Abstract
BACKGROUND
Plant pathogenic bacteria severely impact crop yield and quality. Long‐term use of conventional bactericides has led to resistance, and raised environmental and food safety concerns, necessitating new, efficient and eco‐friendly agents.
RESULTS
This study designed and synthesized a series of novel piperidine‐functionalized formononetin derivatives (N1–N42) and evaluated their antibacterial activity. In vitro assays showed that several compounds strongly inhibited Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac). Notably, thiourea‐piperidine N30 and piperidine hydrochloride N42 were highly effective against Xoo, with median effective concentration (EC50) values of 6.5 and 4.0 μg mL−1, respectively, outperforming the controls thiodiazole copper (TC, 33.3 μg mL−1) and zinc thiazole (ZT, 13.9 μg mL−1). N42 also exhibited potent activity against Xac (EC50 = 7.5 μg mL−1), superior to activities of TC (30.7 μg mL−1) and ZT (58.7 μg mL−1). In vivo pot experiments confirmed that N30 exhibited good efficacy against rice bacterial leaf blight (BLB), whereas N42 showed dual efficacy in controlling both BLB and citrus canker (CC). Further investigation of the antibacterial modes and related phenotypes revealed that N30 and N42 could cause bacterial cell‐envelope damage, decrease biofilm formation and exopolysaccharide (EPS) production, attenuate swarming motility, reduce the activities of extracellular virulence‐related enzymes, and enhance the activities of defense‐related enzymes in host plants.
CONCLUSION
N30 and N42 have good potential to be developed as formononetin‐based antibacterial agents. © 2026 Society of Chemical Industry.
Keywords: antibacterial activity, antibacterial modes, antibacterial phenotypes, formononetin derivatives, piperidine functionalization
A series of piperidine‐functionalized formonononetin derivatives were synthesized. In vitro and in vivo experiments confirmed N30 had potent anti‐Xoo efficacy and N42 dually targeted Xoo and Xac. They disrupted bacterial envelopes, weakened virulence and enhanced plant defenses, showing good antibacterial potential.
1. INTRODUCTION
Plant pathogenic bacteria, as one of the main biological factors causing harm to crops, hinder the sustainable development of agriculture through various means such as damaging crops, raising costs, polluting the environment, and threatening trade security. 1 , 2 , 3 , 4 , 5 , 6 These pathogens are particularly difficult to control owing to their latent infection patterns and ability to form protective biofilms. 7 Among these pathogens, Xoo and Xac are recognized as two of the most notorious phytopathogens, causing BLB and CC, respectively, and resulting in substantial global yield losses and severe economic damage. 8 , 9 Although synthetic pesticides have long been used for disease management, 10 , 11 their overuse has led to pathogen resistance, environmental pollution and food safety risks, 12 , 13 , 14 , 15 driving the urgent demand for eco‐friendly and effective alternatives. 16
Natural products are promising sources for green pesticide development owing to their good biocompatibility and low resistance induction, but their widespread direct application is limited by poor environmental stability, narrow activity spectrum and unsatisfactory physicochemical properties. 17 , 18 In this context, functional modification of natural products by introducing bioactive pharmacophores has become a widely recognized and effective strategy, 19 enabling the efficient construction of novel candidate molecules with potent antibacterial activity and low resistance propensity.
Formononetin, a naturally occurring isoflavonoid widely distributed in Trifolium pratense, Astragalus and licorice, 20 has been extensively investigated for its pleiotropic biological and pharmacological properties in the medical field, with well‐documented antimicrobial, antiviral, anti‐inflammatory and antitumor activities. 21 , 22 , 23 , 24 , 25 However, its agricultural application remains largely unexplored. Owing to its intrinsic drawbacks, including poor water solubility, low bioavailability 26 and weak antibacterial activity, 16 its direct utilization is greatly limited. Accordingly, structural modification of formononetin for agricultural applications is of great significance.
Nitrogen‐containing heterocycles, especially the piperidine moiety, are widely utilized in pharmaceutical and agrochemical molecular design owing to their unique properties. 27 Piperidine derivatives display broad‐spectrum antibacterial, antiviral, herbicidal and insecticidal activities with high selectivity, efficacy and low toxicity, 28 , 29 , 30 thus serving as an ideal scaffold for the functional modification of formononetin. In our previous studies, several formononetin derivatives containing benzyl piperidine were synthesized and showed promising antibacterial activity, 31 yet their structural diversity, antibacterial spectrum and structure–activity relationships (SAR) have remained insufficiently explored. Therefore, further functional modification of formononetin is essential to enrich structural diversity, expand the antibacterial spectrum, and systematically investigate SAR.
Based on the above background, formononetin was employed as the lead compound, and various substituted piperidine rings were introduced at its 7‐hydroxyl position via linkers of different lengths to design and synthesize a series of novel piperidine‐functionalized formononetin derivatives. Subsequently, the in vitro and in vivo antibacterial activities of the target compounds were systematically evaluated against important plant pathogenic bacteria, including Xoo and Xac. Furthermore, their antibacterial modes and related phenotypes were explored with a focus on virulence‐related phenotypes and defense enzyme responses in host plants.
