Anti-Virulence Strategy of Novel Dehydroabietic Acid Derivatives: Design, Synthesis, and Antibacterial Evaluation

Anti-virulence strategies are attractive and interesting strategies for controlling bacterial diseases because virulence factors are fundamental to the infection process of numerous serious phytopathogenics. To extend the novel anti-virulence agents, a series of dehydroabietic acid (DAA) derivatives decorated with amino alcohol unit were semi-synthesized based on structural modification of the renewable natural DAA and evaluated for their antibacterial activity against Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac), and Pseudomonas syringae pv. actinidiae (Psa). Compound 2b showed the most promising antibacterial activity against Xoo with an EC50 of 2.7 μg mL−1. Furthermore, compound 2b demonstrated remarkable control effectiveness against bacterial leaf blight (BLB) in rice, with values of 48.6% and 61.4% for curative and protective activities. In addition, antibacterial behavior suggested that compound 2b could suppress various virulence factors, including EPS, biofilm, swimming motility, and flagella. Therefore, the current study provided promising lead compounds for novel bactericides discovery by inhibiting bacterial virulence factors.


Introduction
Plant diseases are significant threats to crop products worldwide due to the diverse bacterial virulence factors (VFs) promoting pesticide resistance. Rice is the main cash crop worldwide. However, Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial leaf blight (BLB), resulted in 20-60% yield losses during the post-pandemic period [1][2][3]. VFs significantly contribute to the BLB outbreak and are the main cause of pesticide resistance to traditional bactericides.
Instead of existing as planktonic cells, pathogens predominantly survived in the environment containing affluent biofilms, contributing significantly to their pathogenicity in natural environments. Bacterial infections are caused by numerous virulence factors, such as biofilm, extracellular polysaccharide (EPS), swimming motility, flagella, etc. The literature revealed that approximately 80% of bacterial infections involve biofilm that promotes resistance [4][5][6][7]. Meanwhile, EPS is a key element of bacterial biofilm and enhances cell adhesion on the surface of plants [8][9][10]. Additionally, swimming motility during the infection cycle allows bacteria to travel away from the harmful environment and into the plant tissue, which they can quickly colonize [6]. Swimming motion and bacterial flagella are closely related [11,12], as many bacteria use flagellum for swimming motility [10]. It is clear that VFs are important for bacterial pathogens and are sometimes required for infections [13]. Thus, discovering bacterial virulence inhibitors based on natural products is an appealing method for managing persistent bacterial diseases effectively.
The inhibiting bacterial mechanisms of bioactive natural products and their derivatives are well-established [14][15][16][17]. Notably, natural dehydroabietic acid (DAA) and its derivatives infections [13]. Thus, discovering bacterial virulence inhibitors based on natural produ is an appealing method for managing persistent bacterial diseases effectively.
The inhibiting bacterial mechanisms of bioactive natural products and their deriv tives are well-established [14][15][16][17]. Notably, natural dehydroabietic acid (DAA) and its d rivatives exhibit a broad range of biological activities, and it is a significant renewa forestry resource [17]. As shown in Figure 1, some dehydroabietic acid derivatives h outstanding antibacterial and antibiofilm activity [18][19][20][21]. Meanwhile, the amino alcoh unit exists widely in some drugs and antibacterials. Therefore, the above-mentioned co pounds with amino alcohol and DAA moieties demonstrated strong antibacterial acti ties [14,22,23] and were used to develop pesticides to control several plant diseases. In t work, to excavate new anti-virulence agents, a series of amino alcohol-DAA compoun were prepared by adding an amino alcohol moiety to a DAA natural skeleton. The eva ation of the antibacterial mechanism also suggested that the DAA derivative 2b fu tioned as a potential virulence factor inhibitor for regulating rice BLB. Figure 1. Some commercial and reported bioactive structures with an amino alcohol moiety a dehydroabietic acid and the method for producing target molecules [15,[18][19][20][21]24]

Synthesis of DAA Derivatives
According to previous methods [14,[22][23][24], a series of DAA derivatives with ami alcohol moiety were exquisitely synthesized using combinatorial chemistry. The desi concept of the target compounds is shown in Figure 1. Intermediate 1 was obtained introducing epoxybromopropane, and target compounds were synthesized through epoxy ring opening reaction. The detailed experimental protocol for synthesizing co pounds was found in the supporting information (The spectra data of title compound w displayed in Figures S1-56).

