Developing Fermentation Liquid of Bacillus amyloliquefaciens PMB04 to Control Bacterial Leaf Spot of Sweet Pepper

: Sweet pepper is an important vegetable in the world. Bacterial leaf spot, caused by the pathogen Xanthomonas perforans , is a limiting factor that signi ﬁ cantly reduces the quality and yield of sweet peppers. The use of chemical fungicides is currently the main disease-control method for bacterial leaf spot disease. It is important to develop an eco-friendly biocontrol method by using antagonistic microorganisms. Bacillus amyloliquefaciens PMB04 has strong antagonistic e ﬀ ects against pathogens and can inhibit the occurrence of diseases. B. amyloliquefaciens PMB04 has the potential for the development of a disease-control product. Primarily, PMB04 contained a strong inhibitory e ﬀ ect against all isolated X. perforans strains. In the inoculation assay, the severity of bacterial leaf spot disease on sweet peppers was reduced by PMB04 bacterial suspensions. To increase the convenience of ﬁ eld applications in future prospects, the development of the PMB04 fermentation liquid was carried out using di ﬀ erent ratios of brown sugar and yeast extract in a 30 L fermentation tank. The results exhibited that the fermentation liquid of the 3-1 and 2-1 formulas obtained the highest bacterial population in a 30 L fermentation tank. The fermentation liquid of the 0.5-0.5 formula was the most stable formula for two di ﬀ erent conditions in terms of a consistent bacterial population and sporulation. In addition, the 200-fold dilution of the 3-1 and 0.5-0.5 fermentation liquids revealed the best control e ﬃ cacy on bacterial leaf spot disease of sweet peppers. Additionally, the results of the 0.5-0.5 fermentation liquid (PMB4FL) with di ﬀ erent dilution concentrations also demonstrated that the 200-and 500-fold dilutions had the best control e ﬃ cacy. To understand the e ﬀ ect of commonly used copper-containing fungicides on sweet peppers on the application of microbial agent PMB4FL, the e ﬀ ects of copper hydroxide and tribasic copper sulfate on the growth of X. perforans strains and B. amyloliquefaciens PMB04 were assayed. The results exhibited that the above two fungicides did not have any inhibitory e ﬀ ect on the growth of PMB04 but had a strong inhibitory e ﬀ ect on the X. perforans strain. In the follow-up control experiment, the treatment of copper hydroxide had no synergistic e ﬀ ect with PMB4FL to control bacterial leaf spot disease. We concluded that the use of the PMB4FL fermentation liquid alone on the leaves could e ﬀ ectively control the occurrence of bacterial leaf spots in sweet pepper crops.


