Evaluation of the Antibacterial Effects and Mechanism of Action of Protocatechualdehyde against Ralstonia solanacearum

Protocatechualdehyde (PCA) is an important plant-derived natural product that has been associated with a wide variety of biological activities and has been widely used in medicine as an antioxidant, anti-aging and an anti-inflammatory agent. However, fewer reports concerning its antibacterial effects on plant-pathogenic bacteria exist. Therefore, in this study, protocatechualdehyde was evaluated for its antibacterial activity against plant pathogens along with the mechanism of its antibacterial action. PCA at 40 μg/mL was highly active against R. solanacearum and significantly inhibited its growth. The minimum bactericidal concentration and minimum inhibitory concentration values for PCA were 40 μg/mL and 20 μg/mL, respectively. Further investigation of the mechanism of action of PCA via transmission electron microscopy and biological assays indicated that the destruction of the cell structure, the shapes and the inhibition of biofilm formation were important. In addition, the application of PCA effectively reduced the incidence of bacterial wilt on tobacco under greenhouse conditions, and the control efficiency was as high as 92.01% at nine days after inoculation. Taken together, these findings suggest that PCA exhibits strong antibacterial activity against R. solanacearum and has the potential to be applied as an effective antibacterial agent for controlling bacterial wilt caused by R. solanacearum.


Introduction
Ralstonia solanacearum is a rod-shaped Gram-negative plant-pathogenic bacterium that causes bacterial wilt disease [1][2][3]. This soil-borne bacterium can infect more than 200 plant species, mainly in the Solanaceae and Musaceae families [4]. This soil-borne pathogen can also cause typical wilting symptoms by colonization, invasion, survival and growth in the root system and xylem tissue via wounds or natural openings [5,6]. During the development of disease, R. solanacearum cells secrete several virulence factors, including an extracellular polysaccharide, several plant cell wall-degrading enzymes and some type III-secreted effectors [7][8][9][10][11]. The pathogen is widely distributed in tropical, subtropical and some temperate regions and affects significant economic crops, such as tomato, banana, potato, and tobacco. The bacterium can be free-living in soil or in water as a saprophyte after destroying the host [12]. Therefore, R. solanacearum is one of the most destructive pathogens of many economically important plants [3].
To control bacterial wilt, agrochemicals, such as copper derivatives, antibiotics and quaternary ammonium compounds, are conventionally used for crop protection [13,14]. However, the application of these traditional agrochemicals has been proven not very effective in controlling this soil-borne

R. solanacearum Growth Curves with PCA Treatment
In this study, the antibacterial activity of PCA was estimated by growth curves using the turbidimeter test method. The cells were treated with PCA concentrations of 10, 20, 30 and 40 μg/mL, and PCA significantly inhibited the growth of the bacteria at 30 and 40 μg/mL treatment; a PCA concentration of 40 μg/mL almost completely stopped R. solanacearum growth after 26 h incubation ( Figure 2).

Morphology Characterization of R. solanacearum by SEM
SEM images of the morphology of R. solanacearum after inoculation with PCA concentrations of 30 and 40 μg/mL were taken. Under our experimental conditions, the untreated bacteria ( Figure 3A) and 0.1% DMSO-treated ( Figure 3B) bacteria showed typical R. solanacearum morphology-a short rod shape with the integrity of the membrane structure intact and a smooth texture; moreover, no significant difference between these treatments was evident. In contrast, PCA treatment at both concentrations obviously destroyed the surface structure of the bacterial cells and shapes (Figures 3C,D and 4). The shape of the treated cells was longer than the untreated cells and their cellular integrity was lost at a concentration of 30 and 40 μg/mL ( Figures 3C and 4). Additionally, the widths of treated cells were significantly decreased compared to the control groups ( Figure 4). In addition, the cells treated with 40 μg/mL PCA showed greater damage than those treated with 30 μg/mL PCA ( Figures 3D and 4). These results suggested that PCA could damage the cell wall of R. solanacearum and that the damaging effect was greater at a higher concentration.