2. MATERIALS AND METHODS
2.1. Instruments and chemicals
The structures of the target compounds were characterized by nuclear magnetic resonance (NMR) spectroscopy, performed on a ECX500 (500 MHz) or JMTC600 (600 MHz) NMR spectrometer (JEOL, Tokyo, Japan). High‐resolution mass spectrometry (HRMS) data were acquired using a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Morphological analysis of the plant pathogens was performed using a BX53 fluorescence microscope (Olympus, Tokyo, Japan) and a Nova Nano SEM 450 scanning electron microscope (FEI, Hillsboro, OR, USA). Enzymatic activities were assessed on a Multiskan Sky full‐wavelength spectrophotometer from Life Technologies (Singapore). All chemical reagents were commercially acquired: formononetin (98%), substituted isothiocyanates, 1‐boc‐4‐methylaminopiperidine, and congo red were procured from Shanghai Tansoole Biotechnology Co., Ltd (China); 0.1 mol L−1 phosphate‐buffered saline (PBS) buffer, soluble starch and enzyme activity assay kits were obtained from Beijing Solarbio Science & Technology Co., Ltd (China); 2.5% glutaraldehyde and 0.1% crystal violet aqueous solution were sourced from Shanghai Yuanye Bio‐Technology Co., Ltd (China); sodium carboxymethyl cellulose was provided by Tianjin Komiou Chemical Reagent Co., Ltd (China); potassium iodide (KI) and iodine (I2) were purchased from Shanghai Macklin Biochemical Co., Ltd (China); other conventional solvents were supplied by Tianjin Zhiyuan Chemical Reagent Co., Ltd (China).
2.2. Plant pathogens
The tested bacteria, including Xanthomonas oryzae pv. oryzae (Xoo, PXO99A), Xanthomonas axonopodis pv. citri (Xac, 29‐1), Xanthomonas campestris pv. mangiferaeindicae (Xcm, MG19080403), Pseudomonas syringae pv. actinidiae (Psa, G1), Pectobacterium carotovorum subsp. brasiliense (Pcb, MLS19101101), Acidovorax citrulli (Ac, TG24042001), Ralstonia solanacearum (Rs, FQ2003220). Among them, Xoo, Xac and Psa were obtained from the Center for R&D of Fine Chemicals of Guizhou University, whereas the other strains (Xcm, Pcb, Ac, Rs) were generously provided by the Chinese Academy of Tropical Agricultural Sciences.
2.3. Synthesis of intermediate 1−3 and target compounds N1−N42
2.3.1. General procedure for the synthesis of intermediates 1–3
Intermediates 1–3 were synthesized according to the methods reported previously by our group. 31
2.3.2. General procedure for target compounds N1−N40
Intermediate 3 (0.71 mmol) was dissolved in 20 mL dichloromethane under stirring at room temperature for 0.5 h in a 50‐mL round‐bottom flask. Subsequently, various substituted isothiocyanates (0.71 mmol) were added dropwise, and the reaction was continued at room temperature for an additional 0.5 h. After completion, the mixture was diluted with 100 mL water and extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate (Na2SO4) and concentrated under reduced pressure to afford the crude product, which was further recrystallized from 20 mL methanol to yield the target compounds N1−N40 in 48–91% yield.
2.3.3. General procedure for target compounds N41−N42
Intermediate 3 (0.71 mmol) was dissolved in 25 mL methanol with gradual heating to 60 °C in a 100 mL single‐neck round‐bottom flask. After complete dissolution, 1 mL 37% hydrochloric acid (HCl) was added dropwise, and the mixture was stirred for 1–2 h for salt formation. Subsequently, approximately two‐thirds of the methanol was removed under reduced pressure using rotary evaporation. Then, 20 mL ethyl acetate were added, and the mixture was vigorously stirred for another 1–2 h, resulting in the precipitation of a solid. The insoluble solid was isolated by filtration and dried under vacuum to afford the target compounds N41 and N42 in yields of 85 and 90%, respectively.
2.4. Antibacterial activity test
2.4.1. Antibacterial activity in vitro
In vitro antibacterial activities of the target compounds N1–N42 against seven pathogenic bacteria (Xoo, Xac, Xcm, Psa, Pcb, Ac and Rs) were initially evaluated at 100 μg mL−1 using the turbidimetric method as reported in the literature. 32 The negative control was treated with an equal volume of DMSO as that used for dissolving the tested compounds, whereas commercial bactericides TC and ZT were set as positive controls at the corresponding concentrations. The tested bacterial strains were first inoculated into NB medium and cultured with shaking at 180 rpm and 28–30 °C to the logarithmic growth phase [optical density at 595 nm (OD595) = 0.6–0.8]. After sample preparation and bacterial inoculation, all mixtures were incubated under the same shaking conditions for 12–48 h. Subsequently, the antibacterial inhibition rate was calculated according to the determined OD595 values. For highly active compounds, the EC50 values were further determined via the two‐fold dilution method within a concentration range of 3.13–50 μg mL−1. Each concentration was arranged in three technical replicates, and the experiment was repeated three times independently. Detailed experimental procedures are available in section 1 of the Supporting Information.
2.4.2. Antibacterial activity in vivo test
2.4.2.1. Pot Experiment of N30 and N42 against BLB in Vivo
In vivo control efficacy of N30 and N42 against BLB was evaluated using the leaf‐clipping method as described in the literature. 28 N30 and N42 were formulated at 200 and 100 μg mL−1 by initial dissolution in a small amount of DMSO followed by dilution with sterilized deionized water containing 1% Tween‐80. TC and ZT were used as positive control agents at the same final effective concentrations as the tested compounds. The negative control was composed of sterilized deionized water containing 1% Tween‐80, supplemented with an equal volume of DMSO as that used for dissolving the tested compounds. Healthy 8‐week‐old rice plants were selected, with at least three replicates per treatment (12 leaves sampled per pot for measurement). For protective activity: tested and control solutions were sprayed at 10 mL per pot until leaf surfaces, followed by Xoo suspension (OD595 = 0.6–0.8) inoculation via leaf‐clipping after 24 h; for curative activity: inoculation first, then tested and controls solutions were sprayed at 24 h after inoculation. After being cultured in a greenhouse (25 °C, natural sunlight) for 14 days, the treated plants were assessed for disease severity, followed by calculation of disease index and control efficacy. Detailed experimental steps are provided in section 2 of the Supporting Information.
