Synergistic Control of Bacterial Fruit Blotch Using Bacillus velezensis ZY1 and Chemical Bactericides

Abstract

Bacterial fruit blotch (BFB) is a devastating disease caused by Acidovorax citrulli, severely impacting the watermelon and melon industries and leading to significant economic losses. Currently, researchers have not identified any commercial melon varieties with high resistance to BFB, and farmers primarily rely on chemical agents for prevention and control. However, the extensive use of these agents contributes to increased drug resistance among pathogenic bacteria, making it essential to develop environmentally friendly control methods. To explore the feasibility of combining the Bacillus velezensis ZY1 strain with chemical agents for BFB management, we assessed the efficacy of 10 bactericides and the ZY1 strain against Acidovorax citrulli. The results show that Prothioconazole, Zhongsheng Tetramycin Solution, Streptomycin Sesquisulfate, Tetramycin, Zhongshengmycin, and ZY1 exhibited significant inhibitory effects on the growth of Acidovorax citrulli. We determined the biocompatibility of the bactericides with the ZY1 strain using the plate confrontation method and the flat counting method. Zhongsheng Tetramycin Solution, Zhongshengmycin, and Kasugamycin exhibited good compatibility with the ZY1 strain. Additionally, we established the optimal compounding ratio of the bactericide and ZY1 using the Horsfall method. Among these, Zhongshengmycin demonstrated the best performance when combining its efficacy against Acidovorax citrulli with its biocompatibility with the ZY1 strain. The combination of Zhongshengmycin and ZY1 at a volume ratio of 5:5 significantly inhibited Acidovorax citrulli, exhibiting a clear synergistic effect with a synergy index (I_R) value of 1.542. Field tests conducted over 21 days in Beijing greenhouses, Hainan Field facility greenhouses, and Xinjiang showed that the control efficacy of the Zhongshengmycin and ZY1 combination (89.23%) significantly surpassed that of the single agent Zhongshengmycin (80.35%) and the biocontrol bacterium ZY1 (72.12%). Notably, the application rate of Zhongshengmycin in the mixture was only half that of the single agent, resulting in a significant reduction in chemical usage. The combination of Bacillus velezensis ZY1 and Zhongshengmycin not only decreases chemical usage but also significantly enhances control efficacy compared to using Zhongshengmycin alone.

1. Introduction

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is a serious bacterial disease affecting crops worldwide [1]. BFB primarily impacts cucurbit crops and spreads rapidly [2]. Since its discovery in the U.S., BFB has expanded to other regions and countries, currently occurring in nations such as Australia, Brazil, Turkey, Japan, Thailand, Israel, Iran, Hungary, Greece, and in over 10 provinces in China, including Xinjiang, Hainan, Jilin, and Inner Mongolia. This spread has resulted in significant economic losses [2,3]. Currently, no commercially available cultivars exhibit resistance to BFB. The primary strategy for controlling BFB involves the use of chemical agents [4]. However, the long-term excessive use of these chemicals has led to increased resistance among melon crops and has negatively impacted both the environment and public safety [5].

In recent years, biological control has gained increasing attention with the advancement of research in this field [6]. Biocontrol plays a critical role in integrated pest management strategies. The use of biological agents not only reduces reliance on chemical reagents but also avoids leaving harmful residues in the environment. This approach effectively mitigates the environmental impact of chemicals and reduces the risk of phytopathogenic bacteria developing drug resistance [7]. Shi et al. [8] isolated a Bacillus polymyxa G-14 strain from the rhizosphere of melons and conducted greenhouse and field experiments. Their results demonstrated that this bacterial strain significantly inhibited the occurrence and spread of BFB. Furthermore, they found that prophylactic treatment was more effective than curative treatment. Fan et al. [9] showed that Bacillus subtilis 9407 could be used for the biological control of BFB in melons through surfactant-mediated antimicrobial activity and colonization, achieving a biological control efficacy of 61.7%. However, several challenges remain in using single biocontrol agents to manage crop diseases in agricultural production. Diverse environmental conditions and complex soil systems limit the effectiveness of biological control, leading to poor colonization, low survival rates, and unstable biocontrol outcomes [10].

Research into combining biological and chemical agents for disease prevention and control has increased in recent years. This approach compensates for the limitations of biological agents while reducing the need for chemical pesticides [11].

