The R. solanacearum strain GMI1000 was grown in B liquid medium (1% peptone, 0.1% tryptone, 0.1% yeast extract, and 2.5% glucose) or B solid medium supplemented with 1.5% agar at 28°C. The P. syringae and X. campestris pv. vesicatoria were grown in LB liquid medium (1% peptone, 0.5% yeast extract, and 1% NaCl) containing rifampicin (0.005%) or LB solid medium supplemented with 1.5% agar and rifampicin (0.005%) at 28°C.

In this study, all small molecule compounds (HPLC purity >99%) were acquired from TargetMol (Shanghai, China, Supplementary Table S1). These compounds were dissolved in dimethyl sulfoxide (DMSO) to achieve final concentrations of 1 mM or as indicated and subsequently stored at −20°C. Prior to experimentation, the frozen compound solutions were thawed at room temperature for 10 min and then incorporated into the B or B agar media at the designed concentrations. The equivalent volume of DMSO served as a solvent control.

Antibacterial activity screening of compounds was conducted utilizing 1.5-mL test tubes. Specifically, 282 μL of B liquid culture medium was dispensed into each tube, followed by the addition of 15 μL of bacterial suspension of OD₆₀₀ = 0.01. Subsequently, 3 μL of 1 mM compound solution was incorporated to achieve a final concentration of 10 μM. An equivalent volume of DMSO served as a control treatment. The resulting mixtures were incubated at 28°C on a shaking incubator set with 180 rpm for 24 h, after which the OD₆₀₀ values were measured. Each treatment was performed in triplicate to ensure the reliability of the results.

The growth curve of R. solanacearum, P. syringae, or X. campestris pv. vesicatoria was performed as previously described with minor modifications (Xia et al., 2023). In brief, in a 5 mL B or LB liquid medium, 25 μL of a bacterial suspension (OD₆₀₀ = 0.1) was inoculated. Subsequently, varying concentrations of gatifloxacin hydrochloride were added to achieve final concentrations of 0.0156 mg/L, 0.03125 mg/L, 0.0625 mg/L, 0.125 mg/L, and 0.25 mg/L. An equivalent volume of DMSO was utilized as the solvent control group. The mixed culture was incubated at 28°C with continuous shaking at 180 rpm for 24 or 36 h, after which the OD₆₀₀ was measured at 2-or 3-h intervals. Each treatment was conducted in triplicate to ensure statistical reliability and reproducibility of the results.

The evaluation of bactericidal activity was carried out as previously described with minor modifications (Xia et al., 2023). In brief, one single colony of R. solanacearum, P. syringae, or X. campestris pv. vesicatoria was selected from the B or LB agar plate and cultured overnight in a B or LB liquid medium. The following day, the bacterial suspension was subjected to centrifugation to remove the supernatant, and the resulting pellet was resuspended under sterile conditions. The OD₆₀₀ of the suspension was adjusted to 0.1. The bacterial suspension was then aliquoted into 15-mL centrifuge tubes, each containing 3 mL of the suspension. Gatifloxacin hydrochloride was subsequently added to the tubes to achieve final concentrations of 0.25 mg/L, 0.5 mg/L, 1 mg/L, 2 mg/L, or 4 mg/L. An equal volume of DMSO was included as a solvent control. The treated bacterial suspensions were incubated in a shaking incubator at 180 rpm and 28°C. After incubation for either 1 or 2 h, 100 μL of the suspension was diluted in sterile water to create serial dilutions. Following dilution, 5 μL of each dilution was plated onto the B agar plates. The plates were incubated at 28°C for 48 h. After the incubation period, colonies were enumerated to assess the viability of bacteria in each treatment group. The bactericidal efficacy was calculated based on the colony counts.

The analysis of biofilm formation was conducted following the methodology established by Wu et al. (2015). In a 96-well polystyrene microplate, 190 μL of liquid B medium and 10 μL of R. solanacearum suspension adjusted to OD₆₀₀ = 0.1 were added to each well. Subsequently, gatifloxacin hydrochloride was introduced to each well to achieve final compound concentrations of 0.0625 mg/L, 0.125 mg/L, or 0.25 mg/L, while an equivalent volume of DMSO was used as a negative control. The 96-well plate containing the mixture was statically incubated at 28°C for a duration of 24 h. The culture medium was carefully aspirated using a micropipette, and each well was rinsed gently with 200 μL of sterile water to remove any residual medium. Following this, 220 μL of 0.1% crystal violet solution was added, and the wells were stained at room temperature for 30 min. Upon completion of the staining procedure, the crystal violet solution was aspirated, and each well was washed two times with 200 μL of sterile water to eliminate any remaining dye. Subsequently, the 96-well plate was placed in a fume hood to air-dry for 30 min. After drying, 200 μL of 95% ethanol was added to each well to solubilize the crystal violet that had adhered to the biofilm matrix. Finally, the optical density of the liquid in each well was measured at 530 nm using a microplate reader, providing quantitative data on biofilm formation.