Introduction
Sweet pepper is a vegetable crop widely grown in tropical and subtropical areas all over the world, and bacterial leaf spot disease, caused by diverse Xanthomonas spp., threatens its production [1]. When bacterial leaf spot disease of sweet pepper occurs, punctate water-soaked or gangrenous lesions will appear on the leaves and fruits, which will lead to defoliation of the plant and a reduction of the economic benefits of fruits in severe cases [2]. To control this disease, agricultural scientists have developed many different methods. In the treatment of seeds, disinfection with hot water or sodium hypochlorite can be used to reduce the initial inoculum of pathogenic bacteria [1,3]. On the growing plants, copper-containing fungicides, such as copper hydroxide, tribasic copper sulfate or copper oxide, are mainly used, which can effectively reduce the population of pathogenic bacteria in the field. However, under long-term use, the copper-resistant strains can be isolated from the fields [4,5]. Therefore, introducing a new control method that can complement the conventional methods needs to be considered. Under this context, it is extremely feasible to use antagonistic microorganisms to develop microbial agents.
To develop microbial agents for plant disease control, Bacillus spp. has been extensively studied due to its ability to produce antagonistic compounds and promote plant growth [6,7]. Aside from that, this type of bacteria was used in the development of many commercialized and registered microbial preparations because of their excellent characteristics in agricultural applications [8]. Among them, Bacillus amyloliquefaciens PMB04 has been proven to control the fruit blotch of watermelon, black rot of cabbage, and anthracnose of strawberry through its strong antagonistic activity against pathogens [9][10][11]. Liquid-state fermentation is widely used, as it can provide more nutrients and oxygen in a shorter time [12]. Reports reveal that the production of antagonistic compounds from Bacillus spp. can be improved by adjusting the formulation of fermentation liquids, and these fermentation liquids exhibit better biocontrol effects on plant diseases [12,13]. Even in the study of B. amyloliquefaciens PMB05, it has been shown that the adjustment of the fermentation liquid formula can also enhance the function of the strain by intensifying plant immunity and exerting better disease control ability in the field [11,14]. Thus, whether this bacterial strain has a good antagonistic effect on the pathogen of bacterial leaf spot disease on sweet peppers and whether it can be used to establish a fermentation liquid to prevent the occurrence of the disease is worth being investigated. In this study, we first confirmed that B. amyloliquefaciens PMB04 has good antagonistic activity against different strains of X. perforans. Subsequently, the effect of using a distinct brown sugar and yeast powder ratio in the formula, regarded as carbon and nitrogen materials, respectively, on the bacterial population and sporulation, was analyzed after fermentation. These fermentation liquids were used in the soaking treatment to analyze which formula had the best control effect on the bacterial leaf spot disease of sweet peppers. Moreover, the fermentation liquid with the best control effect was also used to analyze the optimal dilutions for the actual application. In order to effectively combine the use of copper-containing fungicides, in addition to analyzing the antibacterial activities of these fungicides against B. amyloliquefaciens PMB04 and X. perforans, the mixed treatment of fermentation liquid and a coppercontaining fungicide was analyzed to determine if there was any synergistic effect on disease control. In this study, we provide evidence that applying the B. amyloliquefaciens PMB04 fermentation liquid on leaves of sweet pepper is effective in reducing bacterial leaf spot disease, and its effect was not affected by its combination with copper-containing fungicides.

Growth Conditions for Plants and Bacteria
The cultivar of the sweet pepper (Capsicum annuum L.) used in this study was Blue Star (Known-You Seed Co., Kaohsiung, Taiwan). The seeds were sown in a 4.5 cm roundhole tray containing sterilized peat moss, and individual 2-week-old seedlings (with 2 true leaves) were transplanted to a 6 cm pot. The seedlings were grown in a growth chamber (Model F-1200, Hipoint, Kaohsiung, Taiwan) at 28 °C under 16 h of light and 8 h of darkness. The 4-week-old seedlings with 4-6 true leaves were used in subsequent experimental analyses.
Bacterial strains of X. perforans, isolated from the field in different regions (collected from the diseased leaves of sweet peppers), were analyzed by using and blasting their 16S rDNA sequences in the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/ (accessed on 12 July 2023)). The amplification was carried out by using the universal primers 27f and 1525r [15]. Subsequently, these strains were also reconfirmed using the specific primer Bs-Xpf/Bs-Xpr of X. perforans [16]. All the bacterial strains, including B. amyloliquefaciens PMB04 [11] and X. perforans, were cultured on nutrient agar (NA) plates at 28 °C for 48 h.

Inhibitory Assay of B. amyloliquefaciens PMB04 against X. perforans Strains
To investigate whether B. amyloliquefaciens PMB04 has a broad-spectrum antagonistic effect against different strains of X. perforans, the bacterial strains isolated from the field in different regions were used in the assay. The assay was carried out using the double-layer method. Firstly, 100 µL of the bacterial suspension (OD600 at 0.3, the bacterial number was approximately 3 × 10 8 CFU/mL), prepared from each pathogenic bacterial strain, was applied to 5 mL of soft nutrient agar (nutrient broth containing 0.7% of agar), and then, the mixture was poured onto the NA plate. Two pieces of 8 mm filter paper discs (Advantec, Irvine, CA, USA) were placed on the agar plate, and 20 µL of the PMB04 bacterial suspension (OD600 at 0.3, the bacterial number was around 3 × 10 8 CFU/mL), or distilled water as a negative control, was dropped onto the filter paper discs. The inhibitory zones were measured at 48 h after incubation at 28 °C. This experiment was carried out with three plates as repeats, and three experimental repetitions were performed for each assay.