2.4.2.2. Pot experiment of N42 against CC in vivo
In vivo control efficacy of N42 against CC was evaluated using the puncture inoculation method as described in our previous literature. 16 N42, ZT and TC were prepared at 200 and 100 μg mL−1 by dissolving in DMSO and diluting with sterilized deionized water containing 1% Tween‐80, following the protocol of the rice pot experiment. Two controls were set: one noninoculated, and the other inoculated with the test bacteria, both treated with sterile deionized water containing 1% Tween‐80 and an equal volume of DMSO. Healthy 2‐year‐old citrus seedlings were used for subsequent treatments. For the protective assay, artificial leaf puncture wounds were covered with tested agent or solvent‐soaked filter paper for 24 h, then overlaid with Xac‐saturated filter paper (OD595 = 0.1) for another 24 h. For the curative assay, Xac‐saturated filter paper was first applied to wounds for 24 h, followed by agent or solvent treatment. All seedlings were cultured in a glasshouse for 14 days. Control efficacy was evaluated by measuring chlorophyll a, b and total chlorophyll contents using the acetone–ethanol extraction method. Each treatment was designed with three biological replicates. Complete experimental procedures are presented in section 3 of the Supporting Information.
2.5. Statistical analysis
spss statistics 20 (IBM, Armonk, NY, USA) was employed for statistical analyses. Data are presented as the mean ± standard deviation (SD). One‐way analysis of variance (ANOVA) was used to compare differences among groups, and Tukey's honestly significant difference (HSD) post hoc test was conducted for multiple pairwise comparisons when ANOVA yielded a significant result (P < 0.05).
2.6. Investigation of antibacterial modes and related phenotypes
2.6.1. SEM observation of bacterial damage induced by N30 and N42
Morphological and structural changes in the pathogenic bacteria treated with N30 and N42 were examined following a method reported previously. 33 Detailed procedures are listed in section 4 of the Supporting Information.
2.6.2. Growth kinetics study of N30 and N42 against Phytopathogens
Growth kinetics of the phytopathogenic bacteria treated with different concentrations of N30 and N42 were analyzed via growth curve measurement, following a method reported previously. 34 The specific experimental procedures can be found in section 5 of the Supporting Information.
2.6.3. Inhibition assays of biofilm formation and EPS production
The inhibitory effects of N30 and N42 on biofilm formation and EPS production on Xoo, as well as the effect of N42 on Xac, were evaluated using crystal violet staining and gravimetric methods reported previously. 32 The specific steps of the experiments are given in section 6 of the Supporting Information.
2.6.4. Extracellular enzyme activity assay
In this study, the effects of N30 and N42 on the extracellular cellulase and amylase activities of Xoo, and of N42 on those of Xac, were determined using methods reported in the literature with minor modifications. 34 Complete experimental procedures are presented in section 7 of the Supporting Information.
2.6.5. Assessment of swarming motility
The effects of highly active compounds on the swarming ability of Xoo and Xac were investigated using a reported method. 35 The detailed experimental steps are included in section 8 of the Supporting Information.
2.6.6. Defense enzyme activity assay
This study assessed the defense enzyme activities in rice plants that were pre‐treated with N30 and N42 before being infected with Xoo using a reported method. 36 Rice plants which had been growing for ≈8 weeks under identical growth conditions were first sprayed with the tested compounds N30, N42, positive controls (TC, ZT) and negative control (CK). All solutions were prepared according to the rice pot experiment protocol and applied at a single concentration of 200 μg mL−1 with a spray volume of 10 mL per plant. After 24 h of treatment, the plants were inoculated with Xoo bacterial suspension using the leaf‐clipping method. Leaf samples were then collected at 1, 3, 5 and 7 days post‐inoculation. The collected samples were immediately flash‐frozen in liquid nitrogen and stored at −80 °C for subsequent analysis. For the assay, the frozen leaf samples were ground into a fine powder in liquid nitrogen. The activities of defense enzymes, such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), were measured using specific activity assay kits (Beijing Solarbio Science & Technology Co., Ltd) strictly following the manufacturer's instructions.
3. RESULTS AND DISCUSSION
3.1. Characterization of target compounds N1−N42
N1−N42 were synthesized according to Scheme 1 . All target compounds were obtained as white solids with yields ranging from 48% to 91%, and their characterization data (NMR and HRMS) are provided in section 9 of the Supporting Information.
Scheme 1.
Synthetic route of the target compounds N1−N42.
3.2. Biological activities
3.2.1. Antibacterial activity in vitro
In this study, a series of piperidine‐functionalized formononetin derivatives (N1–N40) were first designed and synthesized. However, subsequent bioactivity screening revealed that most target compounds readily precipitated from NB medium. This precipitation behavior may reduce the actual effective concentration of the compounds in solutions, thereby potentially weakening their antibacterial activities and introducing certain interference in the comparison of antibacterial potency among different compounds. Inspired by the excellent water solubility of organic hydrochloride salts, we further constructed N41 and N42. As shown in Table 1, the in vitro inhibitory activities of N1–N42 against seven bacterial strains were evaluated at a concentration of 100 μg mL−1. Among them, N30 exhibited 100% inhibition against Xoo, whereas N2 and N17 showed complete inhibition against Pcb, superior to those of the commercial controls TC and ZT. Notably, the piperidine hydrochloride salt N42 displayed 100% inhibition against Xoo, Xac and Xcm, with markedly higher efficacy than those of TC and ZT.
Table 1.