Bacterial biocontrol agents such as Pseudomonas fluorescens, Bacillus subtilis, Bacillus methylotrophicus, and Bacillus megaterium have shown synergistic effects when used in combination with chemical agents to control various diseases, including chili fruit rot and powdery mildew, rice sheath blight, tomato gray mold, tomato root rot, and wheat stripe disease [12,13,14,15,16]. Anand et al. [12] demonstrated that combining Pseudomonas fluorescens (Pf1) with a low concentration of azoxystrobin was highly effective in treating chili fruit rot and powdery mildew. Although Pf1 alone was effective in controlling both diseases, it was less efficacious than using the standard dose of fungicide. Remarkably, combining Pf1 with a 50% reduction in fungicide dosage provided results comparable to using the full dose of fungicide. However, there have been no reports on the combined use of Bacillus subtilis and chemical agents to control BFB in melons. Previously, our laboratory screened and identified a Bacillus velezensis strain ZY1, which exhibits strong antibacterial activity against A. citrulli and shows promising potential for field application [17].

This experiment aims to screen chemical agents compatible with Bacillus velezensis ZY1 and to optimize the compounding ratio of ZY1 and these chemical agents. Additionally, we will conduct field trials in different regions to establish a novel technology for synergistic control of BFB, combining biological agents and chemical bactericides. This study will provide technical support for reducing chemical pesticide usage in melon production.

2. Materials and Methods

2.1. Materials and Bactericides

Melon cultivars: Cucumis melo cv. IVF192 (Beijing greenhouse potted variety), Xizhoumi No. 5 (Hainan field test variety), and Cuibaomi No. 17 (Xinjiang field test variety).

Bacterial strains: Acidovorax citrulli pslb65 (maintained in this laboratory) and Bacillus velezensis ZY1 (maintained in this laboratory).

Bactericides: 10% prothioconazole suspension (Guizhou Daoyuan Biotechnology Co., Ltd., Guizhou, China), 0.3% tetramycin, 1.7% zhongshengmycin soluble liquid (Hailier Pharmaceutical Group Co., Ltd., Qingdao, China), 46% copper hydroxide water-dispersible granules (purchased from Kodiwa Agricultural Science and Technology Co., Ltd., Shanghai, China), 2% zhongshengmycin and 2% kasugamycin wettable powder (Fujian Kaili Biological Products Co., Ltd., Changtai, China), 3% zhongshengmycin wettable powder (Fujian Kaili Biological Products Co., Ltd., Changtai, China), and 4% kasugamycin aqueous solution (Shaanxi Mecro Biotechnology Co., Ltd., Tokyo, Japan), 0.3 Tetramycin aqueous solution (Liaoning Microscience Biotechnology Co., Ltd., Shenyang, China), 72% Streptomycin Sesquisulfate wettable powder (North China Pharmaceutical Hebei Huanuo Co., Ltd., Shijiazhuang, China), 20% Thiobacille Copper Suspension (Zhejiang Longwan Chemical Co., Ltd., Wenzhou, China), 34.5% oxine-copper, 0.5% tetramycin suspension (Qingdao Otis Biotechnology Co., Ltd., Qingdao, China).

2.2. Indoor Toxicity Determination of Test Agents and ZY1 Against pslb65

Toxicity was determined using the plate confrontation method. The pslb65 strain was thawed from −80 °C and cultured on a lysogeny broth (LB: 15 g of tryptone, 10 g of yeast extract, 10 g of NaCl; add 15 g of agar to solid culture medium) plate. After 48–72 h of incubation at 28 °C, single colonies were picked and inoculated into fresh LB medium in centrifuge tubes. The cultures were shaken at 220 rpm and 28 °C for 12–14 h. The concentration of pslb65 was adjusted to approximately 1 × 10⁸ cfu·mL⁻¹ (OD₆₀₀ = 0.3) using a spectrophotometer (EVOLUTION 300 UV-VIS, Thermo Fisher Scientific, Waltham, MA, USA). The pslb65 strain was then mixed with LB medium at a 1:40 ratio to prepare bacterial plates; the final concentration of pslb65 was 2.5 × 10⁶ cfu·mL⁻¹.

All test bactericides were prepared in sterilized water at their highest, optimal, and lowest concentrations; the concentration of the bactericides was formulated according to the recommended instructions for use, as detailed in

Table 1. Bactericides concentrations used for testing.