Effect of B. amyloliquefaciens PMB04 on the Control of Bacterial Leaf Spot Disease
To understand the efficacy of B. amyloliquefaciens PMB04 on the control of bacterial spots, the bacterial suspension of B. amyloliquefaciens PMB04 was first used in this study. To prepare the bacterial suspension of B. amyloliquefaciens PMB04 or X. perforans XL1, the colonies on the plate were washed with sterilized 0.1% carboxymethyl cellulose (CMC, Sigma, St. Louis, MO, USA) solution and then, we further adjusted its OD600 value to 0.3 (about 3.0 × 10 8 CFU/mL). Inoculation assays were performed with modifications from established inoculation protocols [17]. Before inoculation, the whole aboveground part of the 4-6 leaf seedlings was soaked in the bacterial suspension for 30 s, and the treated seedlings were placed in a ventilated area to dry naturally. Then, the inoculation of X. perforans XL1 was performed with its bacterial suspension for 30 s using the same method. The inoculated plants were wrapped in a transparent plastic bag to maintain moisture and placed in a growth chamber at 28 °C for 7 days. The occurrence of symptoms was observed at 14 days post-inoculation. To calculate the disease severity, the second-spread leaves from three individual plants were used to evaluate the disease index. The determination of the disease index was based on the scales of developed symptoms in a 4 cm 2 (2 cm × 2 cm) area in the middle of the leaf (0: no disease symptoms; 1: less than 5 yellowing spots; 2: more than five yellowing spots; 3: more than 5 lesions with necrotic symptoms; 4: healed necrotic lesions; 5: healed necrotic lesions with shot hole.) The disease severity of each leaf was calculated using the following formula: [(0 × N0 + 1 × N1 + 2 × N2 + 3 × N3 + 4 × N4 + 5 × N5)/(5 × number of leaves)] × 100% [18]. The disease severities were calculated from five sets of plants in each treatment.

Effects of Different Fermentation Formulas on Bacterial Population and Sporulation of 30 L Harvested Fermentation Liquid
To understand the effect of the fermentation formula on a bacterial population and the sporulation of B. amyloliquefaciens PMB04 in fermentation liquids, the assay was carried out in a 30 L tank (BTF-B30L, Bio-top Process & Equipment Inc., Nantou County, Taiwan). To begin the fermentation process, a 2% volume of overnight culture of B. amyloliquefaciens PMB04, prepared from Luria-Bertani (LB) broth, was added into the sterilized fermentation formula and further incubated at 37 °C under 120 rpm for 5 days [19]. The fermentation formula (3-1, 2-1, and 1-1) was tested by adjusting the weight percentage of the granulated sugar from 3% to 1% in the case of a 1% yeast extract (Sunright, New Taipei City, Taiwan). In addition, a formula (0.5-0.5) composed of 0.5% brown sugar and 0.5% yeast extract was also used for analysis. In the analysis of the bacterial population of distinct fermentation liquids, samples were taken and determined using the serial dilution method. In terms of the sporulation, the analysis was performed according to the standard method [20]. Afterward, 10 mL of each fermentation liquid was taken and incubated at 70 °C for 30 min in the water bath. The number of surviving endospores in the fermentation liquid was also determined by serial dilution. The sporulation of fermentation liquid was calculated using the following formula: Sporulation (%) = (the number of endospores/total bacterial population) × 100%. Each fermentation liquid was sampled three times as repeats, and a total of three independent analyses were performed.