Antibacterial activities of N1−N42 in vitro at 100 μg mL−1 *
| Compds. | Xoo | Xac | Xcm | Psa | Pcb | Ac | Rs |
|---|---|---|---|---|---|---|---|
| N1 | 62.3 ± 3.7 | 65.1 ± 2.4 | 55.6 ± 1.8 | 58.3 ± 0.5 | 78.6 ± 4.4 | 43.5 ± 1.2 | 66.0 ± 1.3 |
| N2 | 71.6 ± 2.0 | 57.7 ± 1.5 | 50.6 ± 1.9 | 54.2 ± 3.9 | 100 | 56.0 ± 4.7 | 62.5 ± 2.6 |
| N3 | 63.5 ± 1.4 | 54.4 ± 2.1 | 33.0 ± 4.3 | 41.6 ± 1.0 | 68.6 ± 1.4 | 28.1 ± 4.8 | 62.1 ± 2.8 |
| N4 | 75.6 ± 1.9 | 71.2 ± 4.4 | 65.2 ± 2.1 | 60.3 ± 1.7 | 84.5 ± 1.4 | 44.3 ± 4.7 | 69.0 ± 0.2 |
| N5 | 60.2 ± 2.0 | 62.1 ± 2.0 | 53.3 ± 3.1 | 47.4 ± 4.2 | 80.4 ± 3.9 | 42.1 ± 3.7 | 70.2 ± 1.1 |
| N6 | 76.2 ± 1.0 | 57.0 ± 3.1 | 47.3 ± 0.3 | 58.8 ± 0.5 | 76.7 ± 3.7 | 35.4 ± 2.5 | 58.9 ± 3.9 |
| N7 | 56.7 ± 3.3 | 50.7 ± 3.3 | 46.5 ± 2.4 | 49.7 ± 2.1 | 75.0 ± 5.4 | 49.8 ± 3.1 | 59.0 ± 2.1 |
| N8 | 69.5 ± 3.3 | 54.3 ± 1.7 | 53.3 ± 3.3 | 56.8 ± 0.6 | 81.9 ± 2.4 | 35.0 ± 3.1 | 56.2 ± 2.4 |
| N9 | 74.9 ± 1.3 | 73.8 ± 2.3 | 61.6 ± 3.4 | 54.4 ± 4.5 | 80.8 ± 2.1 | 48.8 ± 4.4 | 76.5 ± 4.1 |
| N10 | 56.9 ± 4.1 | 46.2 ± 2.2 | 41.6 ± 0.9 | 48.1 ± 2.5 | 65.4 ± 4.5 | 45.0 ± 4.8 | 52.1 ± 2.1 |
| N11 | 68.2 ± 3.9 | 53.5 ± 0.9 | 49.3 ± 2.6 | 44.9 ± 4.1 | 69.8 ± 3.2 | 36.8 ± 2.5 | 65.6 ± 0.6 |
| N12 | 77.5 ± 0.5 | 70.0 ± 2.4 | 48.6 ± 1.6 | 58.8 ± 1.7 | 74.7 ± 2.5 | 46.1 ± 2.4 | 76.0 ± 0.8 |
| N13 | 56.1 ± 1.2 | 47.1 ± 3.0 | 35.1 ± 2.8 | 50.6 ± 3.4 | 63.4 ± 4.5 | 31.5 ± 3.6 | 48.5 ± 6.1 |
| N14 | 61.9 ± 4.1 | 49.5 ± 1.7 | 43.0 ± 0.7 | 46.1 ± 2.6 | 59.5 ± 6.9 | 31.3 ± 4.0 | 55.7 ± 2.3 |
| N15 | 42.7 ± 3.4 | 34.8 ± 2.8 | 34.5 ± 4.5 | 43.0 ± 1.0 | 58.3 ± 4.4 | 22.6 ± 3.7 | 54.5 ± 2.2 |
| N16 | 58.1 ± 1.2 | 42.1 ± 2.4 | 39.8 ± 3.0 | 48.8 ± 1.4 | 69.5 ± 2.1 | 41.0 ± 2.2 | 52.5 ± 1.3 |
| N17 | 59.5 ± 1.9 | 61.3 ± 4.6 | 42.0 ± 3.1 | 62.9 ± 1.6 | 100 | 68.3 ± 2.9 | 52.9 ± 3.4 |
| N18 | 71.3 ± 1.6 | 52.9 ± 4.4 | 56.5 ± 3.4 | 57.3 ± 1.2 | 78.8 ± 3.6 | 53.1 ± 2.6 | 73.3 ± 4.9 |
| N19 | 54.0 ± 4.7 | 57.6 ± 5.7 | 44.0 ± 2.1 | 49.9 ± 3.2 | 79.1 ± 2.3 | 47.7 ± 3.2 | 63.5 ± 1.7 |
| N20 | 60.2 ± 1.8 | 51.8 ± 3.7 | 29.3 ± 2.1 | 41.3 ± 2.1 | 79.6 ± 2.1 | 31.9 ± 4.0 | 57.9 ± 4.6 |
| N21 | 44.9 ± 2.0 | 48.9 ± 3.7 | 44.0 ± 3.4 | 44.8 ± 4.1 | 73.7 ± 2.1 | 20.1 ± 3.2 | 59.5 ± 0.3 |
| N22 | 57.5 ± 3.2 | 56.9 ± 3.0 | 53.4 ± 2.3 | 52.9 ± 4.9 | 87.7 ± 3.6 | 38.6 ± 1.6 | 58.6 ± 2.2 |
| N23 | 41.3 ± 2.1 | 41.5 ± 1.3 | 26.7 ± 3.3 | 41.3 ± 1.4 | 46.5 ± 1.2 | 24.3 ± 4.7 | 52.4 ± 3.0 |
| N24 | 49.8 ± 2.5 | 61.8 ± 0.6 | 47.1 ± 2.0 | 60.1 ± 1.6 | 84.1 ± 2.2 | 34.9 ± 1.9 | 60.8 ± 4.9 |
| N25 | 40.3 ± 0.6 | 41.0 ± 2.3 | 44.1 ± 1.6 | 44.4 ± 3.6 | 76.5 ± 4.8 | 31.0 ± 0.5 | 66.8 ± 4.0 |
