Difenoconazole	0.04	0.08	0.16	0.32	0.64	1.28
Propiconazole	0.10	0.20	0.40	0.80	1.60	2.40
Carbendazim	0.30	0.40	0.50	0.60	0.70	0.80
Thiram	2.00	4.00	8.00	16.00	24.00	32.00
Azoxystrobin	8.00	16.00	32.00	64.00	128.00	200.00

). Each dosage of each chemical fungicide was replicated three times. Using SPSS 26.0 software, the EC₅₀ values, toxicity regression equations, 95% confidence intervals, and R² values for all six fungicides were derived.

2.2.7. Determination of Toxicity Synergy Between Combinations of Six Chemical Fungicides and Crude Lipopeptide Extract Against the Pathogen

The combination test of crude lipopeptide extract (A) from B. velezensis dhm2 and chemical fungicides (B) was conducted following Horsfall’s method [23]. Mixed gradients were established based on EC₅₀ ratios, with synergistic effects assessed by comparing observed inhibition rates to theoretical values. Eleven ratios were tested according to EC₅₀ dose proportions: 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. Sterile water served as the control, with three replicates per treatment. The inhibitory effects of different mixtures were determined as described in Section 2.2.2. The toxicity ratios were calculated for each combination following Equations (2) and (3). Synergy is observed if the toxicity ratio (TR) surpasses 1.00; additivity if TR equals 1.00, and antagonism if TR is less than 1.00.

$$\mathrm{Theoretical} \mathrm{inhibition} \mathrm{rate} \left(\%\right)=[ {\mathrm{EC}}_{50}\mathrm{of} \mathrm{A} \times \mathrm{proportion} \mathrm{of} \mathrm{A} + {\mathrm{EC}}_{50} \mathrm{of} \mathrm{B} \times \mathrm{proportion} \mathrm{of} \mathrm{B} ]\times 100$$

(2)

$$\mathrm{TR}={\displaystyle \frac{\mathrm{Actual} \mathrm{inhibition} \mathrm{rate}}{\mathrm{Theoretical} \mathrm{inhibition} \mathrm{rate}}}$$

(3)

Crude lipopeptide extract (A) with difenoconazole (7:3) and propiconazole (7:3) (designated as B) were assessed using the Wadley formula to determine the synergistic ratio. The Equations (4) and (5) were utilized for calculations, with a and b represent the mass ratios of agents A and B, respectively. EC₅₀ (th) refers to the theoretical EC₅₀ value of the mixture, while EC₅₀ (ob) denotes the measured EC₅₀ value. A synergistic ratio (SR) < 0.5 indicates an antagonistic effect, 0.5 ≤ SR ≤ 1.5 indicates an additive effect, and SR > 1.5 indicates a synergistic effect [24].

$${\mathrm{EC}}_{50}\left(\mathrm{th}\right)={\displaystyle \frac{\mathrm{a}+\mathrm{b}}{\frac{\mathrm{a}}{{\mathrm{EC}}_{50}\mathrm{A}}+\frac{\mathrm{b}}{{\mathrm{EC}}_{50}\mathrm{B}}}}$$

(4)

$$\mathrm{SR}={\displaystyle \frac{{\mathrm{EC}}_{50}\left(\mathrm{th}\right)}{{\mathrm{EC}}_{50}\left(\mathrm{ob}\right)}}$$

(5)

2.2.8. Pot Assay of Synergistic Combinations for the Control Effect of Cucumber Fusarium Wilt

EC₈₀ values for crude lipopeptide extract, difenoconazole, and propiconazole were determined as 37.52 g L⁻¹, 5.46 mg L⁻¹, and 2.61 mg L⁻¹, respectively, to evaluate their single and combined effects. Cucumber seeds were germinated, and the etiolated seedlings were transplanted into pots (16.0 cm diameter × 14.0 cm height) containing a sterilized soil–substrate mixture (2:1 dry soil to substrate mass ratio). Soil characteristics included organic matter (17.2 g kg⁻¹), total nitrogen (1.0 g kg⁻¹), available phosphorus (12.0 mg kg⁻¹), available potassium (110.0 mg kg⁻¹), and a pH of 6.2. The pots were maintained in a growth chamber under controlled conditions: 12 h light (6000 lx, 26 °C)/12 h dark (20 °C) with 80% relative humidity. On day 9, when the cucumber seedlings had fully expanded cotyledons, a F. oxysporum spore suspension (10⁷ conidia mL⁻¹) was prepared using hemocytometer counting, and 50 mL was applied to each pot. After 24 h, 50 mL of each fungicide (single agent or synergistic combination) was administered, with distilled water as the control. The seedlings were then cultured for an additional 7 d before efficacy assessment. Each treatment consisted of 7 pots and was replicated three times. Disease ratio and control effect were calculated using the Formulas (6) and (7).