R. solanacearum Growth Curves with PCA Treatment
In this study, the antibacterial activity of PCA was estimated by growth curves using the turbidimeter test method. The cells were treated with PCA concentrations of 10, 20, 30 and 40 µg/mL, and PCA significantly inhibited the growth of the bacteria at 30 and 40 µg/mL treatment; a PCA concentration of 40 µg/mL almost completely stopped R. solanacearum growth after 26 h incubation ( Figure 2).

R. solanacearum Growth Curves with PCA Treatment
In this study, the antibacterial activity of PCA was estimated by growth curves using the turbidimeter test method. The cells were treated with PCA concentrations of 10, 20, 30 and 40 μg/mL, and PCA significantly inhibited the growth of the bacteria at 30 and 40 μg/mL treatment; a PCA concentration of 40 μg/mL almost completely stopped R. solanacearum growth after 26 h incubation ( Figure 2).

Morphology Characterization of R. solanacearum by SEM
SEM images of the morphology of R. solanacearum after inoculation with PCA concentrations of 30 and 40 μg/mL were taken. Under our experimental conditions, the untreated bacteria ( Figure 3A) and 0.1% DMSO-treated ( Figure 3B) bacteria showed typical R. solanacearum morphology-a short rod shape with the integrity of the membrane structure intact and a smooth texture; moreover, no significant difference between these treatments was evident. In contrast, PCA treatment at both concentrations obviously destroyed the surface structure of the bacterial cells and shapes (Figures 3C,D and 4). The shape of the treated cells was longer than the untreated cells and their cellular integrity was lost at a concentration of 30 and 40 μg/mL ( Figures 3C and 4). Additionally, the widths of treated cells were significantly decreased compared to the control groups ( Figure 4). In addition, the cells treated with 40 μg/mL PCA showed greater damage than those treated with 30 μg/mL PCA ( Figures 3D and 4). These results suggested that PCA could damage the cell wall of R. solanacearum and that the damaging effect was greater at a higher concentration. Untreated bacteria were treated with sterile water. Bacteria were also treated with DMSO alone at a final concentration of 0.1%. The OD value of each treatment was the average of three replicates.

Morphology Characterization of R. solanacearum by SEM
SEM images of the morphology of R. solanacearum after inoculation with PCA concentrations of 30 and 40 µg/mL were taken. Under our experimental conditions, the untreated bacteria ( Figure 3A) and 0.1% DMSO-treated ( Figure 3B) bacteria showed typical R. solanacearum morphology-a short rod shape with the integrity of the membrane structure intact and a smooth texture; moreover, no significant difference between these treatments was evident. In contrast, PCA treatment at both concentrations obviously destroyed the surface structure of the bacterial cells and shapes ( Figure 3C,D and Figure 4). The shape of the treated cells was longer than the untreated cells and their cellular integrity was lost at a concentration of 30 and 40 µg/mL ( Figures 3C and 4). Additionally, the widths of treated cells were significantly decreased compared to the control groups ( Figure 4). In addition, the cells treated with 40 µg/mL PCA showed greater damage than those treated with 30 µg/mL PCA ( Figures 3D and 4). These results suggested that PCA could damage the cell wall of R. solanacearum and that the damaging effect was greater at a higher concentration.

Effect of PCA on Biofilm Formation in R. solanacearum
After treatment with PCA concentrations of 10, 20, 30 and 40 μg/mL, the biofilm formation of R. solanacearum was determined after 12, 24 and 36 h. The biomass in all treatments increased with time from 12 h to 36 h ( Figure 5). Biofilm formation was significantly greater in the control and DMSO (without PCA) groups than in the treatment groups ranging from 0.11 to 0.21 and 0.10 to 0.20, respectively. Furthermore, the bioactivity of PCA activity was concentration-dependent, and the biofilm formation was gradually suppressed by PCA as the concentrations increased ( Figure 5). A low concentration of 10 μg/mL did not inhibit biofilm formation, but 30 and 40 μg/mL were found to notably inhibit biofilm formation. Compared with the control, biofilm formation after the 40 μg/mL treatment significantly reduced biofilm formation by 48.11% and 38.90%, and by 38.56% and 37.27% after the 30 μg/mL treatment, at 24 and 36 h, respectively ( Figure 6). The results showed that PCA exhibited a strongly inhibitory on R. solanacearum biofilm formation.