Serial Number	Name of the Medicine	Lowest Concentration (mg·mL−1)	Optimal Concentration (mg·mL−1)	Maximum Concentration (mg·mL−1)
1	Prothioconazole	1.11	1.43	2
2	Zhongsheng Tetramycin Solution	1.67	2	2.5
3	Tetramycin Oxine–copper	0.56	0.67	0.83
4	Streptomycin Sesquisulfate	0.83	1	1.25
5	Tetramycin	1.25	1.67	2.5
6	Thiodiazole–copper	1.43	2	3.33
7	Copper hydroxide	0.56	0.67	0.83
8	Zhongshengmycin Kasugamycin	0.67	0.83	1
9	Kasugamycin	0.56	0.67	0.83
10	Zhongshengmycin	0.56	0.67	0.83

. Sterilized and dried neutral filter paper discs (5 mm in diameter) were placed on the bacterial plates, with three discs per Petri dish. Ten microliters of each drug solution was added dropwise to each filter paper disc, with each concentration gradient repeated in three dishes. Sterilized water served as a blank control. The plates were incubated at 28 °C, and the inhibition zones were observed and photographed after 72 h. The diameter of the inhibition zones was measured using the cross-intersection method, and the inhibition rate was then calculated. The EC₅₀ value was determined, with the experiment repeated three times.

The bacterial inhibition ability of Bacillus velezensis ZY1 suspension was determined using the same method. Bacillus velezensis ZY1 was cultured in LB medium for 24 h, and bacterial suspensions were prepared at concentrations of 1 × 10⁶, 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹ cfu·mL⁻¹. The experiment was repeated three times.

2.3. Determination of Biocompatibility of ZY1 with Chemical Agents

A single colony of the ZY1 strain was selected and inoculated into 5 mL of LB medium, then shaken at 28 °C for 12 h. The concentration of ZY1 was adjusted to approximately 1 × 10⁸ cfu·mL⁻¹ (OD₆₀₀ = 0.3) using a spectrophotometer. To each 4 mL of LB liquid medium, 500 μL of ZY1 bacterial suspension (1 × 10⁸ cfu·mL⁻¹) and 500 μL of various chemical agents were added. The mixture was incubated at 28 °C with shaking at 220 rpm for 12–14 h. After incubation, the OD value of each bacterial suspension was measured using a spectrophotometer. Simultaneously, 100 μL of the bacterial suspension was evenly spread onto LB plates and incubated at 28 °C for 48 h. The number of bacterial colonies on each plate was recorded. Sterile water, added in equal volume, served as a blank control (CK). Each experiment was repeated three times.

2.4. Laboratory Toxicity Determination of the Compound Against Acidovorax Citrulli

Based on the results from the indoor toxicity assay (Section 2.2) and the biocompatibility assay (Section 2.3), we selected Zhongsheng Tetramycin Solution and Zhongshengmycin for combination with the ZY1 strain. To identify synergistic ratios, a compound toxicity test was conducted using chemical agents and ZY1 at different ratios, following the method described in Section 2.2. The tested chemical-to-ZY1 ratios were 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10. Inhibition rates and theoretical inhibition rates were calculated using the method described by Cai et al. [18]. Sterile water was used as a negative control. Each treatment was replicated three times. The synergistic effect of combined toxicity was calculated using the Horsfall method [19]. A potentiation ratio (I_R > 1) was considered synergistic, I_R = 1 was considered additive, and I_R < 1 was considered antagonistic.

2.5. Determination of the Efficacy of the Compound Against BFB in Beijing Greenhouse

The experiment was conducted in March 2024 in the glass greenhouse of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing. The melon seeds were treated in the trays in which they were rooted. Two-week-old melon seedlings (Cucumis melo cv. IVF192; Zhongcai Seed Industry Technology Co., Ltd., Beijing, China) were sprayed using a spray bottle with A. citrulli pslb65 at a concentration of 1 × 10⁸ cfu·mL⁻¹, with approximately 20 mL of bacterial suspension per seedling.

The treatments were as follows:

① Single dose of Zhongsheng Tetramycin Solution (EC₅₀ = 93.984 mg/L);

② Single dose of Zhongshengmycin (EC₅₀ = 33.427 mg/L);

③ Kasugamycin;

④ Copper Hydroxide;

⑤ Bacillus velezensis ZY1 suspension (1 × 10⁸ cfu·mL⁻¹);

⑥ Compounding agent [V_{Zhongsheng Tetramycin Solution}:V_ZY1 = 6:4];

⑦ Compounding agent [V_{Zhongshengmycin}:V_ZY1] = 5:5;

⑧ Compound preparation [V_Kasugamycin:V_ZY1] = 5:5;

⑨ Compounding agent [V_{Copper Hydroxide}:V_ZY1] = 5:5;

⑩ Sterile water (negative control).