$$\mathrm{Disease} \mathrm{ratio} \left(\%\right)={\displaystyle \frac{\mathrm{Number} \mathrm{of} \mathrm{diseased} \mathrm{cucumber} \mathrm{seedlings}}{\mathrm{Total} \mathrm{number} \mathrm{of} \mathrm{cucumber} \mathrm{seedlings} \mathrm{tested}}}\times 100$$

(6)

$$\mathrm{Control} \mathrm{effect}(\%)={\displaystyle \frac{\mathrm{Control} \mathrm{disease} \mathrm{ratio}-\mathrm{Treatment} \mathrm{disease} \mathrm{ratio}}{\mathrm{Control} \mathrm{disease} \mathrm{ratio}}}\times 100$$

(7)

2.2.9. Effects of the Optimal Lipopeptide-Difenoconazole Combination on Defense Enzyme Activities in Cucumber Seedlings

In accordance with the methodology detailed in Section 2.2.8, the impact of difenoconazole, crude lipopeptide extract, and their optimal mixture on the activities of the defense enzymes (SOD, POD, and CAT) in cucumber seedlings was assessed. Enzyme activity measurements were conducted at 24, 48, 60, 72, and 84 h post-irrigation with Fusarium wilt spore suspension, following the protocol established by Gao and Cai [25]. A distilled water treatment was used as the control. Each time point was replicated thrice in the experiment.

2.3. Data Analysis

All experiments were conducted with three biological replicates, and data were expressed as mean ± standard error (SE). Statistical analyses were performed using SPSS 26.0 software (IBM Corp., Armonk, NY, USA). Significant differences between treatments were determined by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test, with a significance level set at p< 0.05.

3. Results

3.1. Results of Confrontation Culture Between B. velezensis dhm2 and Cucumber Fusarium Wilt Pathogen

Figure 1 depicts the experimental arrangement in which B. velezensis dhm2 was initially inoculated and incubated for 24 h. Subsequently, it was co-cultured with the cucumber Fusarium wilt pathogen for 120 h. The control group exhibited complete coverage of the plate by the cucumber Fusarium wilt pathogen. In contrast, the co-culture of dhm2 and the pathogen demonstrated inhibition of pathogen growth by dhm2, thereby impeding further expansion. This observation highlights the effective control exerted by B. velezensis strain on the pathogen, resulting in an inhibition rate of 52.27% ± 5.15%.

3.2. Inhibitory Activity of Fermentation Supernatant from Seed Inoculum of B. velezensis dhm2 with Different Bacterial Ages Against the Pathogen

Figure 2 demonstrates a decrease in the inhibitory effect of the fermentation supernatant from dhm2 on the pathogen, with an increase in inoculum age. The highest inhibition rate of 53.70% was observed at a bacterial age of 4 h (p < 0.05). This suggests that as the age of the seed liquid increases, the vitality of bacterial cells gradually diminishes, resulting in a reduction in the production of antifungal compounds. Therefore, under the specified culture conditions, 4 h was determined to be the optimal bacterial age for maximizing the inhibitory effect of the fermentation supernatant.

3.3. Inhibitory Activity of Fermentation Supernatants Against the Pathogen at Different Culture Time

As depicted in Figure 3, the inhibitory efficacy of the fermentation supernatant against F.oxysporum f. sp. cucumerinum exhibited a biphasic trend with prolonged fermentation time. The peak inhibition rate of 66.00% occurred at 60 h (p < 0.05), followed by a gradual decline in inhibition rate. These findings suggest that the optimal production of antibacterial compounds necessitates a specific fermentation duration.

3.4. Inhibitory Activity of Fermentation Supernatants at Different Concentration Against the Pathogen

Figure 4 depicts a notable increase in the in vitro inhibition rate of F. oxysporum f. sp. cucumerinum with the escalating concentration of the fermentation supernatant from B. velezensis dhm2, ranging from 16.70 mL L⁻¹ to 200.00 mL L⁻¹, resulting in an increase from 30.43% to 71.36%. Using SPSS 26.0, a virulence regression equation was established against the pathogen as y = 1.01x + 3.34 (R² = 0.96), yielding an EC₅₀ of 43.53 mL L⁻¹ with a 95% confidence interval of 37.58−49.60 mL L⁻¹.