Effect of PCA on Biofilm Formation in R. solanacearum
After treatment with PCA concentrations of 10, 20, 30 and 40 μg/mL, the biofilm formation of R. solanacearum was determined after 12, 24 and 36 h. The biomass in all treatments increased with time from 12 h to 36 h ( Figure 5). Biofilm formation was significantly greater in the control and DMSO (without PCA) groups than in the treatment groups ranging from 0.11 to 0.21 and 0.10 to 0.20, respectively. Furthermore, the bioactivity of PCA activity was concentration-dependent, and the biofilm formation was gradually suppressed by PCA as the concentrations increased ( Figure 5). A low concentration of 10 μg/mL did not inhibit biofilm formation, but 30 and 40 μg/mL were found to notably inhibit biofilm formation. Compared with the control, biofilm formation after the 40 μg/mL treatment significantly reduced biofilm formation by 48.11% and 38.90%, and by 38.56% and 37.27% after the 30 μg/mL treatment, at 24 and 36 h, respectively ( Figure 6). The results showed that PCA exhibited a strongly inhibitory on R. solanacearum biofilm formation.

Effect of PCA on Biofilm Formation in R. solanacearum
After treatment with PCA concentrations of 10, 20, 30 and 40 µg/mL, the biofilm formation of R. solanacearum was determined after 12, 24 and 36 h. The biomass in all treatments increased with time from 12 h to 36 h ( Figure 5). Biofilm formation was significantly greater in the control and DMSO (without PCA) groups than in the treatment groups ranging from 0.11 to 0.21 and 0.10 to 0.20, respectively. Furthermore, the bioactivity of PCA activity was concentration-dependent, and the biofilm formation was gradually suppressed by PCA as the concentrations increased ( Figure 5). A low concentration of 10 µg/mL did not inhibit biofilm formation, but 30 and 40 µg/mL were found to notably inhibit biofilm formation. Compared with the control, biofilm formation after the 40 µg/mL treatment significantly reduced biofilm formation by 48.11% and 38.90%, and by 38.56% and 37.27% after the 30 µg/mL treatment, at 24 and 36 h, respectively ( Figure 6). The results showed that PCA exhibited a strongly inhibitory on R. solanacearum biofilm formation.

Assessment of R. solanacearum Swarming Motility in the Presence of PCA
Motility was considered to be closely and positively related with biofilm formation. The previous experiment demonstrated that PCA could obviously suppress biofilm formation. We investigated whether PCA could affect the motility of R. solanacearum in a petri dish. The results indicated that PCA could significantly inhibit swarming motility at concentrations ranging from 10 to 40 μg/mL after 24 and 48 h (Figure 7). At 24 h, the diameter of the migration zone was decreased by 2.12-and 2.07-fold compared with the control. Moreover, the inhibitory effect of PCA on the swarming motility after 48 h was more evident and the diameter of the migration zone was reduced by 4.08-fold. This experiment indicated that PCA potently exerted an obvious inhibitory effect on the swarming motility of R. solanacearum in a dose-dependent manner.

Assessment of R. solanacearum Swarming Motility in the Presence of PCA
Motility was considered to be closely and positively related with biofilm formation. The previous experiment demonstrated that PCA could obviously suppress biofilm formation. We investigated whether PCA could affect the motility of R. solanacearum in a petri dish. The results indicated that PCA could significantly inhibit swarming motility at concentrations ranging from 10 to 40 μg/mL after 24 and 48 h (Figure 7). At 24 h, the diameter of the migration zone was decreased by 2.12-and 2.07-fold compared with the control. Moreover, the inhibitory effect of PCA on the swarming motility after 48 h was more evident and the diameter of the migration zone was reduced by 4.08-fold. This experiment indicated that PCA potently exerted an obvious inhibitory effect on the swarming motility of R. solanacearum in a dose-dependent manner.