The concentrations of the bactericides were determined based on their effectiveness in controlling the A. citrulli pslb65 strain in the laboratory. Disease severity was assessed on days 7, 14, and 21 post-inoculation. The disease was classified into six grades according to the extent of BFB (Bacterial Fruit Blotch) symptoms [20,21]—Grade 0: no visible symptoms, Grade 1: leaf blotch area < 25%, Grade 3: leaf blotch area 25–50%, Grade 5: leaf blotch area 50–75%, Grade 7: leaf blotch area 75–100%, and Grade 9: complete plant death. The disease index was calculated using the following formula:

Disease index = 100 × ∑ (number of diseased leaves at each level × relative level value)/(total number of leaves surveyed × 9).

Control effect (%) = [(disease value in negative control − disease value in a treatment)/disease value in negative control] × 100.

Each treatment was replicated three times, with 30 melon seedlings per treatment. The experiment was repeated three times.

2.6. Field Trial

In March and July 2024, we conducted field trials in two different regions, Hainan Province and Xinjiang Uyghur Autonomous Region.

2.6.1. Determination of Field Efficacy of Compound Against BFB in Hainan Province

The experiment was conducted from March to April 2024 in a greenhouse facility located in Chaomei Village, Lingshui County, Hainan Province (18.458899° N, 109.957547° E). The melon cultivar used was Xizhoumi No. 5. At the start of the experiment, the early stage of BFB onset was observed. We did not spray additional pathogens. The test site had been used for melon cultivation for several years. The experimental treatments followed the same protocol as described in Section 2.5. Ten-week-old melon seedlings were spray-inoculated using a backpack hand sprayer until run-off (Instrument model: HD-400 Manufacturer: Linon Singapore), with approximately 20 mL of bacterial suspension per plant. The greenhouse was divided into 40 plots, with four replicates per treatment. Each plot measured approximately 15 m² and contained 40 plants. The bactericide was applied once every 7 days, with three total applications. The types and concentrations of each bactericide were the same as outlined in Section 2.5. After each application, disease severity was assessed using a three-point sampling method, with five plants surveyed per point and all leaves evaluated per plant. The disease index and control effects were calculated as described previously.

2.6.2. Determination of Field Efficacy of Compound Agent Against BFB in Xinjiang

The experiment was conducted in Anning Qu, Urumqi, Xinjiang Uyghur Autonomous Region, from July to August 2024 (43.842563° N, 87.5717991° E). The melon cultivar used was Cuibaomi No. 17. Similar to Section 2.5, the experiment included ten treatments, with three replicates per treatment, and a total of 30 plots, each approximately 22 m². The experimental protocol and bactericide dosage are identical, as outlined in Section 2.5. Five random points were selected within each plot, with five melon plants surveyed per point. Disease severity was assessed using the same method described in Section 2.6.1.

2.7. Data Statistics and Analysis

In the trial, DPS 8.50 software (Hangzhou Ruifeng Information Technology Co., Ltd., Hangzhou, China) was used to perform one-way analysis of variance (ANOVA), and the LSD method was applied to test significant differences in colony diameter, inhibition rate, colony number, disease index, and control effect.

DPS 8.50 software was used to perform regression equations and median effect concentration (EC₅₀) analysis.

3. Results

3.1. Toxicity Analysis of 10 Chemical Agents and ZY1 Against A. citrulli

Five agents exhibited strong inhibitory effects against the A. citrulli pslb65 strain (Figure 1). The antibacterial rates, from highest to lowest, were as follows: Prothioconazole, Streptomycin Sesquisulfate, Zhongshengmycin, Zhongsheng Tetramycin Solution and Tetramycin. The EC₅₀ values ranged from 17.140 to 321.773 mg·L⁻¹ (

Table 2. Toxicity of ten chemical agents tested against Acidovorax citrulli.

Treatment	Eficacy Regression Equation	EC50/(mg·L−1)
Prothioconazole	y = 1.5508x − 1.9137	17.140
Zhongsheng Tetramycin Solution	y = 1.2577x − 2.4816	93.984
Tetramycin Oxine–copper	—	—
Streptomycin Sesquisulfate	y = 1.3627x − 1.8781	23.891
Tetramycin	y = 0.9097x − 2.2811	321.773
Thiodiazole–copper	—	—
Copper Hydroxide	—	—
Zhongshengmycin Kasugamycin	—	—
Kasugamycin	—	—
Zhongshengmycin	y = 0.9621x − 1.4664	33.427

Note: Regression equations were established using DPS for agent concentration, inhibition rate, and observations to derive EC₅₀ values.