3.5. Inhibitory Activity of Crude Lipopeptide Extract at Different Concentration Against the Pathogen

Figure 5 illustrates a dose-dependent correlation between the concentration of the crude lipopeptide extract and its inhibition rate against F. oxysporum f. sp. cucumerinum, with the inhibition rate ranging from 7.12% to 72.60% as the concentration increased from 0.70 g L⁻¹ to 17.40 g L⁻¹. Figure 6 demonstrates a significant reduction in the mycelial growth of F. oxysporum f. sp. cucumerinum following exposure to the crude lipopeptide extract; notably, at a high concentration of 17.40 g L⁻¹, noticeable changes in the pathogen’s colony morphology and color were observed, indicating suppressed growth. Using SPSS 26.0 software, the virulence regression equation of the crude lipopeptide extract against the pathogen was determined as y = 1.46x + 3.54 (R² = 0.95), with the EC₅₀ calculated to be 9.99 g L⁻¹ and a 95% confidence interval ranging from 6.33 to 22.05 g L⁻¹. As observed in the micrographs of Figure 6, the mycelia in the control group exhibited a relatively smooth surface, uniform thickness, and elasticity, with evenly distributed protoplasm within the mycelia, minimal pigment deposition. In contrast, after treatment with the crude lipopeptide extract at 17.40 g L⁻¹, the mycelia exhibited rough and uneven surfaces with extensive pigment deposition, accompanied by phenomena such as swelling, constriction, and even breakage. Growth of branched mycelia was inhibited, and some apical growth points of the mycelia showed enlargement, with a large number of chlamydospores, presenting relatively severe mycelial damage and deformation (Figure 6).

3.6. Inhibitory Activity of Six Chemical Fungicides Against the Pathogen

Table 3. Toxicity assay of six chemical fungicides against F. oxysporum f. sp. cucumerinum.

Fungicide	Toxicity Regression Equation *	EC50 (μg mL−1)	95% Confidence Interval	R2
Prochloraz	y = 0.88x + 6.37	0.03	0.01–0.06	0.99
Difenoconazole	y = 0.66x + 5.36	0.28	0.14–0.70	0.98
Propiconazole	y = 0.98x + 5.43	0.36	0.12–0.72	0.98
Carbendazim	y = 4.66x + 5.79	0.68	0.58–0.87	0.97
Thiram	y = 0.71x + 4.39	7.10	2.60–13.52	0.95
Azoxystrobin	y = 2.35x + 0.72	65.88	47.56–94.43	0.95

* x denotes the logarithmic value of chemical fungicide concentration, whereas y denotes the probit value.

illustrates the susceptibility of F. oxysporum f. sp. cucumerinum to ergosterol biosynthesis inhibitor (EBI) fungicides, such as prochloraz, propiconazole, difenoconazole, and the benzimidazole fungicide carbendazim. Prochloraz exhibited the most potent activity against the pathogen, with an EC₅₀ of 0.03 μg mL ⁻¹, followed by difenoconazole, propiconazole, and carbendazim, with EC₅₀ of 0.28 μg mL ⁻¹, 0.36 μg mL ⁻¹, and 0.68 μg mL ⁻¹, respectively. Thiram, an organosulfur fungicide, exhibited an EC₅₀ of 7.10 μg mL⁻¹, whereas the strobilurin fungicide azoxystrobin displayed weak inhibitory effects, with an EC₅₀ of 65.88 μg mL⁻¹.

3.7. Toxicity Ratios of the Mixed Combinations Between Crude Lipopeptide Extract from Antagonistic Strain dhm2 and Chemical Fungicides

As shown in

Table 4. Toxicity ratios of different mixing proportions between crude lipopeptide extract from antagonistic strain dhm2 and prochloraz, difenoconazole, propiconazole, azoxystrobin, carbendazim, and thiram against F. oxysporum f. sp. cucumerinum.

