Antibacterial activities of coumarin-3-carboxylic acid against Acidovorax citrulli

Coumarin-3-carboxylic acid (3-CCA), previously screened from natural coumarins, was found to possess strong antibacterial activity against Acidovorax citrulli (Ac). In order to further evaluate the activity of this compound against plant bacterial pathogens and explore its potential value as a bactericidal lead compound, the activity of 3-CCA against 14 plant pathogenic bacteria in vitro and in vivo was tested. Results showed that 3-CCA exhibited strong in vitro activities against Ac, Ralstonia solanacearum, Xanthomonas axonopodis pv. manihotis, X. oryzae pv. oryzae, and Dickeya zeae with EC50 values ranging from 26.64 μg/mL to 40.73 μg/mL. Pot experiment results showed that 3-CCA had powerful protective and curative effects against Ac. In addition, the protective efficiency of 3-CCA was almost equivalent to that of thiodiazole copper at the same concentration. The results of SEM and TEM observation and conductivity tests showed that 3-CCA disrupted the integrity of the cell membrane and inhibited polar flagella growth. Furthermore, 3-CCA resulted in reductions in motility and extracellular exopolysaccharide (EPS) production of Ac while inhibiting the biofilm formation of Ac. These findings indicate that 3-CCA could be a promising natural lead compound against plant bacterial pathogens to explore novel antibacterial agents.


. Introduction
The bacterial diseases of crop plants have been major threats to crop growth, which can result in enormous losses in global food production and cause food security problems (Savary et al., 2012;Sundin et al., 2016).For example, bacterial fruit blotch caused by Acidovorax citrulli (Ac) has severely cut production in Cucumis (e.g., as high as 90% of yield loss in several watermelon cultivars) (Bahar and Burdman, 2010;Tian et al., 2015;Preston et al., 2021).Moreover, bacterial diseases caused by Ralstonia solanacearum (Rs), Xanthomonas axonopodis pv.citri (Xac), and Xanthomonas oryzae pv.oryzae (Xoo) are also recognized as destructive diseases that threaten crop production (Tondo et al., 2013;Jia et al., 2020;Qutb et al., 2020).At present, the control of plant bacterial diseases mainly depends on the use of copper pesticides and antibiotics, resulting in environmental pollution and antibacterial resistance (Vanneste et al., 2013;Sundin et al., 2016;Long et al., 2019).
With attention on the threat to human health due to the irrational use of antibiotics, streptomycin was been officially withdrawn from the Chinese market in 2016 (Shang et al., 2020).Therefore, developing environment-friendly pesticides for controlling plant bacterial disease is a major challenge for chemists and biologists.
Coumarin-3-carboxylic acid (3-CCA) is an important derivative of coumarin, which has received great attention due to its diverse biological functions, including antitumor (Zhang et al., 2018), anticancer (Chimenti et al., 2004;Wei et al., 2017;Ji et al., 2021), and other antimicrobial properties (Lin et al., 2012;Liu et al., 2018).Our preliminary screening experiments confirmed that 3-CCA had significant inhibitory activity against some plant bacteria (data not shown).Therefore, we further conducted a bactericidal bioassay of 3-CCA against 14 phytopathogenic bacteria to explore its bactericidal spectrum.Specifically, assessments included in vitro and in vivo antibacterial activity, ultrastructure observation, and changes in pathogenicity-related physiological and biochemical indices.
. Materials and methods

. . In vitro antibacterial assay
The abovementioned 14 plant pathogenic bacterial strains were used for the antibacterial bioassay according to the method described by Li et al. (2014).All strains were first transferred and cultured on LA plates, then single bacterial colonies were inoculated in LB broth at 28 • C and 180 rpm until OD 600 reached 0.6.The experiments were conducted on 96-well plates.Ten microliters of bacterial cultures were inoculated in 190 µL of LB broth in each well that contained 3-CCA with final concentrations of 100, 50, or 25 µg/mL.Acetone (1%) was used as a solvent control.All 96-well plates were incubated at 28 • C and 180 rpm for 12 h.Then, the bacterial growth was investigated by measuring the OD 600 with a microplate reader (Synergy H1, BioTek Instruments Inc., Vermont, USA).
The toxicities of 3-CCA to five plant pathogenic bacteria, including Ac, Dz, Rs, Xam, and Xoo, were tested at concentrations of 100, 50, 25, 12.5, and 6.25 µg/mL with three wells per concentration.The optical density inhibition rate was calculated using the following formula (Li et al., 2022): (the OD 600 values of the solvent control -the OD 600 values of the treatment) × 100%/the OD 600 values of the solvent control.The values of 50% effective concentration (EC 50 ) were calculated by regressing the optical density inhibition rate against the log of bactericide concentrations with SPSS software (version 20.0,IBM Corp., New York, USA).The experiment was repeated three times.