Assessment of R. solanacearum Swarming Motility in the Presence of PCA
Motility was considered to be closely and positively related with biofilm formation. The previous experiment demonstrated that PCA could obviously suppress biofilm formation. We investigated whether PCA could affect the motility of R. solanacearum in a petri dish. The results indicated that PCA could significantly inhibit swarming motility at concentrations ranging from 10 to 40 µg/mL after 24 and 48 h (Figure 7). At 24 h, the diameter of the migration zone was decreased by 2.12and 2.07-fold compared with the control. Moreover, the inhibitory effect of PCA on the swarming motility after 48 h was more evident and the diameter of the migration zone was reduced by 4.08-fold. This experiment indicated that PCA potently exerted an obvious inhibitory effect on the swarming motility of R. solanacearum in a dose-dependent manner.

The Pathogenicity of R. solanacearum in a Greenhouse Treated with PCA
PCA at a concentration of 40 μg/mL had the most effective in vitro antibacterial activity, so we selected this concentration for disease assessment in a pot experiment. Beginning at seven days after inoculation, the incidence of disease was assessed every two days from seven to 19 days. Typical bacterial wilt symptoms were observed nine days after the tobacco plants were inoculated with R. solanacearum (Figure 8

The Pathogenicity of R. solanacearum in a Greenhouse Treated with PCA
PCA at a concentration of 40 μg/mL had the most effective in vitro antibacterial activity, so we selected this concentration for disease assessment in a pot experiment. Beginning at seven days after inoculation, the incidence of disease was assessed every two days from seven to 19 days. Typical bacterial wilt symptoms were observed nine days after the tobacco plants were inoculated with R. solanacearum (Figure 8

Discussion
Bacterial wilt of tobacco is a devastating disease worldwide and its threat is constantly increasing. For the last few years, traditional chemosynthesis pesticides have been used to control soil-borne diseases caused by R. solanacearum. However, many chemical pesticides pollute the environment and lead to the accumulation of residues in crops and soil. Therefore, in pesticide science, it is essential to search for new natural antibacterial agents that are highly efficient and environmentally friendly [32]. The application of natural products isolated from plants and biological agents has been shown to be effective and environmentally friendly against many plant pathogens [33]. In nature, plants contain a large variety of natural products, many of which have evolved to have antimicrobial activity against pathogens that cause bacterial and fungal diseases, such as flavonoids [34], phenols [35], sesqrepenes [36], alkaloids [37], and coumarins [38]. PCA is a water-soluble antioxidant phenolic aldehyde extracted from the roots of Salvia miltiorrhiza, and it has been widely used in many fields of medicine and biological research [39,40]. However, its potential bactericidal activity against soil-borne pathogens has not been intensively investigated. Therefore, in this research, the antibacterial potentials of PCA against R. solanacearum and the mechanism of action were investigated.
PCA is an important biologically active component of some traditional Chinese medicines. Previous studies have indicated that PCA has antioxidant activity, antibacterial activity [41], anticancer activity [42], anti-aging activity [43], and anti-inflammatory activity [44]. Furthermore, PCA is a widely distributed, naturally occurring phenolic acid and has structural similarity with caffeic acid, syringic acid and gallic acid [45]. Therefore, we speculated that these compounds had strongly similar biological functions. Polyphenols are secondary metabolites that are found ubiquitously in many higher plants and play important roles in defending against plant pathogens by suppressing microbial virulence factors, such as inhibiting biofilm formation and reducing adhesion to the host [35]. Lee et al. reported that tea catechins showed a significant antipathogenic effect against Escherichia coli O157:H7 by suppressing biofilm formation and swarming motility [46]. Those results were consistent with the results of this study in which PCA inhibited biofilm formation and the swarming ability of R. solanacearum (Figures 5-7).
PCA, caffeic acid and gallic acid are all polyphenols that have potential antibacterial activity for food and plant diseases. Moreover, these compounds all have a similar chemical structure, consisting of a benzene ring with two or three hydroxyl groups. Their structural similarity and diversity may be involved in determining their anti-tumor, anti-bacterial, and anti-proliferative bioactivity. Farag et al. demonstrated that gallic acid extracted from Acacia arabica and Punica granatum displayed significant antimicrobial activity against R. solanacearum (MIC values 0.5-9 mg/mL) [47]. Methyl gallate also showed a strong inhibitory effect on R. solanacearum at an IC50 of 8.3 mg/L and MIC of 20 mg/L. A disease control trial in planta indicated that methyl gallate could effectively control tomato bacterial