Fungicide	Mixing Proportion (Volume Ratio)
	0:10	1:9	2:8	3:7	4:6	5:5	6:4	7:3	8:2	9:1	10:0
Prochloraz (ob%) *	51.8 ± 0.50 de	40.9 ± 1.00 g	45.07 ± 0.30 f	51.83 ± 0.98 de	54.08 ± 0.56 cd	61.41 ± 2.20 a	56.62 ± 0.56 bc	55.21 ± 0.49 c	57.18 ± 0.28 bc	59.15 ± 1.29 ab	50.70 ± 1.02 e
Prochloraz (TR) #	1.00 ± 0.01 e	0.79 ± 0.02 g	0.87 ± 0.03 f	1.01 ± 0.02 e	1.05 ± 0.01 de	1.20 ± 0.04 a	1.11 ± 0.01 bcd	1.08 ± 0.01 cd	1.12 ± 0.01 bc	1.16 ± 0.02 ab	1.00 ± 0.02 e
Difenoconazole (ob%)	53.94 ± 0.55 d	45.92 ± 1.69 f	49.86 ± 0.28 e	50.14 ± 0.01 de	57.92 ± 0.73 c	60.00 ± 0.28 b	61.41 ± 0.75 ab	62.54 ± 1.22 a	60.56 ± 0.56 a	53.24 ± 1.13 c	48.17 ± 0.75 d
Difenoconazole (TR)	1.00 ± 0.01 d	0.86 ± 0.03 f	0.94 ± 0.01 e	0.96 ± 0.02 de	1.12 ± 0.03 c	1.18 ± 0.01 b	1.22 ± 0.02 ab	1.25 ± 0.04 a	1.23 ± 0.02 a	1.09 ± 0.04 c	1.00 ± 0.03 d
Propiconazole (ob%)	53.33 ± 0.33 e	46.11 ± 0.88 g	47.50 ± 0.33 g	60.28 ± 0.88 bc	60.83 ± 0.58 ab	63.05 ± 0.58 a	60.83 ± 1.16 ab	63.05 ± 0.26 a	58.89 ± 0.67 cd	57.50 ± 0.33 d	51.67 ± 0.58 f
Propiconazole (TR)	1.00 ± 0.01 d	0.87 ± 0.01 e	0.90 ± 0.01 e	1.15 ± 0.01 bc	1.16 ± 0.01 bc	1.21 ± 0.02 a	1.17 ± 0.02 b	1.22 ± 0.01 a	1.15 ± 0.01 bc	1.13 ± 0.01 c	1.00 ± 0.01 d
Azoxystrobin (ob%)	47.93 ± 1.00 cd	51.24 ± 0.58 b	54.82 ± 0.33 a	50.69 ± 0.88 b	54.55 ± 0.33 a	56.20 ± 0.21 a	48.20 ± 0.67 c	47.66 ± 0.88 cde	43.80 ± 0.88 f	45.73 ± 0.33 e	46.01 ± 0.58 de
Azoxystrobin (TR)	1.00 ± 0.02 c	1.07 ± 0.01 b	1.15 ± 0.01 a	1.07 ± 0.02 b	1.16 ± 0.01 a	1.20 ± 0.03 a	1.03 ± 0.01 bc	1.02 ± 0.02 c	0.94 ± 0.02 d	0.99 ± 0.01 c	1.00 ± 0.02 c
Carbendazim (ob%)	52.50 ± 0.48 bc	48.61 ± 0.21 d	45.56 ± 0.87 e	53.61 ± 1.03 b	59.44 ± 1.00 a	61.39 ± 0.28 a	62.22 ± 1.21 a	61.67 ± 0.96 a	61.11 ± 1.19 a	53.33 ± 0.15 bc	50.83 ± 0.48 cd
Carbendazim (TR)	1.00 ± 0.01 c	0.93 ± 0.02 d	0.87 ± 0.01 e	1.03 ± 0.01 c	1.15 ± 0.02 b	1.19 ± 0.01 ab	1.21 ± 0.03 a	1.20 ± 0.02 a	1.19 ± 0.02 ab	1.05 ± 0.01 c	1.00 ± 0.01 c
Thiram (ob%)	52.89 ± 1.15 b	61.43 ± 0.88 a	60.33 ± 2.02 a	54.82 ± 0.00 b	50.13 ± 1.16 c	55.64 ± 0.33 b	47.66 ± 1.45 c	45.73 ± 0.67 de	42.42 ± 0.33 f	43.25 ± 0.67 ef	46.01 ± 0.58 de
Thiram (TR)	1.00 ± 0.02 c	1.18 ± 0.02 a	1.17 ± 0.04 a	1.08 ± 0.01 c	1.00 ± 0.02 c	1.13 ± 0.01 b	0.98 ± 0.03 cd	0.95 ± 0.02 cde	0.90 ± 0.01 e	0.93 ± 0.01 de	1.00 ± 0.02 c

(ob%) *: denotes the actual inhibition rate; (TR) ^#: denotes the toxicity ratio. Data in each row are alphabetically labeled from “a” to a maximum of “g”, indicating descending order. The label “a” represents the highest inhibition rate or toxicity ratio, signifying the most effective combination in the mixed trials series. Additionally, it shows a significant distinction from other treatments at the 0.05 significance level.