).

A: Prothioconazole; B: Zhongsheng Tetramycin Solution; C: Tetramycin Oxine–copper; D: Streptomycin Sesquisulfate; E: Tetramycin; F: Thiodiazole–copper; G: Copper Hydroxide; H: Zhongshengmycin; I: Kasugamycin; J: Zhongshengmycin; K: negative control. The dilution multiples of each chemical agent is located below respective pictures.

The experimental results demonstrated that the biocontrol agent ZY1 significantly inhibited A. citrulli, with the inhibitory effect increasing as the concentration ranged from 1 × 10⁶ to 1 × 10⁹ cfu·mL⁻¹ (Figure 2). The inhibition rates at 1 × 10⁸ and 1 × 10⁹ cfu·mL⁻¹ were significantly higher than those at 1 × 10⁶ and 1 × 10⁷ cfu·mL⁻¹, but no significant difference was observed between 1 × 10⁸ and 1 × 10⁹ cfu·mL⁻¹ (p < 0.05) (

Table 3. Toxicity of Bacillus velezensis ZY1 against Acidovorax citrulli.

ZY1 Concentration/(cfu·mL−1)	Colony Diameter/mm	Inhibition Rate/%
1 × 106	25.11 ± 0.38 c	27.90% c
1 × 107	27.33 ± 1.00 b	30.37% b
1 × 108	30.33 ± 1.20 a	33.70% a
1 × 109	31.67 ± 0.58 a	35.19% a
CK	0	—

Note: Colony diameters are presented as means ± standard error of at least three independent experiments. Data with different lowercase letters indicate significant differences (p < 0.05).

).

3.2. Compatibility Analysis of Biocontrol Agent ZY1 Strain with the Tested Agents

The experimental results revealed significant differences in the biocompatibility of various chemical agents with the ZY1 strain (Figure 3 and

Table 4. Effects of the tested bactericides on the growth of Bacillus velezensis ZY1.

Treatment	Concentration (mg·mL−1)	Bacterial Growth	Colony Number
Prothioconazole	2	×	—
	1.43	×	—
	1.11	×	—
ZhongshengTetramycin Solution	2.5	+	8 ± 2.887
	2	+	157 ± 20.43
	1.67	+++	141 ± 37.501
Streptomycin Sesquisulfate	1.25	+	12 ± 1.732
	1	+	6 ± 1.528
	0.83	×	—
Tetramycin	2.5	+	1 ± 0.577
	1.67	+	4 ± 2.082
	1.25	++	21 ± 13.65
Kasugamycin	0.83	+++	92 ± 21.825
	0.67	+++	91 ± 25.813
	0.56	++	58 ± 16.093
Zhongshengmycin	0.83	++	85 ± 24.88
	0.67	++	81 ± 31.896
	0.56	++	83 ± 13.614
CK		+++	89 ± 35.501

Note: “+++” indicates good growth, “++” indicates normal growth, “+” indicates able growth. “×” indicates no growth. Colony numbers are presented as means ± standard error of at least three independent experiments.

). In plates containing Prothioconazole, the ZY1 strain failed to grow. In plates with Tetracycline and Streptomycin Sesquisulfate, the number of ZY1 colonies significantly decreased, indicating inhibition of growth. However, the ZY1 strain grew normally in media containing Zhongsheng Tetramycin Solution, Zhongshengmycin, and Kasugamycin, suggesting good compatibility with these three agents.

A: Prothioconazole; B: Zhongsheng Tetramycin Solution; C: Streptomycin Sesquisulfate; D: Tetramycin; E: Kasugamycin; F: Zhongshengmycin; G: negative control. The dilution of each chemical agent is included below the respective pictures.

3.3. Indoor Toxicity Test of the Combination of Zhongshengmycin, Zhongsheng Tetramycin Solution, and Biocontrol Agent ZY1

Based on the toxicity of the test agents to the pslb65 strain and the compatibility of the biocompatible bacteria, we selected Zhongsheng Tetramycin Solution, Zhongshengmycin, and ZY1 for compounding.

The combined toxicity test results for the combination of Zhongshengmycin and ZY1 (

Table 5. Co-toxicity of Zhongshengmycin and Bacillus velezensis ZY1 against Acidovorax citrulli.