Discussion
Bacterial wilt of tobacco is a devastating disease worldwide and its threat is constantly increasing. For the last few years, traditional chemosynthesis pesticides have been used to control soil-borne diseases caused by R. solanacearum. However, many chemical pesticides pollute the environment and lead to the accumulation of residues in crops and soil. Therefore, in pesticide science, it is essential to search for new natural antibacterial agents that are highly efficient and environmentally friendly [32]. The application of natural products isolated from plants and biological agents has been shown to be effective and environmentally friendly against many plant pathogens [33]. In nature, plants contain a large variety of natural products, many of which have evolved to have antimicrobial activity against pathogens that cause bacterial and fungal diseases, such as flavonoids [34], phenols [35], sesqrepenes [36], alkaloids [37], and coumarins [38]. PCA is a water-soluble antioxidant phenolic aldehyde extracted from the roots of Salvia miltiorrhiza, and it has been widely used in many fields of medicine and biological research [39,40]. However, its potential bactericidal activity against soil-borne pathogens has not been intensively investigated. Therefore, in this research, the antibacterial potentials of PCA against R. solanacearum and the mechanism of action were investigated.
PCA is an important biologically active component of some traditional Chinese medicines. Previous studies have indicated that PCA has antioxidant activity, antibacterial activity [41], anticancer activity [42], anti-aging activity [43], and anti-inflammatory activity [44]. Furthermore, PCA is a widely distributed, naturally occurring phenolic acid and has structural similarity with caffeic acid, syringic acid and gallic acid [45]. Therefore, we speculated that these compounds had strongly similar biological functions. Polyphenols are secondary metabolites that are found ubiquitously in many higher plants and play important roles in defending against plant pathogens by suppressing microbial virulence factors, such as inhibiting biofilm formation and reducing adhesion to the host [35]. Lee et al. reported that tea catechins showed a significant antipathogenic effect against Escherichia coli O157:H7 by suppressing biofilm formation and swarming motility [46]. Those results were consistent with the results of this study in which PCA inhibited biofilm formation and the swarming ability of R. solanacearum (Figures 5-7).
PCA, caffeic acid and gallic acid are all polyphenols that have potential antibacterial activity for food and plant diseases. Moreover, these compounds all have a similar chemical structure, consisting of a benzene ring with two or three hydroxyl groups. Their structural similarity and diversity may be involved in determining their anti-tumor, anti-bacterial, and anti-proliferative bioactivity. Farag et al. demonstrated that gallic acid extracted from Acacia arabica and Punica granatum displayed significant antimicrobial activity against R. solanacearum (MIC values 0.5-9 mg/mL) [47]. Methyl gallate also showed a strong inhibitory effect on R. solanacearum at an IC 50 of 8.3 mg/L and MIC of 20 mg/L. A disease control trial in planta indicated that methyl gallate could effectively control tomato bacterial wilt [20]. In addition, gallic acid possessed a high antifungal activity against Fusarium solani via the degradation of fungal cell walls [48]. In addition, Zhao et al. demonstrated that flavonoids isolated from the Chinese medicinal plant Dalbergia odorifera exhibited a stronger inhibitory activity against R. solanacearum [16]. In this study, our findings indicated that PCA exhibited a strong inhibitory effect on the growth of R. solanacearum in vitro (Figures 1 and 2), and also could control tobacco bacterial wilt effectively in planta (Figure 9).
The MIC of an antibacterial agent is the lowest concentration required to simply inhibit the growth of bacteria. The MBC is the minimal concentration of an agent that kills a particular bacterium. An antibacterial agent is usually regarded as bacterial if the MBC is not more than four times the MIC [49]. The result reported in this study showed that the MBC (40 µg/mL) was two times the MIC (20 µg/mL) for PCA, which means it does meet this general rule of thumb to be labeled as an antibiotic. However, it should also be noted that some antibiotics caused an aggregation effect of bacterial cells which could have large impacts on the MIC and MBC calculations. Previous studies showed that some flavonoids induce bacterial aggregation [50]. Then further findings demonstrated that the flavonol galangin caused Staphylococcus aureus cells to clump together, and this aggregation effect led to the decreases in bacterial numbers detected in the time-kill assay and the MBC was determined [51]. The decreases in MBC assays may have been caused by bacterial aggregation rather than cell death. In this report, the calculated MIC and MBC of PCA were measured by using the agar dilution technique. The time-kill assay conducted in the present study indicated that 40 µg/mL of PCA could strongly inhibit the growth of the bacteria, and this result was consistent with that measured by the agar dilution assay. Therefore, it could be speculated that PCA could not cause the aggregation of R. solanacearum cells.
In this report, we have shown that PCA could significantly decrease the incidence of tobacco bacterial wilt at concentrations greater than 40 µg/mL in a greenhouse experiment. Although PCA has shown potential as an effective bactericide for the control of bacterial wilt, it is not known whether the control efficiency can be sustained during the tobacco growth cycle in the field. Further research is required to analyze the Ralstonia populations' dynamics, and to explore the optimal application and duration of protection. In addition, a new form of plant-type antibiotic or a new antibacterial drug could be developed via minor modifications based on the structure of PCA. New studies for the further verification of the antibacterial mechanism of PCA against R. solanacearum at the physiological and molecular levels need to be performed. These studies would provide a scientific and theoretical basis for developing novel compounds and improving their antibacterial effects.