| N26 | 63.8 ± 4.0 | 47.0 ± 4.6 | 41.1 ± 3.4 | 52.0 ± 3.9 | 67.0 ± 2.2 | 28.7 ± 4.5 | 58.8 ± 1.6 |
| N27 | 51.0 ± 4.5 | 34.2 ± 1.4 | 42.3 ± 2.7 | 48.3 ± 2.1 | 66.5 ± 3.9 | 38.5 ± 1.7 | 56.5 ± 4.9 |
| N28 | 51.1 ± 3.8 | 41.8 ± 0.6 | 44.4 ± 3.2 | 56.3 ± 1.8 | 74.7 ± 1.8 | 33.2 ± 4.4 | 43.8 ± 0.8 |
| N29 | 43.1 ± 3.7 | 49.8 ± 0.6 | 40.8 ± 1.5 | 48.4 ± 3.1 | 75.7 ± 2.0 | 31.4 ± 1.2 | 39.1 ± 2.3 |
| N30 | 100 | 66.4 ± 2.9 | 51.3 ± 1.2 | 51.7 ± 2.2 | 76.9 ± 3.0 | 48.9 ± 1.6 | 61.8 ± 3.4 |
| N31 | 70.5 ± 1.6 | 57.4 ± 1.1 | 50.8 ± 2.5 | 66.9 ± 0.7 | 71.9 ± 0.7 | 47.3 ± 1.6 | 73.1 ± 1.4 |
| N32 | 67.1 ± 1.1 | 66.5 ± 1.6 | 42.0 ± 3.4 | 55.1 ± 2.0 | 67.2 ± 3.9 | 42.2 ± 0.6 | 62.1 ± 0.7 |
| N33 | 51.8 ± 0.4 | 37.9 ± 1.3 | 32.7 ± 1.5 | 47.9 ± 3.1 | 49.3 ± 2.1 | 27.6 ± 3.7 | 39.3 ± 1.6 |
| N34 | 54.9 ± 3.9 | 41.8 ± 3.6 | 29.5 ± 3.5 | 36.9 ± 1.8 | 52.3 ± 2.9 | 26.5 ± 3.1 | 45.7 ± 8.6 |
| N35 | 49.1 ± 3.3 | 43.4 ± 1.6 | 52.5 ± 4.9 | 55.3 ± 3.6 | 65.9 ± 5.7 | 29.6 ± 4.8 | 45.4 ± 1.0 |
| N36 | 45.0 ± 0.7 | 37.7 ± 2.9 | 27.6 ± 2.0 | 35.1 ± 2.7 | 68.8 ± 4.9 | 19.6 ± 3.5 | 43.8 ± 2.6 |
| N37 | 45.1 ± 4.1 | 38.9 ± 4.7 | 36.0 ± 1.6 | 30.7 ± 1.5 | 70.9 ± 0.4 | 21.1 ± 1.0 | 40.2 ± 2.7 |
| N38 | 63.3 ± 0.2 | 43.9 ± 4.7 | 38.8 ± 4.0 | 39.6 ± 1.8 | 63.7 ± 2.1 | 30.1 ± 4.1 | 42.0 ± 1.8 |
| N39 | 37.2 ± 1.3 | 32.9 ± 3.3 | 36.4 ± 1.5 | 32.7 ± 2.3 | 74.8 ± 2.6 | 23.7 ± 4.9 | 30.6 ± 2.8 |
| N40 | 40.8 ± 2.6 | 27.9 ± 1.6 | 27.0 ± 1.9 | 34.0 ± 0.5 | 74.8 ± 2.7 | 22.5 ± 2.4 | 44.2 ± 0.5 |
| N41 | 99.4 ± 0.2 | 100 | 81.2 ± 1.7 | 96.5 ± 0.6 | 70.4 ± 1.8 | 29.8 ± 2.8 | 37.0 ± 0.2 |
| N42 | 100 | 100 | 100 | 98.6 ± 0.4 | 75.6 ± 1.2 | 43.9 ± 2.8 | 47.9 ± 3.8 |
| FMN | 31.2 ± 3.3 | 44.6 ± 3.1 | 32.9 ± 2.8 | 24.8 ± 2.9 | 21.9 ± 3.4 | 39.4 ± 4.3 | 38.9 ± 3.8 |
| TC † | 73.4 ± 2.6 | 58.6 ± 1.9 | 70.8 ± 3.1 | 69.4 ± 2.7 | 67.8 ± 2.3 | 64.6 ± 3.5 | 93.4 ± 2.9 |
| ZT ‡ | 85.1 ± 3.1 | 72.6 ± 1.4 | 90.5 ± 2.1 | 57.3 ± 1.8 | 46.5 ± 1.5 | 60.8 ± 2.3 | 49.6 ± 2.5 |
The data represent the mean ± SD of three independent experiments.
Thiodiazole copper.
Zinc thiozole.
Based on the screening results presented in Table 1, the EC50 values of highly active compounds was further determined, and the corresponding data are summarized in Table 2. The activity assessment demonstrated that N30, N41 and N42 exhibited potent inhibitory activity against Xoo, with EC50 values significantly lower than those of ZT and TC. Against Xac and Xcm, N41 and N42 also showed greater efficacy, with EC50 values better than those of the commercial controls. Meanwhile, N17 showed the strongest activity against Pcb, with an EC50 value superior to both TC and ZT.
Table 2.