Synthesis of DAA Derivatives
According to previous methods [14,[22][23][24], a series of DAA derivatives with amino alcohol moiety were exquisitely synthesized using combinatorial chemistry. The design concept of the target compounds is shown in Figure 1. Intermediate 1 was obtained by introducing epoxybromopropane, and target compounds were synthesized through an epoxy ring opening reaction. The detailed experimental protocol for synthesizing compounds was found in the supporting information (The spectra data of title compound was displayed in Figures S1-S56).

Inhibitory Effects of Compound 2b on the Xoo-Biofilm Formation and EPS Production
Biofilm, as one of the important VFs, is a significant and highly conserved structure for the bacterial community that acts as a crucial physical barrier against various complex environmental threats, including pH, temperature changes, host defense, and antibiotics [25][26][27]. Consequently, biofilm may significantly increase bacterial resistance [28][29][30][31] and is responsible for 80% of bacterial infections [4,32]. Additionally, EPS is the main component of the biofilm that promotes adherence to host surfaces [33,34]. However, Xanthomonas species such as Xoo and Xanthomonas campestris pv. campestris secreted the EPS known as xanthan gum. Therefore, xanthan gum would be used as a Xoo (a kind of Xanthomonas) indicator for detecting EPS production. To verify the experimental concentration is suitable, namely, that compound 2b displayed anti-virulence activity rather than killing activity, the OD595 value was assayed (Table S2). Notably, when the dosage was 5.40 μg mL −1 , compound 2b did not show any bactericidal activity. Thus, the biofilm formation assay is carried out.

Inhibitory Effects of Compound 2b on the Xoo-Biofilm Formation and EPS Production
Biofilm, as one of the important VFs, is a significant and highly conserved structure for the bacterial community that acts as a crucial physical barrier against various complex environmental threats, including pH, temperature changes, host defense, and antibiotics [25][26][27]. Consequently, biofilm may significantly increase bacterial resistance [28][29][30][31] and is responsible for 80% of bacterial infections [4,32]. Additionally, EPS is the main component of the biofilm that promotes adherence to host surfaces [33,34]. However, Xanthomonas species such as Xoo and Xanthomonas campestris pv. campestris secreted the EPS known as xanthan gum. Therefore, xanthan gum would be used as a Xoo (a kind of Xanthomonas) indicator for detecting EPS production. To verify the experimental concentration is suitable, namely, that compound 2b displayed anti-virulence activity rather than killing activity, the OD 595 value was assayed (Table S2). Notably, when the dosage was 5.40 µg mL −1 , compound 2b did not show any bactericidal activity. Thus, the biofilm formation assay is carried out.
xanthan gum biosynthesis process decreased with increasing concentrations. Therefore, compound 2b had the potential to inhibit bacterial biofilm formation.   xanthan gum biosynthesis process decreased with increasing concentrations. Therefore, compound 2b had the potential to inhibit bacterial biofilm formation.

The Inhibition Effect of Swimming Motility
Most phytopathogenic bacteria, including Ralstonia solanacearum and Xanthomonas, display good swimming motility [35], with bacterial swimming being the fastest mode of motility [12]. Swimming motility enables bacteria to sense environmental changes, avoid harmful environmental stressors, and move toward nutrients, consequently markedly enhancing bacterial fitness [36,37]. Figure 6 showed that swimming diameter decreased with increasing doses. The swimming diameters at concentrations of 0, 1.35, 2.70, 5.40, and 10.8 µg mL −1 were 13.8, 11.5, 7.7, 3.6, and 0 mm, respectively; bacterial swimming motility was gradually weakened. Compound 2b inhibited bacterial motility levels, lowered fitness, and decreased infections.