. . In vivo antibacterial assay
The protective and curative activity of 3-CCA against melon bacterial fruit blotch was assayed using a potted experiment (Li et al., 2013).The dissolved compound in acetone was diluted to final concentrations of 50, 100, and 200 µg/mL with distilled water containing 0.1% Tween-20.Seedlings of melon (hybrid melon: cv.Yangjiaomi, purchased from China Vegetable Seed Technology Co., LTD.) with two true leaves were planted in a greenhouse (25-28 • C) for 10-15 days and were randomly assigned to two groups.
For the purpose of conducting a curative activity test, the cotyledons and leaves of melons in the first group were inoculated with the bacterial suspension of Ac (OD 600 = 1).After inoculation for 24 h, cotyledons and leaves were sprayed with tested agents (200, 100, and 50 µg/mL) or a standard bactericide (20% thiodiazole copper SC at 100 µg/mL).For preventive activity assay, the inoculation was conducted 24 h after performing the same spraying procedure.Control seedlings within each group were inoculated with the same bacteria suspension and treated with sterile distilled water containing the same dose of acetone and Tween-20.The pots were arranged in a randomized block design with three replicates, and six melon seedlings with cotyledons injured with a sterile syringe were used per replicate.
For the development of melon bacterial fruit blotch, the inoculated melon seedlings were incubated for 7 days under the standard conditions (16 h light at 25 ± 2 • C and 60 ± 5% RH + 8 h dark at 20 ± 2 • C and 75 ± 5% RH), and then disease indexes were evaluated at 7 days of post-inoculation according to the coverage of the necrotic area that developed in leaves using a scale as described in Tian et al. (2015) with some modifications: namely Grade 0, healthy, asymptomatic leaf; Grade 1, the disease area accounts for approximately 5% of the leaf area; Grade 3, the disease area accounts for 6-25% of the leaf area; Grade 5, the disease area accounts for 26-50% of the leaf area; Grade 7, the disease area accounts for 51-75% of leaf area; and Grade 9, the disease area accounts for 76-100% of leaf area.

Disease index =
(The number of diseased leaves in each grade × corresponding grade value) total number of leaves investigated × the highest disease grade value × 100 Control effect (%) = Disease index in the control − Disease index in the treated group Disease index in the control × 100

. . Growth curve
The effect of 3-CCA on the growth of Ac in vitro was determined using the method described by Silva-Angulo et al. ( 2015) with minor modifications.The bacteria were cultured in LB broth at 180 rpm at 28 • C for 18 h, then collected and inoculated into LB medium containing 3-CCA to obtain the final concentrations of 200, 100, 50, 25, 12.5, and 6.25 µg/L.Acetone (1%) was used as a solvent control.The bacteria in 96-well plates were incubated at 28 • C with shaking at 180 rpm for 60 h, followed by the measurement of the optical density at 600 nm (OD 600 ) every 12 h.Three independent growth assays were conducted for each concentration.

. . Bacterial morphology
Scanning electron microscopy (SEM) was performed to evaluate the effect of 3-CCA on the morphologic changes in Ac (Wang et al., 2016).Cultures (OD 600 = 0.6) were centrifuged at 5,000 rpm for 5 min at 4 • C and then washed three times with 0.1 mol/L of phosphate buffer (pH 7.2).The agents were added to the bacterial suspension to obtain a final concentration of 50 µg/mL.An equal volume of solvent was considered control.Then, the bacterial suspension was incubated at 28 • C with shaking at 180 rpm for 24 h.The cells were collected after centrifugation at 4 • C at 5,000 rpm for 5 min and then washed three times with 0.1 mol/L of phosphate buffer (pH 7.2).Samples were fixed in a 2.5% glutaraldehyde solution for 4 h at 4 • C and washed with phosphate buffer three times (5 min for each time).The cells were dehydrated by a sequential grade of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 15 min for each concentration, followed by a dehydration of 100% ethanol for 20 min.After freeze-drying for 8 h and coating with gold, the morphology of the samples was observed using an FEI Phenom desktop scanning electron microscope (FEI Company, Eindhoven, Netherlands).
Transmission electron microscopy (TEM) was used to examine the flagella formation of Ac according to the method described by Liu et al. (2021) with minor modifications.Ten milliliters of bacterial suspensions (OD 600 = 0.1) were added to an LA medium containing 3-CCA (0 or 50 µg/mL) and then incubated at 28 • C for 24 h.Thin sections, including the cells, were placed on a copper grid coated with carbon film, stained with 1% phosphotungstic acid two times (15 s for each time), rinsed with sterile water, and then stood for 1 h.After completely drying, the samples were observed under an HT7700 transmission electron microscope (Hitachi Ltd., Tokyo, Japan).