Control Effect of B. amyloliquefaciens PMB04 Fermentation Liquids on Bacterial Spot of Sweet Pepper
To understand the effects of the formulation differences on the control of bacterial leaf spot disease on sweet pepper, a distinct B. amyloliquefaciens PMB04 fermentation liquid was applied in the inoculation assay. The 200-fold-diluted fermentation liquids were used as the initial test concentration [19]. For each treatment, the 4-week-old seedlings were soaked in a 200-fold dilution of fermentation liquid for 30 s. A total of 0.1% of CMC was used as the blank treatment. After the water film on the leaves was dried, the treated seedlings were then soaked in the bacterial suspension of X. perforans XL1 for 30 s. Then, the disease severity was determined, as described above, at 14 days post-inoculation.

Sensitivity of B. amyloliquefaciens PMB04 and X. perforans XL1 to Copper-Containing Fungicides
To evaluate whether commonly used copper-containing fungicides have the potential to be used together with fermentation liquid in the field, the effects of copper-containing fungicides on the growth of B. amyloliquefaciens PMB04 and X. perforans XL1 were assayed in nutrient broth. Before the assay, bacterial suspensions of B. amyloliquefaciens PMB04 and X. perforans XL1 at OD600 0.3 were prepared. A total of 100 µL of bacterial suspension was added into 5 mL of nutrient broth containing 0.54 mg ai mL −1 of tribasic copper sulfate (NUFARM GmbH & Co KG, St. Peter-Strass, Australia, 500×) or 0.27 mg ai mL −1 of copper hydroxide (Corteva Agriscience, Houston, TX, USA, 2000×). Sterilized water was used in the blank treatment as a negative control. The OD600 values were determined after the mixture was incubated at 28 °C under 200 rpm for 12 h and 24 h. The experiment was performed with three repeats for each treatment.

Effect of B. amyloliquefaciens PMB04 Fermentation Liquid Filtrate against X. perforans
To realize whether the fermentation liquid of B. amyloliquefaciens PMB04 has a better inhibitory effect of inhibiting X. perofrans than the culture broth, this experiment was conducted with the filtrates obtained from the fermentation liquid (0.5-0.5, named PMB4FL) and culture broth for analysis. To obtain the filtrates, all the materials were centrifuged at 8000× g for 10 min and further filtered with a 0.22 µm filter. A total of 500 µL of filtrate was added into 500 µL of the X. perforans XL1 bacterial suspension. After incubation at 28 °C for 8 h, 1 mL of the mixture was transferred to a new microtube and stained with 1.5 µL of SYTO 9 (Thermo, Waltham, MA, USA) in the dark for 30 min. The images were observed under a specific filter (Excitation/Emission: 465-495 nm/515-555 nm) under a fluorescent microscope (Leica, Wetzlar, Germany). The images were used to calculate the fluorescence intensities by using the ImageJ software (https://imagej.nih.gov/ij/ (accessed on 1 March 2023)). In each treatment, 10 images were taken as repeats.

Effect of Copper Hydroxide on B. amyloliquefaciens PMB04 Fermentation Liquid in the Control of Bacterial Leaf Spot of Sweet Pepper
To understand whether the copper-containing fungicides would affect the controlled effect of the B. amyloliquefaciens PMB04 fermentation liquid on bacterial leaf spot disease on sweet pepper, the copper hydroxide copper agent and the fermentation liquid (PMB4FL) were used in the assay. Before inoculation, the 4-week-old seedlings were soaked in the 200-fold-diluted PMB4FL, 2000-fold-diluted copper hydroxide, or the mixture containing 200-fold-diluted PMB4FL and 2000-fold-diluted copper hydroxide for 30 s. The treatment with 0.1% of CMC was used as the blank treatment. After the water film on the leaves was dried, the treated seedlings were then soaked in the bacterial suspension of X. perforans XL1 for 30 s to perform the inoculation. The disease severity was determined as described above at 14 days post-inoculation.