, the optimal combinations were identified using the Horsfall method. The mixture of crude lipopeptide extract and prochloraz/azoxystrobin at a 5:5 ratio demonstrated the highest toxicity ratio (TR) of 1.20. Similarly, combinations with difenoconazole/propiconazole at a 7:3 ratio yielded the highest TR values of 1.25 and 1.22, respectively. The mixture with carbendazim at a 6:4 ratio achieved a TR of 1.21, while the combination with thiram at a 1:9 ratio exhibited a TR of 1.18. Therefore, the combinations of crude lipopeptide extract with difenoconazole/propiconazole at a 7:3 ratio were selected for subsequent Wadley tests.

Based on the Horsfall screening results, the crude lipopeptide extract–fungicide combinations demonstrating relatively high toxicity were selected for subsequent Wadley testing in combination with propiconazole and difenoconazole, respectively. As presented in Figure 7 and Figure 8, when the concentration of the optimal crude lipopeptide extract–propiconazole combination increased from 500 to 4000 μg mL⁻¹, the inhibition rates against F. oxysporum f. sp. cucumerinum ranged from 16.81% to 66.40%. Using the Wadley formula, the theoretical EC₅₀ value of the crude lipopeptide extract–propiconazole mixture was determined to be 6993.08 μg mL⁻¹. The actual toxicity regression equation for the mixture against F. oxysporum f. sp. cucumerinum, calculated using SPSS 26.0, was y = 1.67x − 0.52 (R² = 0.97), yielding an actual EC₅₀ value of 2052.21 μg mL⁻¹. The SR of 3.41 (>1.50) confirmed a synergistic effect.

According to the Wadley test, when the concentration of the optimal combination of crude lipopeptide extract and difenoconazole increased from 500 to 4000 μg mL⁻¹, the inhibition rates against F. oxysporum f. sp. cucumerinum ranged from 26.86% to 79.17% (Figure 7 and Figure 9). The theoretical EC₅₀ of the mixture was 6993.12 μg mL⁻¹. Based on experimental measurements, the toxicity regression equation of the mixture against F. oxysporum f. sp. cucumerinum, calculated using SPSS 26.0 software, was y = 1.47x + 0.49 (R² = 0.99). The actual EC₅₀ was 1190.26 μg mL⁻¹, with SR of 5.88 (>1.50), confirming a synergistic effect.

3.8. Control Efficacy of Optimized Combinations Against Cucumber Fusarium Wilt in Pot Trial

Root drenching experiments were performed 24 h post pre-inoculation with a spore suspension of F. oxysporum f. sp. cucumerinum, utilizing various treatments, including individual agents and optimized mixed combinations, as detailed in

Table 5. Control effect of different treatments on cucumber Fusarium wilt in pot trail.

Item	Control Effect (%) *
Crude lipopeptide extract	39.29 ± 3.60 d
Propiconazole	42.86± 5.83 d
Difenoconazole	53.57 ± 3.57 c
Mixture of crude lipopeptide extract and Propiconazole	64.29 ± 4.13 b
Mixture of crude lipopeptide extract and Difenoconazole	80.85 ± 2.76 a

* The letters “a” to “d” assigned to the data indicate a descending order of control efficacy. The combined application of crude lipopeptide extract and difenoconazole demonstrated the highest inhibition rate, marked as “a”, with statistically significant differences compared to other treatments (p < 0.05). In contrast, the crude lipopeptide extract alone showed the lowest efficacy, labeled as “d”, and exhibited no significant difference from propiconazole alone (p > 0.05).

and Figure 10. The findings revealed that the combined application of crude lipopeptide extract and difenoconazole resulted in a notable 80.95% efficacy in controlling cucumber Fusarium wilt, demonstrating the highest survival rate of cucumber seedlings (p < 0.05). Subsequently, the combination of crude lipopeptide extract and propiconazole displayed a control efficacy of 64.29%. In comparison, the control efficacies of the three individual agents—crude lipopeptide extract, propiconazole, and difenoconazole—against potted cucumber Fusarium wilt were 39.29%, 42.86%, and 53.57%, respectively. Notably, difenoconazole exhibited the most effective control efficacy among the individual agents.