Chemicals and Bacterial Strains
PCA (HPLC ě 98%) used in this study was purchased from the Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). PCA was dispersed in 20% dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL and diluted in sterile distilled water (ddH 2 O) to the desired concentration. R. solanacearum (phylotype I, race1, biovar 3) was used throughout the study [52].

Determination of the MIC and the MBC
The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of PCA was calculated by agar dilution assay at different concentrations (10,20,30,40 µg/mL) in petri dishes. One hundred microliters of a R. solanacearum suspension adjusted to 1ˆ10 5 cfu/mL was spread directly onto each antibiotic-containing agar dilution plate. Plates with 1% DMSO but without PCA and without any additives were used as controls. The plates were incubated at 30˘1˝C. The MIC was determined as the lowest concentration at which no colony formation was observed after cultured 48 h. The MBC was defined as the lowest concentration of PCA that prevented the growth of bacteria after cultured 96 h. All assays were independently repeated at least three times.

Antimicrobial Assay
The antimicrobial activity of PCA was evaluated by examining the OD growth curves as follows. PCA dissolved in DMSO was added into 100 mL of beer extract broth obtain a final concentrations of 10, 20, 30 or 40 µg/mL, and the control culture was supplemented with 1% DMSO alone. The medium was inoculated with 100 µL of a freshly cultured suspension of R. solanacearum (1ˆ10 9 cfu/mL). The cultures were incubated at 180 r/min for 36 h at 30˝C, and cell growth was monitored spectrophotometrically (the optical density at 600 nm was recorded at 2 h intervals). All treatments were determined in triplicated and calculated to obtain an average value.

Bacterial Morphology
The morphology of R. solanacearum cells in the presence of PCA was evaluated using a scanning electron microscope. R. solanacearum at the logarithmic growth phase was diluted into a 10 8 cfu/mL suspension of beer extract broth. PCA was added to the bacterial suspension to reach a final concentration of 30 µg/mL and 40 µg/mL. The bacterial suspension was then shaken at 180 r/min and 30˝C for 12 h. The cells were collected by centrifugation at 6000 rpm for 5 min, washed three times with 0.1 mol/L pH 7.0 phosphate buffer, and were then fixed in a 2.5% glutaraldehyde solution at 4˝C overnight. After fixation, the cells were dehydrated in a graded ethanol series (1 mL; 30%, 50%, 70%, 85% and 95%) with two changes every 5 min. The final cells were resuspended in Tert-butanol and fixed on the smooth surface of aluminum foil for observing after spraying with gold. Suspensions with 0.1% DMSO added and an untreated control were used.