. . Swimming assay
Swimming assay of Ac was implemented according to the method of Di Bonaventura et al. ( 2008) with slight modifications.Cultures of Ac (OD 600 = 0.6) were prepared.The LB with 0.3% agar powder was completely dissolved via heating in a microwave oven.After the temperature decreased to 40 • C, 3-CCA was added to the culture medium at the final concentrations of 200, 100, 50, 25, and 12.5 µg/mL.An equal volume of acetone was used as a solvent control.The agent-contained medium was poured into sterile Petri plates.After solidification, 5 µL of bacterial suspension was dropinoculated at the center of semisolid medium plates and incubated at 28 • C for 48 h.Swimming motility was evaluated by measuring the diameter of the longest bacterial circles.The motility assays were performed three times.

. . Cell membrane permeability
The cell membrane permeability was determined by the detection of electrolyte leakage in Ac according to the method of Diao et al. ( 2014) with minor modifications.Cultures of Ac (20 mL) in the logarithmic growth phase were centrifuged for 20 min at 2,000 rpm.After removing the supernatant, the bacterial cells were rinsed and resuspended in 20 mL of sterile water.The 3-CCA dissolved in acetone was diluted to concentrations of 200, 100, 50, 25, 12.5, and 6.25 µg/mL with bacterial suspension.Acetone (1%) was used as a solvent control.Thereafter, the electric conductivities of these mixtures at 0, 2, 4, 6, and 8 h were measured and recorded  (Kenyon et al., 1985).The experiment was repeated three times, with three replicates for each concentration.

. . EPS content
EPS content was determined according to the method of Shi et al. (2015).The acetone solution containing agents was diluted with bacterial suspension (OD 600 = 0.6) to final concentrations of 100, 50, 25, 12.5, and 6.25 µg/mL and then incubated at 28 • C with shaking at 180 rpm for 72 h.An equal volume of acetone was used as a solvent control.The supernatant was collected after centrifugation at 3,000 rpm for 20 min at 4 • C and then mixed with three volumes of absolute ethanol and incubated overnight to precipitate EPS.After centrifugation, the pellets were oven-dried at 70 • C to a constant weight.Assays were repeated three times, and each assay was conducted in triplicate.

. . Biofilm formation assay
The effect of 3-CCA on biofilm formation was assessed according to a previous description (Du et al., 2018) with some modifications.The Ac was cultured in LB broth for 18 h at 180 rpm and 28 • C. The cultures were then centrifuged and adjusted to an OD 600 = 1.The gradient dilutions of 3-CCA were added into sterilized borosilicate glass tubes at concentrations of 100, 50, 25, 12.5, and 6.25 µg/mL (diluted with culture medium).Bacterial cultures and agent dilution buffer (100 µL of each) were added and considered negative controls.All the tubes were then incubated statically at 28 • C for 7 days.Then, remove the contents from each tube gently and wash the glass tubes three times with sterile deionized water.After that, the biofilms contained in the glass tubes were stained with 0.1% crystal violet solution for 1 h, rinsed three times with deionized water to diminish the unbound crystal violet, and then stained with 10% glacial acetic acid for 1 h.The solution of 200 µL from each tube was transferred to 96-well plates to measure the OD 600 values.The inhibition rate (%) was calculated as: (the OD 600 values of the control-the OD 600 values of the treatment)×100/the OD 600 values of the control.Six test tubes were prepared for each concentration treatment, and the experiment was repeated three times.

. . Statistical analysis
Results were expressed as means ± standard error (SE) of three independent repeated experiments.Data were analyzed with a variance analysis using SPSS software (version 20.0,IBM Corp., Frontiers in Microbiology frontiersin.org Armonk, NY, USA).Duncan's multiple range test was conducted to compare statistical significance at a P < 0.05.Graphs were generated with Sigma Plot (version 12.5, Systat Software Inc., San Jose, CA, USA).
Based on the antibacterial screening results, the EC 50 values of 3-CCA against these five sensitive bacteria were evaluated.The results demonstrated that the EC 50 values of 3-CCA against Ac, Xam, Rs, Xoo, and Dz were 26.64, 33.17, 31.62, 35.98, and 40.73 µg/mL, respectively (Table 1). .