EC50 values of several highly active compounds against Xoo, Xac, Xcm and Pcb
| Bacteria | Compd. | n | R | Toxic regression equation | r | EC50 (μg mL−1)* |
|---|---|---|---|---|---|---|
| Xoo | N30 | 4 | 2‐CH3‐Ph | y = 1.0694× + 4.1282 | 0.9929 | 6.5 ± 0.4c |
| N41 | 3 | / | y = 2.8711× + 2.9079 | 0.9791 | 5.4 ± 0.7c | |
| N42 | 4 | / | y = 2.4646× + 3.5036 | 0.9768 | 4.0 ± 0.3c | |
| TC † | / | / | y = 1.7343× + 2.3600 | 0.9874 | 33.3 ± 2.1a | |
| ZT ‡ | / | / | y = 1.1939× + 3.6338 | 0.9960 | 13.9 ± 1.2b | |
| Xac | N41 | 3 | / | y = 1.9822× + 3.2090 | 0.9988 | 8.0 ± 1.4c |
| N42 | 4 | / | y = 2.6469× + 2.6819 | 0.9744 | 7.5 ± 0.9c | |
| TC † | / | / | y = 2.2295× + 1.6840 | 0.9823 | 30.7 ± 2.4b | |
| ZT ‡ | / | / | y = 1.1204× + 3.0187 | 0.9851 | 58.7 ± 3.1a | |
| Xcm | N41 | 3 | / | y = 1.8028× + 2.9047 | 0.9666 | 14.5 ± 1.1b |
| N42 | 4 | / | y = 1.2867× + 3.6519 | 0.9831 | 11.2 ± 0.7b | |
| TC † | / | / | y = 1.0300× + 3.2881 | 0.9797 | 45.9 ± 2.3a | |
| ZT ‡ | / | / | y = 1.2299× + 2.8736 | 0.9694 | 53.6 ± 1.7a | |
| Pcb | N2 | 3 | 4‐Cl‐Ph | y = 0.5684× + 4.1660 | 0.9833 | 29.3 ± 1.5bcd |
| N4 | 3 | 2‐Cl‐Ph | y = 0.3966× + 4.3357 | 0.9783 | 47.3 ± 1.2abc | |
| N5 | 4 | 4‐F‐Ph | y = 0.9123× + 4.0250 | 0.9759 | 11.7 ± 0.6e | |
| N8 | 4 | 4‐CH3‐Ph | y = 0.7063× + 4.0568 | 0.9744 | 21.6 ± 0.9cde | |
| N9 | 4 | 3‐CH3‐Ph | y = 0.5975× + 4.2046 | 0.9796 | 21.4 ± 1.7cde | |
| N17 | 3 | 4‐NO2‐Ph | y = 0.5514× + 4.4359 | 0.9734 | 10.5 ± 0.5e | |
| N22 | 4 | 4‐Cl‐Ph | y = 0.7158× + 4.0372 | 0.9881 | 22.1 ± 1.1cde | |
| N24 | 4 | 2‐Cl‐Ph | y = 0.8609× + 3.9501 | 0.9743 | 16.6 ± 0.7de | |
| N39 | 4 | 2,4‐di‐F‐Ph | y = 0.4556× + 4.1635 | 0.9698 | 68.6 ± 3.4ab | |
| TC † | / | / | y = 0.7463× + 3.4632 | 0.978 | 84.2 ± 3.7a | |
| ZT ‡ | / | / | y = 0.6811× + 3.9913 | 0.9645 | 30.3 ± 2.2bcd |
Note: The toxic regression equation is defined as y=ax+b, in which: x: common logarithm value of tested compound concentration (x=lgC); y: Probit value (probability unit) converted from experimentally determined bacterial inhibition rate; a and b: empirical regression coefficients obtained by linear fitting of x and y.
The EC50 values represent the mean ± SD of three independent experiments; Different lowercase letters indicate statistically significant differences between means at P ≤ 0.05.
Thiodiazole copper.
Zinc thiozole.
3.2.2. SAR analysis of antibacterial activities in vitro
Based on the integrated analysis of data from Tables 1 and 2, the SAR of the target compounds against phytopathogenic bacteria was systematically summarized, and preliminary trends were concluded. First, N41 and N42 bearing a hydrochloride moiety tended to present remarkably improved antibacterial activities against Xoo, Xac and Xcm, with EC50 values ranging from 4.0 to 14.5 μg mL−1, which were superior to those of thiourea‐piperidine formononetin derivatives (N1–N40) and the control agents ZT and TC. These results suggest that hydrochloride modification can improve antibacterial efficacy. Secondly, the electronic properties and substitution position of the benzene rings appear to correlate with compound activity in a chain‐length‐dependent manner. For unsubstituted benzene rings or those substituted with halogen or nitro groups, derivatives with a carbon chain length of n = 3 tended to show generally higher inhibitory effects against the tested phytopathogens than those with n = 4. When a strong electron‐withdrawing group (e.g. —CF 3, —OCH3) was attached at the para‐position of the benzene ring, compounds with n = 4 typically showed higher inhibition rates than those with n = 3. Interestingly, when these groups were at the meta‐position, the n = 3 compounds generally remained more active. For electron‐donating substituents (e.g. —CH3), the compounds with n = 3 generally exhibited enhanced activity when such substituents were positioned at the 3‐ or 4‐site of the benzene ring. By contrast, ortho‐substitution led to higher inhibition rates for n = 4 compounds. Thirdly, carbon chain length may result in differences in antibacterial activity against various pathogens: the n = 4 series tended to be more potent against Xoo (N30, 100% inhibition), whereas the n = 3 series appeared to perform better against Pcb and Rs (N2, N17; both 100% inhibition of Pcb).