Biofilm Formation Assay
The biofilm formation assay was performed by crystal violet staining as described by Petters et al. with slight modifications [53]. A polystyrene microtiter plate assay was used to quantify biofilm formation by R. solanacearum. Five centrifuge tubes were sterilized, and to each tube the 5 mL mixed cultures were added (5 µL of inoculum (OD 600 « 1.0) mixed with a final PCA concentration of 10 µg/mL, 20 µg/mL, 30 µg/mL and 40 µg/mL in beer extract broth and without PCA as a control). Biofilm growth was initiated by inoculating 200 µL of mixed cultures into individual wells of a 96-well microtiter plate. The plates were sealed with plastic wrap and incubated without shaking for 12, 24 and 36 h at 30˝C. At the end of the incubation period, the liquid medium was removed and immediately washed three times with distilled water. Each well was stained with 0.1% crystal violet for 30 min. After staining, each well was washed three times with distilled water to remove excess stain. The crystal violet was removed from the complex with 200 µL of 95% ethanol, and the absorbance values of biofilms was measured on a microplate reader at OD 490nm . Each treatment had three replications, and the experiment was performed three times.

Swarming Assay
The swarming assay was based on Englert et al. [54] with slight modifications. A semisolid medium containing 0.35% agar [55] supplemented with PCA at various concentrations (10, 20, 30 and 40 µg/mL) was prepared. Then, the petri dishes were air-dried for 30 min on a bacteria-free work bench. An overnight culture of R. solanacearum (OD 600 « 0.8) was collected and washed twice using sterile water at 6000 rpm for 5 min and resuspended in sterile water. The bacterial suspension (2 µL) was drop-inoculated at the center of semisolid medium plates. The colony diameters were measured in both the vertical and the horizontal direction on each plate in triplicate after incubation for 24 h and 48 h at 28˝C. The results were expressed as the mean of three independent experiments.

Effect of PCA on Seedling Tobacco Incubated with R. solanacearum
Pot experiments were conducted to appraise the effect of PCA for controlling tobacco bacterial wilt. First, 5 mL of a freshly culture of a R. solanacearum inoculum solution at 10 7 cfu/mL was inoculated in the rhizosphere. Then, a 10 mL solution of PCA (40 mg/L) or streptomycin (40 mg/L) was irrigated to the tobacco roots, which were inoculated with R. solanacearum two days later. A mixture consisting of DMSO with water in the proper proportion was poured on the control tobacco seedlings. The seedlings were cultivated in the greenhouse at a temperature of 30˘1˝C with a relative humidity of 85% to 90%. The incidence of bacterial wilt was monitored every two days, from seven days to 19 days after inoculation. In this experiment, each treatment had three replicates and each replicate included 20 tobacco plants. The disease rate was quantified as 0 = no symptoms; 1 = a small number of wilting leaves or a side with a streak spot; 2 = a diseased side with over half wilting leaves or a stem with a black steak spot under the top of the tobacco plant; 3 = a diseased side with over two-thirds wilting leaves or a stem with a black steak spot up to the top of the tobacco plant; and 4 = the entire plant died. To determine disease index and the control efficiency, we used the following formula: where n i = the number of plants with respective disease index, v i = disease index (0, 1, 2, 3, 4), and N = the total number of plants used in each treatment.
Control efficiency (%) " CK´T CKˆ1 00p%q (2) where T = the disease index of treatment, CK = the disease index of control group.

Conclusions
In conclusion, this study evaluated the antibacterial activity of plant-derived protocatechualdehyde (PCA), which is the major active ingredient of S. miltiorrhizae against R. solanacearum. The results of this study indicate that the PCA had the strongest antibacterial activity and could be a potential antibacterial agent. The biological activity of PCA is largely due to its disruptive effect on the cell structure and shapes. Moreover, PCA could significantly reduce biofilm formation and suppress the swarming motility of R. solanacearum. Experiments conducted in the greenhouse showed that PCA also significantly reduced the incidence of bacterial wilt of tobacco. Overall, this is the first report concerning the antibacterial effects of PCA on plant-pathogenic bacteria. Further studies are expected to assess its control efficacy in the field and to explore its optimal application. Moreover, further structural modification and design based on PCA could improve the biological activity and contribute to the development of new antibacterial drugs against infections of R. solanacearum.