VZhongshengmycin:VZY1	Colony Diameter/mm	Actual Inhibition Rate/%	Theoretical Inhibition Rate/%	IR
0:10	25.22 ± 1.07 bc	28.03% bcd	28.03%	—
1:9	24.22 ± 2.84 c	26.91% cd	27.01%	0.996
2:8	24.22 ± 1.95 c	26.91% cd	25.98%	1.036
3:7	28.11 ± 4.22 b	31.23% abc	24.96%	1.251
4:6	29 ± 1.86 ab	32.22% ab	23.93%	1.346
5:5	31.78 ± 0.19 a	35.31% a	22.91%	1.542
6:4	30.33 ± 1.21 ab	33.70% a	21.88%	1.540
7:3	25.11 ± 1.58 bc	27.90% bcd	20.86%	1.337
8:2	21.22 ± 2.99 c	23.58% d	19.83%	1.189
9:1	16.56 ± 0.51 d	18.40% e	18.81%	0.978
10:0	16 ± 0.33 d	17.78% e	17.78%	—

Note: Formula used for IR: I_R = Eab/Eth, where Eab is the actual inhibition rate of the compound, and Eth is the theoretical inhibition rate of the compound. I_R > 1 indicates a synergistic effect, I_R = 1 indicates an additive effect, and I_R < 1 indicates an antagonistic effect. Theoretical inhibition rate = (V_{Zhongshengmycin} × actual inhibition rate) + (V_ZY1 × actual inhibition rate). Colony diameters are presented as means ± standard error of at least three independent experiments. Data within each column with different lowercase letters indicate significant differences (p ≤ 0.05).

) indicated that the compound ratios exhibiting synergistic effects were V_{Zhongshengmycin}:ZY1 = 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2, with actual inhibition rates of 26.91%, 31.23%, 32.22%, 35.31%, 33.70%, 27.90%, and 23.58%, respectively. Among these, the highest synergistic effect and inhibition rate occurred at a volume ratio of 5:5. Therefore, we selected V_{Zhongshengmycin}:ZY1 = 5:5 as the optimal compounding ratio.

The combined toxicity test results for the combination of Zhongsheng Tetramycin Solution and ZY1 (

Table 6. Co-toxicity of Zhongsheng Tetramycin Solution and Bacillus velezensis ZY1 against Acidovorax citrulli.

VZhongsheng Tetramycin Solution:VZY1	Colony Diameter/mm	Actual Inhibition Rate/%	Theoretical Inhibition Rate/%	IR
0:10	24.89 ± 1.02 ab	27.66% ab	27.66%	—
1:9	25.55 ± 1.35 ab	28.39% a	25.92%	1.095
2:8	24.22 ± 2.59 ab	26.91% ab	24.17%	1.113
3:7	24.33 ± 1.45 ab	27.04% ab	22.43%	1.206
4:6	20.22 ± 1.26 b	22.47%b	20.68%	1.807
5:5	23.39 ± 3.27 ab	25.99% ab	18.94%	1.372
6:4	27.22 ± 0.39 a	30.25% a	17.20%	1.759
7:3	24.33 ± 2.6 ab	27.04% ab	15.45%	1.750
8:2	23.56 ± 6.45 ab	26.17% ab	13.71%	1.901
9:1	22.56 ± 1.39 b	25.06% ab	11.96%	2.095
10:0	9.2 ± 0.17 c	10.22% c	10.22%	—

Note: Formula used for I_R: I_R = Eab/Eth, where Eab is the actual inhibition rate of the compound, and Eth is the theoretical inhibition rate of the compound. I_R > 1 indicates a synergistic effect, I_R = 1 indicates an additive effect, and I_R < 1 indicates an antagonistic effect. Theoretical inhibition rate = (V_{Zhongsheng Tetramycin Solution} × actual inhibition rate) + (V_ZY1 × actual inhibition rate). Colony diameters are presented as means ± standard error of at least three independent experiments. Data within each column with different lowercase letters indicate significant differences (p ≤ 0.05).

) showed that the compound ratios with synergistic effects were V_{Zhongsheng Tetramycin Solution}:ZY1 = 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The actual inhibition rates for these ratios were 28.39%, 26.91%, 27.04%, 22.47%, 25.99%, 30.25%, 27.04%, 26.17%, and 25.06%, respectively. Based on the results of the synergistic effect and actual antibacterial rate, we observed a significant synergistic effect at a volume ratio of 6:4, which also yielded the highest actual antibacterial rate. Thus, we concluded that the optimal ratio for compound selection was V_{Zhongsheng Tetramycin Solution}:ZY1 = 6:4.