The Inhibition Effect of Swimming Motility
Most phytopathogenic bacteria, including Ralstonia solanacearum and Xanthomonas, display good swimming motility [35], with bacterial swimming being the fastest mode of motility [12]. Swimming motility enables bacteria to sense environmental changes, avoid harmful environmental stressors, and move toward nutrients, consequently markedly enhancing bacterial fitness [36,37]. Figure 6 showed that swimming diameter decreased with increasing doses. The swimming diameters at concentrations of 0, 1.35, 2.70, 5.40, and 10.8 μg mL −1 were 13.8, 11.5, 7.7, 3.6, and 0 mm, respectively; bacterial swimming motility was gradually weakened. Compound 2b inhibited bacterial motility levels, lowered fitness, and decreased infections.

The Inhibition Effect of Xoo-Flagellum Assembly
The flagellum is the important bacterial organelle responsible for swimming motility [38], comprising the filament, hook, and basal body [38]. The ability of bacterial cells to move toward beneficial environments and escape harmful environmental stressors, and the swimming motility mediated by the bacterial flagellum, play a significant role in the bacterial infection cycle, increasing the probability of cells interacting with hosts' surfaces [39,40]. The ability was extremely beneficial for enhancing search potency, enabling bacteria to seek advantages and avoid disadvantages. It revealed that a bacterium's virulence toward its host was significantly influenced by flagellum-mediated swimming motility [41,42]. Swimming motility, chemotaxis, and host cell invasion increased the likelihood of bacteria interacting with host organism surfaces during the infection [13]. Furthermore, the mutation of flagella-related genes resulted in a loss of motility, reduction in bacterial colonization, downregulation of host cell immunity, decrease the virulence, and reduction in pathogenicity [13,42]. Thus, the flagellum, which also contains swimming motility, initial attachment, tissue invasion, and biofilm formation, contributes to bacterial virulence and infection [13,42].
As shown in Figure 7,

The Inhibition Effect of Xoo-Flagellum Assembly
The flagellum is the important bacterial organelle responsible for swimming motility [38], comprising the filament, hook, and basal body [38]. The ability of bacterial cells to move toward beneficial environments and escape harmful environmental stressors, and the swimming motility mediated by the bacterial flagellum, play a significant role in the bacterial infection cycle, increasing the probability of cells interacting with hosts' surfaces [39,40]. The ability was extremely beneficial for enhancing search potency, enabling bacteria to seek advantages and avoid disadvantages. It revealed that a bacterium's virulence toward its host was significantly influenced by flagellum-mediated swimming motility [41,42]. Swimming motility, chemotaxis, and host cell invasion increased the likelihood of bacteria interacting with host organism surfaces during the infection [13]. Furthermore, the mutation of flagella-related genes resulted in a loss of motility, reduction in bacterial colonization, downregulation of host cell immunity, decrease the virulence, and reduction in pathogenicity [13,42]. Thus, the flagellum, which also contains swimming motility, initial attachment, tissue invasion, and biofilm formation, contributes to bacterial virulence and infection [13,42].
As shown in Figure 7, the percentage of flagellum assembly was 0.78, 0.49, and 0.26 at doses of 0, 0.27, and 1.35 µg mL −1 . These findings show compound 2b strongly interfered with the flagellum assembly process at concentrations 0.27 and 1.35 µg mL −1 . Moreover, bacterial flagellum assembly interfered similarly at a dose of 0 µg mL −1 (served as a control). The primary cause was that some bacteria had mature flagellum while others were at the initiation or growth stage. The assembly and disassembly of the flagellum, a dynamic nanostructure, was coupled with the cell cycle [43]. Therefore, we can infer that the cells in the initiation and growth stages did not complete the assembly of their flagella. These findings suggested compound 2b might significantly interfere with bacterial flagellum assembly and cause virulence downregulation. Moreover, bacterial flagellum assembly interfered similarly at a dose of 0 μg mL −1 (served as a control). The primary cause was that some bacteria had mature flagellum while others were at the initiation or growth stage. The assembly and disassembly of the flagellum, a dynamic nanostructure, was coupled with the cell cycle [43]. Therefore, we can infer that the cells in the initiation and growth stages did not complete the assembly of their flagella. These findings suggested compound 2b might significantly interfere with bacterial flagellum assembly and cause virulence downregulation.