. E cacy of -CCA for controlling Ac
The present results showed that 3-CCA had both protective and curative effects against Ac (Figure 2).The protective effect was superior to the curative effect (Figure 2).The protective and curative effects of 3-CCA at 200 µg/mL against Ac were 61.88% and 53.19% (Table 2), which were better than those of the positive control thiodiazole copper.
.   to the solvent control, which was obviously dependent on concentrations.The mean values of the lag phase at 60 h are significantly inhibited by 3-CCA at concentrations of 100 and 200 µg/mL.

. . The e ect of -CCA on the cell morphology of Ac
The morphological changes in Ac after treatment with 3-CCA were investigated via SEM and TEM.As shown in Figure 4, the control bacterial cells maintained their uniformity in shape, showing a plump and smooth form.Comparatively, the surfaces of the bacteria were rough and wrinkled after they were treated with 50 µg/mL of 3-CCA (Figure 4).TEM results displayed that the polar flagella of Ac in solvent control grew normally, but some of the flagella were broken after being exposed to 50 µg/mL 3-CCA (Figure 4).

. . E ect of -CCA on the bacterial motility of Ac
The effect of 3-CCA on the bacterial motility of Ac was determined by plate inhibition assay.As shown in Figure 5, the swimming diameters of the colonies decreased along the increases in agent concentrations, in which the swimming diameters of Ac exposed at 12.5 to 100 µg/mL of 3-CCA being 18.79% to 88.81% lower than those in control.No swimming motility was observed when the concentration of 3-CCA increased to 200 µg/mL.

. . The e ect of -CCA on the cell membrane permeability of Ac
The electric conductivity values increased with increasing concentrations and the extension of incubation time, indicating that 3-CCA promoted the increase in membrane permeability of Ac in a dose-and time-dependent manner (Figure 6).When the treatment concentrations ranged from 25 to 200 µg/mL, the electric conductivities were significantly higher than those of the solvent control group.3-CCA at 6.25 or 12.5 µg/mL exerted a minimal effect on membrane permeability compared to other concentrations.

. . The e ect of -CCA on the EPS content of Ac
The EPS production was decreased to varying degrees after the cells of Ac were treated with 3-CCA at different concentrations.Compared with solvent control, the production of EPS of Ac treated with 3-CCA at all concentrations was significantly reduced (P < 0.05) (Figure 7).

. . The e ect of -CCA on the biofilm formation of Ac
Compared with the solvent control, 3-CCA resulted in remarkable inhibition of biofilm formation, and the inhibitory effect was promoted along with increased concentrations of 3-CCA Frontiers in Microbiology frontiersin.org(Figure 8).The average inhibition rates of the 3-CCA treatment at 100 and 50 µg/mL were 76.48% and 62.13%, respectively (Figure 8).