Data Analysis
Statistical analyses were performed using SPSS Statistics software for Windows, version 25 (IBM Corp, Armonk, NY, USA). Analysis of variance (ANOVA) and post hoc tests (Tukey's HSD) were used to analyze the significant differences between treatments in the assays (p < 0.05).

Inhibitory Effect of Bacillus amyloliquefaciens PMB04 against Xanthomonas perforans Strains
Before all the analyses, the 16S rDNA sequences obtained by amplifying the XL1, XL2, XL3, XL4 and G1 strains isolated from the field were blasted. All strains were identified as X. perforans based on their sequences that shared more than 99.16% of their identity with the X. perforans strains (Supplementary Materials). A positive reaction amplicon of 197 bp could be amplified from all strains after the PCR reaction using the specific primer of X. perforans. To test whether B. amyloliquefaciens PMB04 can broadly inhibit the strains of X. perforans (isolated from the diseased tissue of sweet pepper obtained from different fields), a confrontation assay was performed. The results showed that all X. perforans strains were inhibited by B. amyloliquefaciens PMB04, especially the XL1, XL2, XL4 and G1 strains ( Figure 1). The confrontation assay was performed with the double-layer agar method. The top layer was mixed with the bacterial suspension of each X. perforans strain and then poured on the nutrient agar plates. After a paper disc was placed on the top layer, 20 µL of B. amyloliquefaciens PMB04 bacterial suspension was applied to the paper disc. Panel (A) reveals the quantification of the inhibitory zone. Blank indicates the treatment with sterilized water as a negative control. Different letters above columns indicate significant differences between different bacterial pathogens based on Tukey's HSD test (p < 0.05). Panel (B) shows the permeabilized inhibitory zones of different X. perforans strains produced by B. amyloliquefaciens PMB04 on the NA plate.

Control Efficacy of Bacillus amyloliquefaciens PMB04 Bacterial Suspension on Bacterial Leaf Spot Disease in Sweet Pepper
To confirm whether the strong antagonistic activity of B. amyloliquefaciens PMB04 to X. perforans strains would be able to control bacterial leaf spots, its bacterial suspension was pretreated on sweet pepper. The results showed that the symptom development of bacterial leaf spots was inhibited by B. amyloliquefaciens PMB04. In addition, the disease severity when treated with the PMB04 treatment was significantly reduced to 48.33%, compared to 73.33% of the blank treatment. Meanwhile, the control efficacy was 34.10% ( Figure 2).

Figure 2.
Effect of Bacillus amyloliquefaciens PMB04 bacterial suspension on the control of bacterial leaf spot in sweet pepper. The assay was performed using the soaking method, as described in Materials and Methods. Before inoculation, the seedlings were soaked in a bacterial suspension of B. amyloliquefaciens PMB04 for 30 s. Then, the air-dried seedlings were soaked in a bacterial suspension of X. perforans XL1 for 30 s for the inoculation. Panel (A) reveals the disease severity after inoculation. The * indicates a significant difference compared with the blank treatment, as assessed using a t-test (p < 0.05). Panel (B) shows the visual symptoms of bacterial leaf spots reduced by B. amyloliquefaciens PMB04 on sweet pepper.

Effects of Formulated Differences on the Population and Sporulation of Bacillus amyloliquefaciens PMB04 Fermentation Liquids from a 30 L Fermenter
To realize the effect of brown sugar concentrations in the formulations on the fermentation of B. amyloliquefaciens PMB04, 3% to 1% brown sugar was applied in the basal formula with 1% of yeast extract to evaluate the cell production and sporulation of B. amyloliquefaciens PMB04 in the fermentation liquids. The results exhibited that the cell population of the B. amyloliquefaciens PMB04 in 3-1, 2-1 and 1-1 fermentation liquids were 1.00 × 10 9 , 9.01 × 10 8 and 3.50 × 10 8 CFU/mL, respectively. In addition, in the 0.5-0.5 fermentation liquid, the cell population was 4.83 × 10 8 CFU/mL. The 3-1 and 2-1 formulations exhibited the highest population of PMB04 than other formulations in the fermentation liquid. Moreover, the surviving cells, after heating, exhibited that the sporulation ratios of B. amyloliquefaciens PMB04 reached 100% in all the fermentation liquids (Table 1).