Cell Membrane Morphology Analysis
The cell membrane plays a role in nutrient intake, biomacromolecule transportation, and signal transduction, as well as being a considerable barrier to complicate external environmental stresses [31]. Thus, the cell membrane plays a significant role in these physiological and biochemical processes. A loss in bacterial cell membrane integrity results in increased membrane permeability, which impacts cell physiology and metabolism, leading to cytoplasm leakage and cell death [32,[44][45][46][47]. Additionally, the membrane reportedly plays a crucial role in preserving cell homeostasis, with a loss of membrane integrity leading to the end of the cellular life cycle [30,48]. Figure 7 indicated that compound 2b affected flagella assembly but not the morphology of cellular membranes. In addition, the

Cell Membrane Morphology Analysis
The cell membrane plays a role in nutrient intake, biomacromolecule transportation, and signal transduction, as well as being a considerable barrier to complicate external environmental stresses [31]. Thus, the cell membrane plays a significant role in these physiological and biochemical processes. A loss in bacterial cell membrane integrity results in increased membrane permeability, which impacts cell physiology and metabolism, leading to cytoplasm leakage and cell death [32,[44][45][46][47]. Additionally, the membrane reportedly plays a crucial role in preserving cell homeostasis, with a loss of membrane integrity leading to the end of the cellular life cycle [30,48]. Figure 7 indicated that compound 2b affected flagella assembly but not the morphology of cellular membranes. In addition, the properties of VF inhibitors did not hinder cellular development and proliferation at a low dose of compound 2b. Therefore, SEM technology was used to analyze the morphology of cell membranes. Figure 8 showed the morphology observation results: bacterial morphology was unchanged after treatment with varying doses of 0, 25, and 50 µg mL −1 and showed a smooth surface and rod-shaped structure. As a result, compound 2b only affected biological processes related to bacterial VFs and did not affect the structure of bacterial cell membranes or interfere with normal cellular growth and proliferation. properties of VF inhibitors did not hinder cellular development and proliferation at a low dose of compound 2b. Therefore, SEM technology was used to analyze the morphology of cell membranes. Figure 8 showed the morphology observation results: bacterial morphology was unchanged after treatment with varying doses of 0, 25, and 50 μg mL −1 and showed a smooth surface and rod-shaped structure. As a result, compound 2b only affected biological processes related to bacterial VFs and did not affect the structure of bacterial cell membranes or interfere with normal cellular growth and proliferation.

Pathogenicity of the Xoo Interacted with Compound 2b
Several VFs strongly correlate with the pathogenicity of infections [49]. VFs secreted by different bacteria are crucial for promoting cell colonization and enhancing pathogenicity during infection [50,51]. Pathogens use several VFs to overcome the host's defense system [52]. Thus, bacterial pathogenicity depends on their ability to secrete numerous VFs [53]. R. solanacearum uses various VFs to infect plants and cause a withering phenomenon. EPS, also known as xanthan gum in Xoo, lipopolysaccharides, extracellular enzymes (including amylase, endoglucanase, polygalacturonate lyase, and protease), and biofilm are some significant VFs that have been found in many bacteria [54,55]. Additionally, bacterial motility and flagella facilitate cell colonization and adhesion [55]. Erwinia amylovora develops many VFs to overcome the plant immune system and facilitate infection [56]. The bacterium Xanthomonas campestris pv. campestris encodes for type III secretion system-dependent transcription activator-like effectors, among other VFs [54]. Massive VFs may interfere with the host's vascular system and cause wilting symptoms [57]. Cell and tissue damage caused by Staphylococcus aureus and Nocardia adhesion and invasion are important pathogenetic factors [26]. A thorough analysis revealed that antibacterial peptides reduced bacterial pathogenicity by inhibiting VFs activity [58]. Disrupting the secretion and assembly of VFs has been associated with several anti-virulence compounds, including those that inhibit biofilm formation, lower EPS production, and interfere with initial bacterial adhesion [59]. The mechanism of VFs in host infection has been gradually explored and excavated thanks to advancements in molecular biology techniques and a comprehensive understanding of VFs. Targeting VFs would be a desirable and practical method to eliminate or reduce bacterial pathogenicity and weaken resistance by interfering with virulence biosynthesis processes as opposed to cell death because VFs are crucial for bacterial infections [6,60,61].
Thus, the analysis of Xoo-pathogenicity was performed based on the results of the above-mentioned completed experiments, and compound 2b significantly disrupted Xoovirulence biological processes. As shown in Figure 9, the Xoo cells suspension was coincubated with compound 2b for one day at the different doses of 0 (referred to as the control), 0.27, 1.35, and 2.70 μg mL −1 . The rice plant was then inoculated with the above-