3.9. Effects of the Combination of Crude Lipopeptide Extract and Difenoconazole on Stress-Resistant Enzyme Activities in Cucumber Seedlings

Figure 11 illustrates the fluctuation in superoxide dismutase (SOD) activity over time following the inoculation of potted cucumber seedlings with F. oxysporum f. sp. cucumerinum and subsequent root drenching with agents 24 h later. The SOD activity peaked at 72 h, at which point the mixed combination showed increases of 0.94% and 4.59% compared to the crude lipopeptide extract alone and difenoconazole alone, respectively, exhibiting an initial rising trend followed by a decline. The mixed combination of crude lipopeptide extract and difenoconazole displayed the maximum SOD activity at 315.76 U g⁻¹ FW min⁻¹. Throughout the observation period, except at 24 h, the dominant mixed combination showed the highest SOD activity, followed by the crude lipopeptide extract, difenoconazole, and the control, which had the lowest enzyme activity. Notably, at 60 h and 72 h, the effects of the crude lipopeptide extract and the dominant mixed combination were comparable and superior to those of difenoconazole alone.

Figure 12 illustrates the pattern of peroxidase (POD) activity across different treatments, which showed an initial increase followed by a decrease over time. Notably, the combination of crude lipopeptide extract and difenoconazole exhibited the highest POD activity at all the time points tested. The peak activity for both the control and all treatments occurred at 48 h. Specifically, the POD activity of the crude lipopeptide extract-difenoconazole mixture peaked at 281.63 U g⁻¹ FW min⁻¹, which was 45.77% higher than that of the sole crude lipopeptide extract treatment and 22.43% higher than that of the sole difenoconazole treatment. This peak value was also 2.76 times higher than that of the control (p < 0.05). By 60 h and 72 h, no significant differences in POD activity were observed between the sole treatment with crude lipopeptide extract and the sole treatment with difenoconazole.

Figure 13 illustrates a consistent increase in catalase (CAT) activity over time for both the control group and various treatments. Specifically, the control group exhibited the highest enzyme activity at 24 h (p < 0.05), whereas the combination of crude lipopeptide extract and difenoconazole demonstrated the highest activity at all other time points. Notably, the CAT activity of the mixture peaked at 84 h, reaching 23.39 U g⁻¹ FW min⁻¹, which was 11.10% higher than that of the sole crude lipopeptide extract treatment and 5.87% higher than that of the sole difenoconazole treatment.

4. Discussion

F. oxysporum f. sp. cucumerinum (FOC) demonstrates robust saprophytic activity in soil and produces abundant chlamydospores, rendering it a classic polycyclic pathogen. Continuous monoculture intensifies the incidence of cucumber Fusarium wilt, presenting a significant threat to global cucumber production [26]. Developing effective eco-friendly control strategies for this disease is essential for sustainable agricultural practices [27]. This study examines the application of microbial metabolites and their synergistic interactions with reduced chemical fungicides to improve disease suppression. The results offer important insights for both Fusarium wilt management and environmental conservation.

B. velezensis has demonstrated notable efficacy in biocontrol against crop diseases and growth promotion, underscoring its potential for integrated disease management and sustainable agriculture [28]. Research indicates substantial variability in the inhibitory effects of the same biocontrol strain across Fusarium species. For example, B. velezensis RC218 exhibited inhibition rates of 45.3% against F. graminearum FRC1 and 28.4% against FRC6, compared to 58.4% and 31.6% against F. poae FRC42 and FRC50, respectively—findings later confirmed in greenhouse pot experiments [29]. These results emphasize the need for targeted screening of biocontrol strains against Fusarium isolates from diverse environments. In this study, a dual-culture assay revealed that B. velezensis dhm2—previously isolated from maize rhizosphere soil—achieved a 52.27% inhibition rate against FOC. This preliminary finding suggests the strain’s inhibitory potential, supporting further investigation into its fermentation metabolites for biocontrol applications. However, the results also highlight the need for additional screening to identify more effective biocontrol strains against this pathogen, thereby improving disease management in agricultural production.