3.2.3. Antibacterial activity in vivo
3.2.3.1. Antibacterial efficacy of N30 and N42 against BLB in vivo
The in vivo curative and protective activities of N30 and N42 against BLB were determined by the leaf‐clipping method to further evaluate the practical disease control potential of the target compounds, with results summarized in Tables 3 and 4, and in Fig. 1. Both compounds exhibited favorable control efficacy against BLB, with the curative and protective activities of N42 slightly superior to those of N30. Specifically, N30 exhibited curative activities against BLB were 1.41‐ and 1.61‐fold those of TC, and 1.34‐ and 1.30‐fold those of ZT at 200 and 100 μg mL−1. Its protective activities achieved 1.15‐ and 1.18‐fold those of TC, and 1.07‐ and 1.47‐fold those of ZT. At these concentrations, N42 exhibited even higher curative activities, which were 1.45‐ and 2.15‐fold those of TC, as well as 1.43‐ and 1.70‐fold those of ZT. Its protective activities also were superior, at 1.21‐ and 1.78‐fold those of TC, and 1.17‐ and 1.48‐fold those of ZT. These results suggest that both N30 and N42 show good prospects for further development as potential antibacterial candidates against BLB.
Table 3.
Curative and protective activities of N30 against bacterial leaf blight of rice
| Treatment | Compd. | 14 days after inoculation | |||
|---|---|---|---|---|---|
| 200 μg mL−1 | 100 μg mL−1 | ||||
| Disease index (%) | Control efficacy (%)* | Disease index (%) | Control efficacy (%)* | ||
| Curative | N30 | 39.6 | 53.9 ± 2.7a | 57.8 | 32.6 ± 3.3a |
| TC † | 52.9 | 38.3 ± 4.5b | 68.5 | 20.2 ± 4.9b | |
| ZT ‡ | 51.2 | 40.3 ± 2.7b | 64.4 | 25.0 ± 2.0b | |
| CK § | 85.8 | — | 85.8 | — | |
| Protective | N30 | 36.4 | 55.5 ± 4.7a | 57.9 | 29.3 ± 3.1a |
| aa | TC † | 42.4 | 48.2 ± 2.6a | 62.9 | 24.9 ± 1.9ab |
| ZT ‡ | 39.4 | 51.9 ± 4.7a | 65.6 | 19.9 ± 2.9b | |
| CK § | 81.9 | — | 81.9 | — | |
Control efficacy values represent the mean ± SD of three independent experiments; Different lowercase letters indicate statistically significant differences between means at P ≤ 0.05.
Thiodiazole copper.
Zinc thiozole.
Negative control.
Table 4.
Curative and protective activities of N42 against bacterial leaf blight of rice.
| Treatment | Compd. | 14 days after inoculation | |||
|---|---|---|---|---|---|
| 200 μg mL−1 | 100 μg mL−1 | ||||
| Disease index (%) | Control efficacy (%)* | Disease index (%) | Control efficacy (%)* | ||
| Curative | N42 | 35.1 | 56.5 ± 2.3a | 46.4 | 42.4 ± 1.5a |
| TC † | 49.2 | 39.0 ± 3.0b | 64.7 | 19.7 ± 4.0b | |
| ZT ‡ | 48.8 | 39.4 ± 4.5b | 60.5 | 24.9 ± 2.4b | |
| CK § | 80.6 | / | 80.6 | / | |
| Protective | N42 | 31.7 | 58.3 ± 4.7a | 46.1 | 39.3 ± 3.9a |
| TC † | 39.4 | 48.1 ± 3.4b | 39.4 | 22.1 ± 4.8b | |
| ZT ‡ | 38.2 | 49.7 ± 1.6ab | 38.2 | 26.6 ± 3.2ab | |
| CK § | 75.9 | / | 75.9 | / | |
Control efficacy values represent the mean ± SD of three independent experiments. Different lowercase letters indicate statistically significant differences between means at P ≤ 0.05.
Thiodiazole copper.
Zinc thiozole.
Negative control.
Figure 1.
Curative and protective activities of N30 and N42 against bacterial leaf blight of rice in vivo. (A,C) Curative activities of N30 and N42, respectively; (B,D) Protective activities of N30 and N42, respectively. Treatments included N30, N42, positive controls thiodiazole copper (TC) and zinc thiazole (ZT) at 200 and 100 μg mL−1, and a negative control (CK, sterilized deionized water containing 1% Tween‐80 and an equal volume of DMSO matching that used to dissolve the tested compounds). Disease symptoms were photographed at 14 days after inoculation. Representative images from three independent experiments are shown for each treatment; quantitative results and statistical analyses are provided in Table 3.
3.2.3.2. Effects of N42 against CC in vivo
Furthermore, the in vivo control efficacy of N42 against CC was evaluated via pot experiments, with results summarized in Table 5 and Fig. 2. N42 exhibited curative activities that were 1.91‐ and 1.56‐fold those of TC, and 1.20‐ and 0.94‐fold those of ZT at 200 and 100 μg mL−1, respectively. Meanwhile, its protective activities were 2.57‐ and 2.19‐fold those of TC, and 1.69‐ and 1.64‐fold those of ZT, respectively. These results suggested that N42 may exhibit certain development value and research prospect for the control of CC, and deserves further investigation.
Table 5.
Curative and protective activities of N42 against citrus canker.
| Treatment | Compds. | 14 days after inoculation | |||
|---|---|---|---|---|---|
| 200 μg mL−1 | 100 μg mL−1 | ||||
| Ct (mg g−1) | Control efficacy (%)* | Ct (mg g−1) | Control efficacy (%)* | ||
| Curation | N42 | 1.3449 | 53.0 ± 1.1a | 1.1391 | 30.1 ± 2.7a |
| TC † | 1.1176 | 27.7 ± 1.7c | 1.0421 | 19.3 ± 1.5b | |
| ZT ‡ | 1.2649 | 44.1 ± 0.3b | 1.1562 | 32.0 ± 1.0a | |
| Water | 1.7672 | / | 1.7672 | / | |
| CK § | 0.8687 | / | 0.8687 | / | |
| Protection | N42 | 1.5143 | 83.3 ± 3.7a | 1.2126 | 48.6 ± 2.5a |
| TC † | 1.0717 | 32.4 ± 2.0c | 0.9831 | 22.2 ± 1.1c | |
| ZT ‡ | 1.2195 | 49.4 ± 3.3b | 1.0483 | 29.7 ± 1.2b | |
| Water | 1.6595 | / | 1.6595 | / | |
| CK § | 0.7901 | / | 0.7901 | / | |
Control efficacy values represent the mean ± SD of three independent experiments. Different lowercase letters indicate statistically significant differences between means at P ≤ 0.05.