Pathogenicity of the Xoo Interacted with Compound 2b
Several VFs strongly correlate with the pathogenicity of infections [49]. VFs secreted by different bacteria are crucial for promoting cell colonization and enhancing pathogenicity during infection [50,51]. Pathogens use several VFs to overcome the host's defense system [52]. Thus, bacterial pathogenicity depends on their ability to secrete numerous VFs [53]. R. solanacearum uses various VFs to infect plants and cause a withering phenomenon. EPS, also known as xanthan gum in Xoo, lipopolysaccharides, extracellular enzymes (including amylase, endoglucanase, polygalacturonate lyase, and protease), and biofilm are some significant VFs that have been found in many bacteria [54,55]. Additionally, bacterial motility and flagella facilitate cell colonization and adhesion [55]. Erwinia amylovora develops many VFs to overcome the plant immune system and facilitate infection [56]. The bacterium Xanthomonas campestris pv. campestris encodes for type III secretion system-dependent transcription activator-like effectors, among other VFs [54]. Massive VFs may interfere with the host's vascular system and cause wilting symptoms [57]. Cell and tissue damage caused by Staphylococcus aureus and Nocardia adhesion and invasion are important pathogenetic factors [26]. A thorough analysis revealed that antibacterial peptides reduced bacterial pathogenicity by inhibiting VFs activity [58]. Disrupting the secretion and assembly of VFs has been associated with several anti-virulence compounds, including those that inhibit biofilm formation, lower EPS production, and interfere with initial bacterial adhesion [59]. The mechanism of VFs in host infection has been gradually explored and excavated thanks to advancements in molecular biology techniques and a comprehensive understanding of VFs. Targeting VFs would be a desirable and practical method to eliminate or reduce bacterial pathogenicity and weaken resistance by interfering with virulence biosynthesis processes as opposed to cell death because VFs are crucial for bacterial infections [6,60,61].
Thus, the analysis of Xoo-pathogenicity was performed based on the results of the above-mentioned completed experiments, and compound 2b significantly disrupted Xoovirulence biological processes. As shown in Figure 9, the Xoo cells suspension was coincubated with compound 2b for one day at the different doses of 0 (referred to as the control), 0.27, 1.35, and 2.70 µg mL −1 . The rice plant was then inoculated with the abovementioned cell suspension using the leaves clipping method. Subsequently, the Xooinoculated rice plant was cultured for 14 days. Finally, samples from the control treatment showed more lathy lesions with a length of 11.5 cm. The lesion lengths of other treatments, 0.27, 1.35, and 2.70 µg mL −1 were 8.7, 5.9, and 2.9 cm, respectively. As a result of interfering with the manufacture of multiple bacterial VFs, compound 2b demonstrated the ability to suppress Xoo-VFs and strongly reduce bacterial pathogenicity. Compound 2b would be a potent virulence inhibitor to manage rice BLB successfully.
Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 10 of 17 mentioned cell suspension using the leaves clipping method. Subsequently, the Xoo-inoculated rice plant was cultured for 14 days. Finally, samples from the control treatment showed more lathy lesions with a length of 11.5 cm. The lesion lengths of other treatments, 0.27, 1.35, and 2.70 μg mL −1 were 8.7, 5.9, and 2.9 cm, respectively. As a result of interfering with the manufacture of multiple bacterial VFs, compound 2b demonstrated the ability to suppress Xoo-VFs and strongly reduce bacterial pathogenicity. Compound 2b would be a potent virulence inhibitor to manage rice BLB successfully.