The efficacy of gatifloxacin hydrochloride in controlling bacterial wilt in tomatoes was assessed using a naturalistic soil-soaking method. A 10-mL solution of 4 mg/L gatifloxacin hydrochloride was irrigated to the roots of 4-week-old tomato plants, with an equivalent volume of DMSO serving as a negative control. After 24 h, a 10 mL suspension of R. solanacearum (OD₆₀₀ = 0.1) was inoculated. The inoculated plants were maintained in a greenhouse at 28°C under a light cycle of 14 h in light and 10 h in dark conditions. Symptoms of wilting were recorded daily. Disease severity was evaluated using a scoring system ranging from 0 to 4: 0 indicated no wilting symptoms; 1 represented 1 to 25% leaf wilt; 2 indicated 26 to 50% leaf wilt; 3 represented 51 to 75% leaf wilt; and 4 indicated 76 to 100% leaf wilt.

In brief, 25 μL of a suspension of P. syringae and X. campestris pv. vesicatoria at an OD₆₀₀ of 0.1 was added to 5 mL of LB liquid medium, followed by the addition of different amounts of gatifloxacin hydrochloride to achieve final concentrations of 0.015 mg/L and 0.5 mg/L, respectively. An equal volume of DMSO was used as a negative control. After mixing, the liquid was incubated for 24 h at 28°C with shaking at 180 rpm. The OD₆₀₀ values of the bacterial suspensions in different treatment groups were measured. Each treatment was performed with three technical replicates.

Overnight cultures of P. syringae were subjected to centrifugation and subsequently resuspended in a 10 mM MgCl₂ solution, with the OD₆₀₀ of the resuspended culture adjusted to 0.1. This bacterial suspension was uniformly applied to the foliage of 4-week-old tomato plants, followed by the administration of a working concentration of 4 mg/L of gatifloxacin hydrochloride solution. A corresponding volume of DMSO dilution was employed as the control treatment. The inoculated plants were maintained in a growth chamber at 28°C, with 70% relative humidity and a photoperiod of 14-h light and 10-h dark. Phenotypic assessments of the inoculated leaves were conducted 8 and 10 days post-inoculation. For each treatment, three leaf disks measuring 0.5 cm² were collected and homogenized in 1 mL of sterile water. A 100 μL aliquot of this homogenate was subsequently diluted in sterile water to generate serial dilutions. Each diluted suspension was plated in 10 μL volumes onto the LB agar plates containing rifampicin. Following a 48-h incubation at 28°C, bacterial titers for each treatment were quantified, ensuring a minimum of six replicates per experimental group.

The data were analyzed with either Excel or GraphPad Prism using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) or one-way analysis of variance (ANOVA) for multiple comparisons (p < 0.05).

We previously screened a plant-derived compound library and identified harmine as an antibacterial agent against R. solanacearum, with a minimum inhibitory concentration of 120 mg/L (Xia et al., 2023). To further identify natural compounds that can inhibit R. solanacearum growth, we screened a library consisting of 57 microorganism-derived small molecule compounds and 1 fully synthetic small molecule compound and tested their antibacterial activity against R. solanacearum at a concentration of 10 μM. Many of those compounds inhibited the growth of R. solanacearum, with the inhibitory rate varying from 40 to 97% (Figure 1; Supplementary Table S2). Among those bioactive compounds, four compounds, namely, gatifloxacin hydrochloride, paromomycin sulfate, fervenulin, and 2,3-butanediol, exhibited a very strong inhibitory effect on the growth of R. solanacearum in a liquid medium, with an inhibitory rate higher than 60% (Figure 1). Gatifloxacin hydrochloride showed the best inhibitory effect, with an inhibitory effect rate of 96%. Other compounds, such as indoleacetic acid and 3-methyluridine, also exhibited an inhibitory effect on the growth of R. solanacearum, though the inhibitory effect was not as strong as that of the above-mentioned four compounds. In summary, the screening identified several small molecule compounds as novel antibacterial agents against R. solanacearum.

To further explore the inhibitory effect of gatifloxacin hydrochloride on R. solanacearum growth, we assessed its effectiveness across a concentration range of 0.0156 mg/L to 0.25 mg/L. The growth curves from 12 to 24 h in liquid culture revealed that all tested concentrations significantly inhibited the growth of R. solanacearum (Figure 2). In particular, growth was almost completely suppressed at concentrations of 0.25 mg/L and 0.125 mg/L. The inhibitory effect decreased as the concentration of gatifloxacin hydrochloride was lowered, with the inhibition rate dropping to 45% at 0.0156 mg/L.

After confirming that gatifloxacin hydrochloride inhibits the growth of R. solanacearum, we next investigated whether it could exhibit bactericidal activity. To test this, R. solanacearum was first cultured in a liquid medium and then treated with various concentrations of gatifloxacin hydrochloride, ranging from 0.25 mg/L to 1 mg/L. Following 1-h and 2-h treatments, the bacterial cells were harvested, resuspended in sterile water, and plated on agar to assess viable bacterial counts. Compared to the DMSO control, treatment with 0.25 mg/L of gatifloxacin hydrochloride resulted in an 89% reduction in bacterial viability at both 1 and 2 h, while 1 mg/L killed 99% of the bacteria (Figure 3). These findings indicate that gatifloxacin hydrochloride exhibits potent bactericidal activity against R. solanacearum.