The age of the seed inoculum and fermentation duration significantly impact the types and quantities of metabolites generated by biocontrol bacteria. In this research, we conducted an optimization study for B. velezensis dhm2. The optimal seed inoculum age was determined to be 4 h, with continued fermentation for 60 h post-inoculation identified as the ideal condition for producing antimicrobial metabolites, resulting in an inhibition rate of 66.00%. This outcome showed a substantial improvement compared to the inhibition rate observed prior to optimization. Interestingly, our findings differ from those of Li et al., who reported that metabolites from B. velezensis FX-6 exhibited the highest efficacy against tomato Fusarium wilt after 72 h of fermentation [30]. This discrepancy suggests that metabolic profiles differ among various strains of B. velezensis. It is postulated that the types and quantities of lipopeptide bioactive compounds produced by biocontrol bacteria undergo dynamic changes as the fermentation period progresses. Jemil et al. employed LC/MSD-TOF analysis to demonstrate that B. methylotrophicus DCS1 consistently produced novel crude lipopeptide extracts in the fermentation medium over time. Their results indicated that bacillomycin D and fengycin were the primary fermentation metabolites during the first 24–48 h, whereas surfactin, iturin, and other lipopeptides accumulated by 72 h. Notably, the crude lipopeptide extract produced by 72 h fermentation exhibited maximum antifungal activity against pathogenic fungi [31]. The production of metabolites by biocontrol bacteria is significantly influenced not only by fermentation conditions but also by the composition of the culture medium. Studies have shown that B. velezensis FJAT-46737 produces increased levels of iturin, fengycin, and surfactin in media containing high organic nitrogen content, leading to enhanced antimicrobial activity [32]. This underscores the crucial impact of medium composition on lipopeptide production. Thus, optimizing the fermentation medium for dhm2 is essential for enhancing lipopeptide yield in future research endeavors.

The biocontrol efficacy of lipopeptides derived from Bacillus spp. against plant pathogens is well established [33,34,35]. Mihalache et al. [36]. demonstrated that lipopeptides like fengycin, surfactin, and mycosubtilin, exhibit potent antifungal activity against the F. oxysporum f. sp. iridacearum, the causal agent of iris root rot. Notably, mycosubtilin—either alone or in combination with surfactin—demonstrated strong antimicrobial activity, with a minimum inhibitory concentration (MIC) of just 5 µg mL⁻¹. This value is significantly lower than the MIC (500 µg mL⁻¹) of the conventional fungicide thiophanate-methyl, highlighting the superior disease-suppressive potential of crude lipopeptide extracts compared to synthetic fungicides. The synergistic effect of crude lipopeptide extracts with antimicrobial agents represents a promising strategy for broadening their application spectrum. In this study, the combined application of crude lipopeptide extracts and difenoconazole demonstrated 80.85% pot control efficacy, representing a 50.92% and 105.78% increase over difenoconazole and crude lipopeptide extracts alone, respectively. These findings suggest that combining crude lipopeptide extracts with chemical fungicides holds significant potential for managing cucumber Fusarium wilt while overcoming the inherent limitations of live bacterial agents [37,38]. Similarly, while cinnamon extract alone inhibited F. odoratissimum by 44.5%, its combination with B. tequilensis-derived lipopeptide extracts increased inhibition to 54.2%, marking a 21.8% improvement [39]. The “crude extract + chemical agent” approach proposed in this study not only enhances antimicrobial efficacy but also reduces chemical fungicide dosages, offering a targeted solution for controlling diverse F. oxysporum strains. The lipopeptide extracts from B. velezensis dhm2 and B. tequilensis significantly enhanced the antifungal effects of chemical fungicides or other active compounds, demonstrating their potential practical application. However, it remains unclear whether this enhancement is due to a single lipopeptide component or the synergistic action of multiple lipopeptides. The literature suggests that the iturin family of lipopeptides plays a vital role in controlling plant fungal diseases by inhibiting fungal plant pathogens. For example, B. subtilis SQR9 combats cucumber Fusarium wilt by producing lipopeptide antibiotics like bacillomycin [40], while B. velezensis GB03 shows substantial inhibition of F. oxysporum f. sp. strigae mycelial growth. Structural identification using LC-MS/MS revealed bacillomycin D as the primary active component against F. oxysporum [41]. Bacillomycin belongs to the iturin family of lipopeptides. Nonetheless, further research is needed to clarify the structural characteristics and synergistic roles of the main antifungal lipopeptides produced by dhm2 in this study.