Effects of B. amyloliquefaciens PMB04 Fermentation Liquids on the Control of Bacterial Spot
To evaluate the distinct B. amyloliquefaciens PMB04 fermentation liquids on the control of bacterial leaf spot, a 200-fold dilution of each fermentation liquid was first used in the inoculation assay. The results showed that all the B. amyloliquefaciens PMB04 fermentation liquids significantly reduced the occurrence of bacterial leaf spot disease. In contrast, compared to the disease's severity in the blank treatment (76.67%), those with the 3-1, 2-1 and 1-1 fermentation liquids were 44.44%, 47.50%, 55.56% and 42.50%, respectively ( Figure 3). The control efficacy of the 3-1, 2-1 and 1-1 fermentation liquids were 42.03%, 38.04%, 27.53% and 44.57%. The 3-1 and 0.5-0.5 fermentation liquids exhibited the best control efficacy on bacterial leaf spots of sweet pepper. Since the 0.5-0.5 fermentation liquid (PMB4FL) has shown superior control efficacy on bacterial leaf spot disease of sweet pepper, the assay was further carried out using dilutions with different concentrations. The results showed that treatments with 200×, 500× and 1000× dilutions of PMB4FL reduced the occurrence of bacterial leaf spot, with their disease severities being 46.67%, 54.67% and 63.75%, respectively (Figure 4). Furthermore, the control efficacy of 200×, 500× and 1000× dilutions had a disease severity of 39.13%, 28.70% and 16.85%, respectively. Compared to the blank treatment, the 200× dilution was the most effective treatment in disease control.

Effect of B. amyloliquefaciens PMB04 Fermentation Filtrate on the Survival of X. perforans Cells
To confirm whether the B. amyloliquefaciens PMB04 fermentation liquid with the best control efficacy would affect the cell survival of X. perforans, the filtrates from PMB4FL or LB were applied for analysis. The green fluorescence, indicating the surviving cells, was not notably different in the treatment of the LB culture filtrate compared with the blank at 8 h after treatment; however, there was less fluorescence in the treatment of the PMB4FL filtrate ( Figure 5A). After quantification, the results showed that the relative fluorescence of the PMB4FL filtrate treatment was significantly lower than that of the blank and LB culture filtrate treatments ( Figure 5B).

Inhibitory Effects of Copper-Containing Fungicides against B. amyloliquefaciens PMB04 and X. perforans XL1
To understand the potential impact of the copper-containing fungicides commonly used to control bacterial spot of Solanaceae crops on the application of microbial agents in the field, their effects on the bacterial growth of B. amyloliquefaciens PMB04 and X. perforans were first determined for evaluation. The results showed that the treatment of tribasic copper sulfate or copper hydroxide had no effect on the growth of B. amyloliquefaciens PMB04 compared to the blank treatment. However, both tribasic copper sulfate and copper hydroxide have a superior inhibitory effect on the growth of X. perforans XL1 ( Table  2).

Effects of Copper Hydroxide on PMB4FL in the Control of Bacterial Leaf Spot in Sweet Pepper
To understand the effect of copper-containing fungicides on the control of PMB4FL fermentation liquid, copper hydroxide was used as a model for the control analysis. The results showed that the disease severity of the blank treatment was 77.78%, the disease severities of 200× dilution of PMB4FL alone, 2000× dilution of copper hydroxide alone, the mixture containing 200× dilution of PMB4FL and 2000× dilution of copper hydroxide were 50.00%, 68.75% and 55.56%, respectively ( Figure 6). Only the treatment with PMB4FL alone and the treatment with the mixture of PMB4FL and copper hydroxide could significantly reduce the occurrence of disease, which was 34.78% and 27.54% control efficacy. Although the copper hydroxide treatment has a 10.33% control efficacy, there is no significant difference in the disease severity compared with the blank treatment.