3.4. Control Effects of Chemical Agents and B. velezensis ZY1 on BFB in Greenhouses

In this study, we evaluated the control effects of four chemical agents and the biocontrol agent ZY1 in a greenhouse setting, as well as the combination of both tools. The compounding ratios for the four combinations were as follows: V_{Zhongshengmycin}:ZY1 = 5:5; V_{Zhongsheng Tetramycin Solution:}ZY1 = 6:4; V_Kasugamycin + V_ZY1 = 5:5; V_{Copper Hydroxide} + V_ZY1 = 5:5.

As shown in Figure 4 combining three chemical agents (Zhongshengmycin, Zhongsheng Tetramycin Solution, and Kasugamycin) with ZY1 resulted in a higher control effect than single-agent treatments. However, the combination of Copper Hydroxide and ZY1 exhibited a lower control effect than Copper Hydroxide alone. It can be seen from Table S1, the combination of Zhongshengmycin and ZY1 demonstrated the best preventive and therapeutic effects, with disease indices for BFB of 3.45, 8.20, and 9.88 at 7, 14, and 21 days, respectively. The control effects on days 7, 14, and 21 were 87.47%, 79.41%, and 81.92%, respectively, significantly higher than those of the single-agent treatments of Zhongshengmycin and ZY1 (p < 0.05). The synergistic ratio of Streptomycin and ZY1 on day 21 was I_R = 1.255. Photographs of the 21-day control effect of different treatment groups on melon can be viewed in Figure S1.

3.5. Control Effects of Chemical Agents and Bacillus velezensis ZY1 on BFB in the Fields

3.5.1. Field Trial in Hainan Province

The control effects of all four chemical agents combined with ZY1 were higher than those of single-agent treatments with ZY1. The 21-day control effects, ranked from highest to lowest, were Zhongshengmycin + ZY1, Copper Hydroxide + ZY1, Zhongsheng Tetramycin Solution + ZY1, and Kasugamycin + ZY1 (Figure 5). It can be seen from Table S2, the efficacy at days 7, 14, and 21 for Copper Hydroxide + ZY1, Zhongsheng Tetramycin Solution + ZY1, and Kasugamycin + ZY1 did not significantly differ from the single-agent treatments with ZY1. In contrast, the control effects of the combination of Zhongshengmycin and ZY1 at 7, 14, and 21 days were 87.28%, 87.32%, and 90.34%, respectively. These values were significantly higher than those for the single-agent treatments of Zhongshengmycin and ZY1 (p < 0.05). The 21-day synergistic ratio between Zhongshengmycin and ZY1 was I_R = 1.049. Photographs of the 21-day control effect of different treatment groups on melon can be viewed in Figure S2.

3.5.2. Field Trial in Xinjiang Uygur Autonomous Region

The control effects of the four bactericides combined with ZY1 were superior to those of the single-bactericide and ZY1 treatments. The 21-day control effects, ranked from highest to lowest, were Zhongshengmycin + ZY1, Zhongsheng Tetramycin Solution + ZY1, Copper Hydroxide + ZY1, and Kasugamycin + ZY1. The efficacy at 7, 14, and 21 days for Copper Hydroxide + ZY1 and Kasugamycin + ZY1 did not differ significantly from the single-agent treatments with ZY1. The 7- and 14-day efficacy of Zhongsheng Tetramycin Solution + ZY1 was not significantly different from the single-agent treatments with ZY1, but the 21-day efficacy was significantly higher than that of ZY1 alone (Figure 6). It can be seen from Table S3, the control effects of Zhongshengmycin + ZY1 at 7, 14, and 21 days were 88.99%, 87.96%, and 89.23%, respectively, significantly exceeding those of the single-agent treatments of Zhongshengmycin and ZY1. Additionally, the 21-day synergistic ratio between Zhongshengmycin and ZY1 was I_R = 1.170. Photographs of the 21-day control effect of different treatment groups on melon can be viewed in Figure S3.

4. Discussion

BFB can affect crops throughout their entire growth and development, leading to wilting, rotting, and even plant death. This significantly impedes the sustainable development of watermelon and melon industries in various countries [22]. At present, chemical agents are mainly used in the market to control BFB. For example, Dong [23] used Streptomycin Sesquisulfate and No. 1 bactericide to control BFB, and Chen [24] used Copper Hydroxide and agricultural Streptomycin Sesquisulfate to control BFB. However, the excessive use of chemical agents exacerbates issues such as pathogen resistance and pesticide residues in the environment [25]. Biocontrol agents, known for their environmentally friendly properties, have gained increasing attention. Khedher et al. [26] showed that Bacillus subtilis V26 can be a good prevention and control of tuber dry rot on potatoes. However, biological control often acts more slowly and is more susceptible to environmental factors, limiting its widespread use compared to chemical agents [27]. Therefore, combining chemical and biological control offers an advantageous approach. Therefore, we used agents that have been reported and have better effects on the market to control BFB for testing.