To assess the impact of gatifloxacin hydrochloride on the biofilm formation of R. solanacearum, a standard polyvinyl chloride (PVC) assay was employed. Gatifloxacin hydrochloride was tested at concentrations ranging from 0.0625 mg/L to 0.25 mg/L, and biofilm levels were quantified in its presence and absence. Biofilm formation was notably decreased in all gatifloxacin hydrochloride treatments when compared to the DMSO control. Specifically, gatifloxacin hydrochloride at 0.0625 mg/L reduced biofilm formation by 58% (Figure 4A). With the increase in gatifloxacin hydrochloride concentration, the biofilm levels were lower. Gatifloxacin hydrochloride at 0. 25 mg/L reduced biofilm formation by 80% (Figure 4A). We further visualized the biofilm with crystal violet. Different concentrations of gatifloxacin hydrochloride treatments substantially reduced the purple color compared to the DMSO control, indicating inhibition of biofilm formation by gatifloxacin hydrochloride (Figure 4B). Taken together, these results suggest that gatifloxacin hydrochloride reduces the biofilm formation by R. solanacearum.

Given that gatifloxacin hydrochloride shows strong antibacterial activity against R. solanacearum and reduces biofilm formation, we hypothesized that this small molecule compound could delay bacterial wilt disease development caused by this pathogen. R. solanacearum has a wide range of host plants, with Solanaceae plants, particularly tomato, being the most common hosts (Vailleau and Genin, 2023). We treated 4-week-old tomato plants with either DMSO or 4 mg/L of gatifloxacin hydrochloride, followed by inoculating with R. solanacearum using the natural soil drenching method. Compared to DMSO control, gatifloxacin hydrochloride treatment significantly delayed bacterial wilt disease development (Figures 5A,B). At 10 days post-inoculation, the disease index of DMSO-treated plants was 4, while the disease index of gatifloxacin hydrochloride-treated plants was 2.5. We further calculated the control efficiency of gatifloxacin hydrochloride against bacterial wilt. Gatifloxacin hydrochloride treatment led to 37.5% control efficiency in tomatoes (Figure 5C). These results suggest that gatifloxacin hydrochloride has the potential to be an effective antibacterial agent for the control of bacterial wilt caused by R. solanacearum.

The identification of broad-spectrum antibacterial agents is pivotal for the management of plant diseases. To ascertain the broad-spectrum antibacterial potential of gatifloxacin hydrochloride, we initially assessed its inhibitory impact on P. syringae, a prominent bacterial pathogen based on its academic significance and economic impact (Mansfield et al., 2012). At a concentration of 0.015 mg/L, gatifloxacin hydrochloride induced only a marginal inhibition of P. syringae growth, whereas a concentration of 0.5 mg/L nearly completely arrested its growth (Figure 6A). Subsequently, we evaluated the inhibitory effect of gatifloxacin hydrochloride on X. campestris pv. vesicatoria. Consistent with the results observed for P. syringae, 0.5 mg/L of gatifloxacin hydrochloride substantially inhibited the growth of X. campestris pv. vesicatoria in liquid culture (Figure 6B). Then, we performed a growth curve for both P. syringae and X. campestris pv. vesicatoria in the absence or presence of gatifloxacin hydrochloride. Supplement with different concentrations of gatifloxacin hydrochloride significantly suppressed the growth of P. syringae and X. campestris pv. vesicatoria in a liquid medium in a 24-h time interval (Figures 6C,D). We further tested the bactericidal activity of gatifloxacin hydrochloride against P. syringae and X. campestris pv. vesicatoria. Compared to the DMSO control, treatment with 1 mg/L of gatifloxacin hydrochloride killed 98% of the P. syringae, and 4 mg/L of gatifloxacin hydrochloride killed approximately 90% of the X. campestris pv. vesicatoria (Figure 7), indicating a strong bactericidal activity against both pathogens. These findings suggest that gatifloxacin hydrochloride possesses broad-spectrum antibacterial activity against plant pathogenic bacteria.

To elucidate whether gatifloxacin hydrochloride could impede disease progression caused by P. syringae, we inoculated tomato leaves with P. syringae and subsequently treated them with 2 mg/L of gatifloxacin hydrochloride. In comparison with the DMSO control, the development of bacterial spots was significantly slower in the gatifloxacin hydrochloride-treated leaves at 8 and 10 days post-inoculation (Figure 8A). In addition, we quantified the bacterial load in the inoculated tomato leaves and found that treatment with gatifloxacin hydrochloride markedly reduced bacterial titers (Figure 8B). In summary, these results collectively indicate that gatifloxacin hydrochloride exhibits broad-spectrum antibacterial activity against plant pathogenic bacteria and holds promise as a potential agent for the control of bacterial diseases in plants.