Discussion
With increasing attention to food safety, the use of beneficial microorganisms to control plant diseases is accepted by the public as a way to produce safe agricultural products. It is also a very important topic in agricultural science today. Among the beneficial microorganisms that can be applied, Bacillus spp. has attracted much attention because of its ability to produce endospores to withstand adverse environmental conditions, such as high and low temperatures, chemicals and ultraviolet light [21]. In addition, many bacterial strains in this genus have been reported to produce a variety of secondary metabolites that destroy the cells of pathogenic bacteria or enhance a plant's defense responses to help plants resist diseases [22][23][24]. Currently, many studies in the world have proved that Bacillus spp. strains can effectively reduce the occurrence of various bacterial and fungal diseases by their antagonistic activity [6,25,26], indicating that the metabolites produced by Bacillus spp. are an important mechanism for disease control. In previous reports, it was proved that the antagonism of Bacillus amyloliquefaciens PMB04 to the plant pathogens Acidovorax citrulli, Xanthomonas campestris pv. campestris and Colletotrichum gloeosporioides can be effective in controlling watermelon fruit blotch, cabbage black rot, strawberry anthracnose and mango anthracnose, respectively [9][10][11]19]. Since sweet pepper is an important vegetable crop in central and southern Taiwan, the occurrence of bacterial leaf spot disease caused by Xanthomonas perforans often causes tremendous losses to farmers. Therefore, this study intends to explore the control effects of B. amyloliquefaciens PMB04 on bacterial leaf spot disease of sweet pepper crops and to investigate whether the subsequent adjustment of the fermentation formula can further increase the controlled effect of this disease. In this study, PMB04 showed superior antagonistic activity against most of the X. perforans strains isolated from the field. In many reports, the B. amyloliquefaciens strains have demonstrated that this species has a good ability for plant colonization [27][28][29]. Thus, we suggested that the B. amyloliquefaciens PMB04 may also be able to maintain its population in plants and exert its antagonistic effects. In this study, it was demonstrated that B. amyloliquefaciens PMB04 has a good survival ability on sweet pepper leaf surfaces. From the aforementioned results, it can be speculated that B. amyloliquefaciens PMB04 should have the potential to control bacterial leaf spot disease of sweet pepper crops. In the biocontrol assay, by using the bacterial suspension of B. amyloliquefaciens PMB04, we demonstrated that B. amyloliquefaciens PMB04 can significantly reduce the occurrence of bacterial leaf spot disease.
The application potential of beneficial microorganisms is often limited due to an insufficient number of bacteria or time-consuming cultivation. Using fermentation technology for cultivation can not only increase the number of microbial populations rapidly but also increase the production of enzymes or antagonistic substances [12,[30][31][32]. Among them, liquid fermentation can provide nutrients and oxygen supply directly to the microbial strains within a shorter time frame; thus, it is widely used. In this study, brown sugar and yeast powder were used as the basis of the formulation to explore the effects of different proportions of the formulation on the control of B. amyloliquefaciens PMB04 against bacterial leaf spot disease in sweet pepper. Our results showed that in the four formulations designed in this study, the sporulation rate of all fermentation liquids reached 100%, among which formula 3-1 could obtain the highest bacterial population. Based on these results, we speculated that the bacterial population could indeed be increased under the higher sugar supply, and most of the supply of these nutrient sources can be used up by B. amyloliquefaciens PMB04 to enter the process of endospore formation.