As a biocontrol bacteria, the compatibility of B. velezensis with bactericides is the primary reason for their combined use. These synergistic effects demonstrate the potential compatibility between biocontrol microorganisms and chemical fungicides [28,29]. Peng et al. [16] showed that Bacillus subtilis NJ-18 was extremely tolerant to the fungicides Jinggangmycin, Flutolanil, and Difenoconazole. Peng et al. [30] showed that Bacillus subtilis B-001 can grow well in Saisentong. In this study, the growth of ZY1 was unaffected by Zhongshengmycin and Zhongsheng Tetramycin Solution, indicating that ZY1 was tolerant to them. In addition, according to previous results, the suppression of A. citrulli pslb65 infection by B. velezensis ZY1 indicates its role as a microbial biocontrol agent against BFB. Therefore, it is feasible to use B. velezensis ZY1/Zhongshengmycin and Zhongsheng Tetramycin Solution combination to control BFB.

At present, there are more and more reports on the combination of biocontrol agents and bactericides. Liu et al. [31] showed that Bacillus subtilis and Sodium Bicarbonate were used to control ring rot in pears, and the highest inhibition rate of the disease reached 80.91%. Peng et al. [32] showed that the combination of NJ-18 and Jinggangmycin reduced lesion length by 35% and 20%. This shows that combination therapy is more effective than single treatments, which is consistent with the results of our experiments. Field trials conducted in Hainan Province and the Xinjiang Uygur Autonomous Region confirmed that the Zhongshengmycin-ZY1 combination achieved control effects of 90.34% and 89.23%, respectively. These results were significantly higher than the control effects of 86.24% and 80.35% for the single-dose Zhongshengmycin treatments and notably higher than the control effects of ZY1 alone, which were 85.91% and 72.12%, respectively. Therefore, it can be seen that the combination of ZY1 and Zhongshengmycin is a promising approach for the prevention and control of BFB. However, the inhibition zones of ZY1 are higher than that of bactericides, but the control effect of ZY1 in field experiments is lower than that of bactericides. According to our analysis, on the one hand, ZY1 has a very good growth state and the best bacteriostatic state on LB plates. On the other hand, some chemical bactericides have poor dispersibility on LB plates. This may be the cause of this phenomenon.

In disease management strategies, the combination of biocontrol agents and bactericides reduces the dose of bactericides compared with either treatment alone [33]. Arjona-Lopez et al. [34] showed that Fluazinam and rhizobia could effectively control avocado white root rot and reduce the amount of Fuazinam by 10 times. In our trial, the combination of Zhongshengmycin (EC₅₀ = 155.05 mg·L⁻¹) and ZY1 (1 × 10⁸ cfu·mL⁻¹) exhibited the most significant field control effect on A. citrulli when the volume ratio was 5:5, and greenhouse and field controls are the most effective. Compared with the treatment of ZY1 or the bactericides alone, investigation on the efficacy of ZY1 integrated with a reduced dose of bactericides to half of the normal dose effectively reduced the incidence of BFB. Furthermore, the reduced level of bactericide decreased the amount of bactericide residues in food and thereby lowered environmental pollution as well as the risk of bactericide resistance. This can help protect the environment and mitigate the development of drug resistance [32,35]. In addition, under complex environments such as bactericide failure due to resistant strains of pathogen, the combination of biological and chemical control may provide stable disease control [36,37,38]. Previous research has demonstrated that the control of plant disease can be more stable via the combined application of bactericides and Bacillus spp. than either alone [39]. It can be seen that a judicious combination of biological and chemical controls is essential to enhance the advantages while reducing their limitations. In our study, the combination of biological control agent ZY1 with bactericides could reduce the severity of BFB effectively and improve the reliability of disease control compared to the treatment of ZY1 or bactericides alone. Of course, there are still some questions, such as how to explain and prove that ZY1 is active on the leaves in the presence of the bactericides. This needs to be evaluated in our future trials.

Our study indicated that the combination of B. velezensis ZY1 and Zhongshengmycin enhances the efficiency of disease control, which will be a new strategy for controlling BFB.