In Vivo Anti-Xoo Effect of Compound 2b Controlling Bacterial Disease at 200 μg mL −1
Although target compound 2b demonstrated excellent antibacterial activity and an alluring anti-VFs mechanism, the preservation of the crops was our ultimate goal. Therefore, the antibacterial activity of compound 2b was investigated in vivo to verify its anti-Xoo activity. Compound 2b demonstrated excellent curative and protective activities, and the control efficiency was 48.6% and 61.4%, respectively ( Figure 10 and Table 3). Compound 2b exhibited significant antibacterial activity in vitro and exceptional control efficiency in vivo. Although target compound 2b demonstrated excellent antibacterial activity and an alluring anti-VFs mechanism, the preservation of the crops was our ultimate goal. Therefore, the antibacterial activity of compound 2b was investigated in vivo to verify its anti-Xoo activity. Compound 2b demonstrated excellent curative and protective activities, and the control efficiency was 48.6% and 61.4%, respectively ( Figure 10 and Table 3). Compound 2b exhibited significant antibacterial activity in vitro and exceptional control efficiency in vivo.   Although compound 2b showed remarkable antibacterial activity in vitro and in vivo, its phytotoxicity for the target crop remained unknown. Therefore, the phytotoxicity of compound 2b was assessed at doses of 0, 200, and 500 μg mL −1 . As shown in Figure 11 and Table S3, compound 2b did not affect the normal growth of rice leaves and did not cause any lesions or necrosis. Furthermore, as found in Figure S57, predicting results suggested that the title compounds exhibited acceptable physicochemical properties. Thus, it would be a highly effective and low-risk green pesticide option. Although compound 2b showed remarkable antibacterial activity in vitro and in vivo, its phytotoxicity for the target crop remained unknown. Therefore, the phytotoxicity of compound 2b was assessed at doses of 0, 200, and 500 µg mL −1 . As shown in Figure 11 and Table S3, compound 2b did not affect the normal growth of rice leaves and did not cause any lesions or necrosis. Furthermore, as found in Figure S57, predicting results suggested that the title compounds exhibited acceptable physicochemical properties. Thus, it would be a highly effective and low-risk green pesticide option.  Although compound 2b showed remarkable antibacterial activity in vitro and in vivo, its phytotoxicity for the target crop remained unknown. Therefore, the phytotoxicity of compound 2b was assessed at doses of 0, 200, and 500 μg mL −1 . As shown in Figure 11 and Table S3, compound 2b did not affect the normal growth of rice leaves and did not cause any lesions or necrosis. Furthermore, as found in Figure S57, predicting results suggested that the title compounds exhibited acceptable physicochemical properties. Thus, it would be a highly effective and low-risk green pesticide option. Figure 11. The phytotoxicity assessment of compound 2b on rice leaves after co-culturing it for seven days at 0, 200, and 500 µg mL −1 .