Further use of B. amyloliquefaciens PMB04 fermentation liquids from different formulations to evaluate the control efficacy on bacterial leaf spot disease in sweet pepper showed that all formulations could significantly reduce the occurrence of bacterial leaf spot, and there were no differences between the formulations in the control efficacy. The results indicated that even if the 3-1 formulation could increase the bacterial population after fermentation, such an increase cannot improve the control efficacy against diseases. From this result, it can be speculated that the control efficacy is mainly due to the antagonistic compounds in the fermentation liquids, and it can be further speculated that the formula (0.5-0.5) of the lowest nutrient sources used in this study can effectively produce enough antagonistic substances to control bacterial leaf spot disease. In this study, the follow-up analysis of the 0.5-0.5 formula (PMB4FL) also proved that the PMB4FL fermentation liquid could effectively control the occurrence of bacterial leaf spot disease by using the 200-or 500-fold dilution. This result is similar to the recommended dilutions for tomato bacterial wilt and lemon canker in our previous fermentations prepared with other B. amyloliquefaciens strains [14,33]. In addition, we used the technique of living cell staining to prove that the number of X. perforans living cells was reduced by the filtrate of PMB4FL, and this result can be further speculated in that the X. perforans cells can be killed by the antagonistic substances in the fermentation liquid. Many studies have shown that lipopeptide compounds, such as iturin, fengycin and surfactin, are widely found in Bacillus spp. The inhibitory activities of these compounds against different bacterial pathogens all play an important role in disease control [7,22,33,34]. After the whole genome sequence of B. amyloliquefaciens PMB04 was sequenced (Accession: PRJNA751681), the preliminary prediction results of secondary metabolites by antiSMASH showed that this strain contained existing related genes for producing bacillibactin, bacillaene, bacilysin and fengycin. However, as for what type of antagonistic substances, this will still need to be further investigated.
Enhancing the chances of applying microbial agents in the field requires collaboration with conventional fungicides. Although the causal agents of bacterial leaf spot, X. perforans, may have copper resistance genes that exhibit copper resistance [35], copperbased fungicides are still the main recommended treatment against bacterial leaf spot disease of Solanaceae plants.
Because the mode of action of copper-containing fungicides is a multi-site contact activity, it can widely inhibit the growth of microorganisms [36]. In the use of PMB4FL to control bacterial leaf spot disease of sweet pepper, it is worth evaluating whether it will be interfered with by copper-containing fungicide applications. The results of the analyses of copper hydroxide and tribasic copper sulfate in the NB culture medium showed that these two fungicides still had significant inhibitory effects on the growth of the tested X. perforans strain; however, they had no inhibitory effect on the growth of B. amyloliquefaciens PMB04. The results of the control efficacy assay showed that copper hydroxide had no significant control effect on sweet pepper bacterial leaf spot. Previously, Obradovic et al. demonstrated that bacterial leaf spot disease can be reduced by the application of copper hydroxide [37]. We hypothesize that the main reason for the difference between our results and the literature may be due to the differences in bacterial strains. In addition, we speculated that, although the growth of X. perforans can be inhibited in a liquid medium, it may not be able to show ideal inhibitory activity on the leaves to reduce the disease. However, the addition of copper hydroxide could not increase or decrease the effect of the PMB4FL fermentation liquid on the control of bacterial leaf spots. The result is similar to that achieved by Korsten et al., that Bacillus subtilis and copper oxychloride were used together to control avocado diseases, and copper oxychloride could not increase the control effect of Bacillus subtilis [38]. From these results, it can be inferred that B. amyloliquefaciens PMB04 should be tolerant to copper, and the PMB4FL fermentation liquid can be applied alone in the field in the future to exert the effect of controlling bacterial leaf spot disease in sweet pepper.

Conclusions
In this study, we confirmed that B. amyloliquefaciens PMB04 has good antagonistic activity against the X. perforans strains. We have also established a formula that requires only very streamlined nutrient sources to obtain a PMB4FL fermentation liquid that is quite excellent in terms of bacterial population and sporulation. At the same time, the PMB4FL fermentation liquid can effectively control bacterial leaf spot disease in sweet pepper at a concentration of 200-fold dilution. More importantly, this study proves that the application of PMB4FL fermentation liquid will not be impaired by the application of a copper-containing fungicide, and its control effect has the potential to be applied in the field.