The study revealed that the crude lipopeptide extract derived from B. velezensis dhm2 demonstrated significant inhibitory effects against FOC, the pathogen responsible for cucumber Fusarium wilt. Previous studies have suggested that the antimicrobial action of lipopeptides is primarily attributed to their ability to disrupt cell membranes [42]. Treatment of FOC with the crude lipopeptide extract from dhm2 resulted in observable morphological changes in the hyphae, including roughness, distortion, localized protrusions or depressions, and pigment deposition, providing visual evidence of its antibacterial mechanism. Studies on B. amyloliquefaciens BO7 have shown that mutants with modified cell membrane structures displayed heightened sensitivity to lipopeptides, indirectly implying a correlation between lipopeptide effectiveness and cell membrane integrity [43]. Based on our findings, we posit the central hypothesis that the dhm2 lipopeptide disrupts FOC membrane integrity, increasing membrane permeability to facilitate the penetration of chemical fungicides. Specifically, triazole fungicides like difenoconazole and propiconazole target ergosterol synthesis, a crucial membrane component. Consequently, it is suggested that the combined application of the crude lipopeptide extract from our study and difenoconazole results in a dual membrane damage effect by concurrently targeting “physical disruption + biosynthesis inhibition” [44]. This multi-component synergistic mechanism of membrane damage not only enhances control efficacy but also mitigates the risk of pathogen resistance, offering a novel approach for environmentally friendly FOC management.

The study demonstrated that the crude lipopeptide extract from B. velezensis dhm2 significantly boosted defense enzyme activity in cucumber seedlings, akin to previous findings with B. amyloliquefaciens DHA6 in watermelon and B. amyloliquefaciens FBZ24 in tomatoes [45,46]. Moreover, the optimal combination of crude lipopeptide extract and difenoconazole treatment notably increased SOD activity to 315.76 U·g⁻¹ FW·min⁻¹ at 72 h, peaked POD activity at 281.63 U·g⁻¹ FW·min⁻¹ at 48 h, and raised CAT activity to 23.39 U·g⁻¹ FW·min⁻¹ at 84 h in cucumber seedlings. These values represented enhancements of 0.94%, 45.77%, and 11.10%, respectively, compared to lipopeptide treatment alone. Particularly noteworthy was the substantial increase in POD activity, surpassing that of individual biological or chemical treatments, underscoring the synergistic benefits of their combined application. Further analysis demonstrated that this synergistic system possesses distinctive advantages in defense mechanisms. The combination not only enhances defense enzyme activity but also establishes a dual defense strategy comprising “systemic resistance and direct bactericidal effects.” This is achieved through lipopeptides disrupting pathogen membrane structures and fungicides inhibiting ergosterol synthesis. In contrast to the Trichoderma-methyl thiophanate combination reported by Abd-El-Khai et al. [47], which improves kidney bean disease resistance solely by increasing peroxidase activity, this system differs from the singular mode of B. subtilis ATCC21556, which only induces lettuce defense genes via lipopeptides. Instead, it integrates immediate control and sustained protection through temporal coordination: fungicides rapidly suppress acute infections, while lipopeptides persistently activate long-term host defenses [48]. This may explain why the synergistic combination exhibits significantly higher pot control efficacy (80.85%) compared to individual treatments. This comprehensive approach offers an eco-friendly and sustainable solution for managing diseases in cucumber and other vegetable crops. Future research will focus on optimizing fermentation conditions, formulation development, and field application techniques for the biocontrol strain dhm2 to ensure consistent plant disease control efficacy.

5. Conclusions

B. velezensis dhm2 effectively controlled cucumber Fusarium wilt, with a 52.27% inhibition rate in confrontation culture. Optimal inhibitory activity of the fermentation supernatant was achieved using a 4 h seed inoculum followed by 60 h fermentation. The EC₅₀ of the crude lipopeptide extract against the pathogen was 9.99 g L⁻¹, while prochloraz, the most potent chemical fungicide tested, had a lower EC₅₀ of 0.03 μg mL⁻¹. Notably, combining the crude lipopeptide extract with six chemical fungicides yielded synergistic effects, with the difenoconazole (7:3) and propiconazole (7:3) mixtures exhibiting the highest toxicity ratios (1.25 and 1.22, respectively) via the Horsfall method and significant synergism as confirmed by the Wadley test (ratios of 5.88 and 3.41). This combined treatment also markedly enhanced defense enzyme activities (SOD, POD, CAT) in cucumber seedlings. A key novelty of this study lies in the practical implication of utilizing crude lipopeptide extracts—avoiding costly purification steps—for synergistic interaction with chemical fungicides. This strategy achieved an 80.85% control efficiency in practical application while reducing chemical fungicide usage, addressing critical challenges in sustainable agriculture by lowering environmental risks associated with excessive chemical inputs. However, the current findings are based primarily on laboratory and pot experiments. A notable limitation is the need for further validation through greenhouse and field trials to confirm efficacy under complex agricultural conditions, including strain persistence, environmental adaptability, and long-term stability of the synergistic effect.