Instruments and Chemicals
Thin-layer chromatography plates were used to monitor organic reaction processes (Yantai Jiangyou Silica Development Co., Ltd., Silica HSGF254, Shandong, China) [51]. Bruker AG-400 (Switzerland) and JEOLECX-500 (Japan) were used to measure the 1 H and 13 C nuclear magnetic resonance spectra of the DAA derivatives using CDCl 3 (Anhui Zesheng Technology Co., Ltd, Energy-Chemical, China) or DMSO-d 6 (Anhui Zesheng Technology Co., Ltd, Energy-Chemical, China) as the solvent and internal standard, respectively. Related chemical shifts and coupling constants (J) were represented as parts per million and hertz, respectively. A Thermo Scientific Q Exactive UItiMate 3000 instrument was used to determine the High-resolution mass spectrometry (HRMS) of DAA derivatives. FEI Talos, F200C electron microscope (FEI, USA) images were collected using a transmission electron microscope (TEM) at a voltage of 200 kV. The morphology of phytobacteria was investigated using an FEI Nova NanoSEM 450 (FEI, USA) instrument. DAA (purity > 75%) was used as the starting material purchased from Anhui Zesheng Technology Co., Ltd, Energy-Chemical, China.

Antibacterial Activity Evaluation In Vitro and In Vivo
Analyses of the biological activity of the target molecules against the three plant bacteria Xoo, Xac, and Psa were performed in vitro and in vivo [62,63].

Xoo-Biofilm Formation and EPS Production Analysis
A Xoo-biofilm formation assay using the crystal violet staining method was performed to assess the antibacterial biofilm function of compound 2b [14,62,63]. Initially, a 96-well plate with 200 µL of nutrient broth medium was used, and the bacterial cell suspension was adjusted to 0.1 (OD 595nm ). Different doses of compound 2b were added, and the mixture was incubated at 28°C for 72 h. Following that, 200 µL of medium from each well was aspirated and washed three times with sterile water. Subsequently, bacteria were fixed with 200 µL of Carnoy's fluid for 30 min and stained with 1% crystal violet staining solution for 15 min. The crystal violet solution was then removed from the 96-well plate, and the residue was dissolved using 95% ethanol. Finally, based on the phenol-sulfuric acid standard curve, the OD 470 nm value was measured to determine the inhibitory effect of biofilm formation and EPS production.

Swimming Motility Assay
An examination of bacterial swimming motility was performed to assess the inhibitory effect of compound 2b on Xoo cell motility. Based on previous studies but with a slight modification, Xoo cells suspension was adjusted to 0.2 (OD 595nm ), and 2 µL of cell suspension was inoculated in the center of motility plates (0.3% beef extract, 0.5% peptone, 0.1% yeast powder, 1% glucose, 0.5% agar powder, and pH 7.2) with various doses (0, 1.35, 2.70, 5.40, and 10.8 µg mL −1 ) of compound 2b at 28°C for 72 h. Finally, the swimming diameters for three biological replicates were observed and measured [62].

Pathogenicity Assay
A pathogenicity assay assessed the bacterial virulence after 24 h of interaction between Xoo cells and compound 2b at various doses (0, 0.27, 1.35, and 2.70 µg mL −1 ). Subsequently, the Xoo cell suspension was adjusted to 0.5 (OD 595 nm ), and three biological replicates of rice leaves were inoculated using the leaf-clipping method [14,62]. The leaf lesion lengths were observed and measured after fourteen days, and the one-way analysis of variance was used to evaluate the lesion length data.

Conclusions
The rice BLB caused by Xoo secreting many bacterial VFs is a sustained global danger to agricultural products. Our completed research indicates that compound 2b would be a desirable and potent bactericide candidate for preventing rice BLB by specifically targeting Xoo-VFs. Initially, DAA was believed to be a forestry resource with significant added value due to its wide range of biological activities. Subsequently, many novel DAA derivatives were ingeniously partially synthesized, and their anti-Xoo properties were evaluated in vitro. All biological analyses showed that novel DAA derivatives containing amino alcohol fragments had remarkable anti-virulence properties that functioned by inhibiting a variety of bacterial VFs, including EPS, biofilm, swimming motility, and flagella. In vivo, compound 2b showed excellent curative and protective properties, with minimal phytotoxicity at 200 and 500 µg mL −1 . Finally, we made some preliminary speculations about the mechanism by which 2b inhibited flagella and swimming motility, interfered with EPS secretion and cell adhesion, prevented biofilm formation and bacterial colonization, and decreased bacterial pathogenicity.