. Discussion
The occurrence of diseases caused by plant-pathogenic bacteria has been one of the most important problems in plant protection (Mansfield et al., 2012).With the enhancement of consumers' awareness of environmental protection and increasing demand for safe agricultural products, the development of new chemicals with high efficiency and environmental friendliness is imminent.3-CCA is a coumarin compound and possesses diverse pharmacological activities, including anticoagulant, antioxidant, antibacterial, and antifungal properties (Barot et al., 2015).In the present study, the in vitro antibacterial effect of 3-CCA against 14 pathogenic bacteria was evaluated.The results showed that 3-CCA had broadspectrum antimicrobial activity against almost all tested bacteria, with inhibition rates ranging from 31.03% to 93.11%.According to the result of the toxicity assay, the EC 50 values of 3-CCA against five tested bacteria ranged between 26 and 41 µg/mL, which was in accordance with the report of Chen et al. (2016), who demonstrated the antibacterial effect of coumarins against Rs.The results of pot experiments showed excellent protective and curative effects of 3-CCA on melon bacterial fruit blotch disease.These findings suggest that 3-CCA could be a potential bactericide to control plant disease.
Although the compound 3-CCA has shown inhibitory activity against most of the pathogens used in our study, its activity against Ac was significantly higher than other bacterial species (Figure 1).Thus, the present study illustrates the mode of action of 3-CCA against Ac.Generally, the mode of mechanism of some bactericides involves the disruption of membrane integrity (Feng et al., 2021).Many natural bactericidal components, such as Biochanin A (Hu et al., 2021), daphnetin (Yang et al., 2016), and methyl gallate (Fan et al., 2013), have been tested to inhibit the growth of bacteria by destroying the cell membranes.In this study, we found that the bacteria treated with 3-CCA had a seriously wrinkled surface along with increasing conductivity in a dose-dependent way.In addition, 3-CCA was found to disrupt flagellar growth and block bacterial motility.As the main organelle of bacterial motility, the bacterial flagellum is a quite complex nanomachine that contributes to both bacterial adherence and biofilm formation (Khider et al., 2018;Zhou et al., 2020).Flagellar motility has been demonstrated to play an important role in the initial stage of bacterial biofilm formation (Houry et al., 2010).Without the flagella, Agrobacterium tumefaciens cannot form biofilms (Sato et al., 2005;Waege et al., 2010;Ding et al., 2017).EPS are major components of bacterial biofilm and play a crucial role in the formation and adhesion of bacterial biofilm (Gacesa, 1998).The present study  if there is a similar mode of action on the other pathogens in our future study.Moreover, we found that 3-CCA could inhibit the biofilm formation of Ac at lower concentrations (6.25-25 µg/mL).In phytopathogenic bacteria, biofilm and EPS act as essential pathogenicity factors that are closely related to the pathogenic forces (Lim et al., 2017).The formation of bacterial biofilms can enhance the resistance of bacteria to bactericides, decrease their drug sensitivity, and improve their adaptability and tolerance to the external environment, which increases the difficulty of controlling bacterial diseases (Vestby et al., 2020).Inhibiting the formation of pathogen biofilm has recently been considered a novel strategy for disease control (Harding et al., 2019).Prithiviraj et al. (2005) found that salicylic acid could inhibit biofilm formation in the roots of Arabidopsis thaliana.Harding et al. (2019) discovered that treatment with oxysilver nitrate effectively reduced bacterial biofilm formation, indicating that this compound could be used as a candidate for controlling seed-transmitted bacterial diseases.In this study, 3-CCA remarkably inhibited the biofilm formation of Ac in a concentration-dependent manner, which was consistent with the report of Yang et al. (2016), who confirmed an inhibition of hydroxycoumarins on the biofilm formation of Rs.In addition, one of the roles of coumarin compounds could be to block bacterial quorum sensing (Reen et al., 2018).Hence, we proposed that the inhibition of biofilm formation by bacteria by 3-CCA could be associated with the block of quorum sensing, which needs further study for validation.The findings suggested that 3-CCA might be a promising lead compound for further structure optimization to develop novel antibacterial agents for controlling plant diseases.However, some researchers reported that excess consumption of coumarins may adversely affect our health because they are easily absorbed from the intestines into the lymph and blood, causing cirrhosis of the liver (Kowalczyk et al., 2020), so the toxicity of 3-CCA or novel chemicals designed based on this lead compound to other non-target organisms should also be carried out in future.
In summary, 3-CCA showed a broad-spectrum and efficient bactericidal effect against plant bacterial pathogens, including Ac. 3-CCA exerted an inhibitory effect against Ac by disrupting the integrity of cell membranes and preventing biofilm formation.The results suggest that 3-CCA may be a potential natural lead compound.The antibacterial molecular mechanisms of 3-CCA and its structural optimization need to be further investigated to explore novel antibacterial agents.

FIGURE
FIGUREAntibacterial activities of -CCA against di erent bacterial species.Values presented are means ± standard errors from three independent experiments.Di erent letters on top of the bars within each dosage group indicate significant di erences in the inhibition rate against di erent bacterial species according to Duncan's multiple range test at a P < . .

A
FIGUREIn vivo protective and curative e ects of -CCA against bacterial fruit blotch caused by Ac using potted melon plants in the greenhouse.The experiments were performed three times.

FIGURE
FIGURE Bacterial growth curve of Ac in the presence of -CCA at concentrations ranging from . to µg/mL.The solvent was indicated as a negative control.Values presented are means ± standard errors from three independent experiments.*P < ., **P < ., and ***P < .(Bonferroni's multiple comparisons test).

FIGURE
FIGURESEM and TEM observations of the Ac treated with -CCA.

FIGUREE
FIGURE E ects of -CCA at di erent concentrations on the swimming motility of Ac.Values presented are means ± standard errors from three independent experiments.Di erent letters on top of the bars indicate that the means are significantly di erent according to Duncan's multiple range test at a P < . .

FIGUREEFIGUREE
FIGURE E ects of -CCA at di erent concentrations on the EPS production of Ac.Data represent means ± SE of three independent experiments.Di erent letters on top of the bars indicate that the means are significantly di erent according to Duncan's multiple range test at a P < . .

TABLE
In vivo protective and curative e ects of -CCA against Ac.Means in a column followed by different letter are significantly different (P < 0.05) according to Duncan's multiple range test.