Thiodiazole copper.
Zinc thiozole.
Negative control.
Figure 2.
Curative and protective activities of N42 against citrus canker in vivo. (A) Curative activity of N42; (B) Protective activity of N42. Treatments included N42, positive controls TC and ZT at 200 and 100 μg mL−1, and a negative control (CK, sterilized deionized water containing 1% Tween‐80 and an equal volume of DMSO matching that used to dissolve the tested compounds). Disease symptoms were photographed at 14 days after inoculation. Representative images from three independent experiments are shown for each treatment; quantitative results and statistical analyses are provided in Table 4.
3.3. Antibacterial modes and related phenotypes
3.3.1. Morphological alterations in Xoo and Xac induced by highly active compounds
SEM observation of morphological and structural alterations in pathogenic bacteria under drug treatment provides crucial phenotypic evidence for elucidating antibacterial mode and evaluating bactericidal efficacy. 37 The SEM results showed that the pathogenic bacteria Xoo and Xac had intact morphology, smooth surfaces, full structures and were evenly distributed, presenting normal rod‐like shapes in the untreated groups [Fig. 3(A),(D),(G)]. However, the pathogenic bacteria Xoo and Xac exhibited significant changes in morphology and structure in the experimental groups treated with N30 [Fig. 3(B),(C)] and N42 [Fig. 3(E),(F),(H),(I)]. As the drug concentration increased from 25 to 50 μg mL−1, the bacterial cells gradually presented obvious wrinkling, deformation, surface depression and structural damage. These morphological changes may imply possible cell envelope impairment, which is likely to induce the release of intracellular substances, interfere with normal physiological metabolism and further inhibit bacterial growth.
Figure 3.
SEM images showing the morphological changes of bacterial cells treated with N30 or N42. (A–C) Xoo treated with N30 at concentrations of (A) 0 μg mL−1 (control), (B) 25 μg mL−1 and (C) 50 μg mL−1. (D–F) Xoo treated with N42 at concentrations of (D) 0 μg mL−1 (control), (E) 25 μg mL−1 and (F) 50 μg mL−1. (G–I) Xac treated with N42 at concentrations of (G) 0 μg mL−1 (control), (H) 25 μg mL−1 and (I) 50 μg mL−1. Scale bars for all panels: 2 μm.
3.3.2. Antibacterial kinetics of N30 and N42 against Xoo and Xac
The growth curves of Xoo treated with N30, and of Xoo and Xac treated with N42, are shown in Fig. 4. At low concentrations (1.56 and 3.13 μg mL−1), the growth trends of the bacterial strains were largely similar to those of the blank control but still showed a slight inhibitory effect. The slope of the logarithmic growth phase was slightly reduced, and the final stationary phase cell density also was lower than that of the control group. These observations suggest that N30 and N42, without fully inhibiting bacterial growth, moderately attenuate bacterial proliferation at this concentration range. In the medium concentration groups (6.25 and 12.5 μg mL−1), the bacterial cell density during the logarithmic growth phase decreased significantly, indicating a pronounced inhibitory effect of the compounds at these concentrations. These results imply that N30 and N42 effectively disrupt bacterial proliferation, thereby hindering normal cell growth and division. In the high concentration group (25.0 μg mL−1), the OD595 value of the bacterial suspension showed almost no change throughout the entire monitoring period, with the growth curve appearing as an approximately horizontal line, comparable to the initial inoculation concentration. This indicates that N30 and N42 strongly inhibited bacterial growth and proliferation, thus preventing further expansion of the bacterial population at 25.0 μg mL−1.
Figure 4.
Growth curves of Xoo and Xac treated with different concentrations of N30 and N42. (A) N30 against Xoo; (B) N42 against Xoo; (C) N42 against Xac. Treatments included N30 and N42 at concentrations of 0, 1.56, 3.13, 6.25, 12.5 and 25 μg mL−1. Error bars represent the SD of three independent biological replicates.
3.3.3. Effects of N30 and N42 on biofilm formation and EPS production
Biofilm formation by phytopathogenic bacteria acts as a critical defensive barrier 38 and promotes disease development by enhancing bacterial adhesion, providing physicochemical protection and regulating virulence gene expression. As the major matrix component of biofilms, EPS supports biofilm formation, shields bacteria from stresses including desiccation, antibiotics, and host immunity, and concentrates nutrients for survival. 39 , 40 , 41 This study investigated the effects of N30 and N42 on biofilm formation and EPS production, as shown in Fig. 5. Quantitative assay results revealed that N30 and N42 exerted a concentration‐dependent manner on both biofilm formation and EPS production in phytopathogens. For biofilm formation, N30 and N42 inhibited Xoo gradually over 0.625–10 μg mL−1, with maximum inhibition rates of 89.3% and 87.5%, respectively. N42 also inhibited Xac biofilm formation in the range of 1.25–20 μg mL−1, with an inhibition rate up to 87.6%. For EPS production, N30 and N42 inhibited Xoo across 1.56–25 μg mL−1, with maximum inhibition rates of 56.6% and 72.6%, respectively. N42 also inhibited EPS production in Xac, with inhibition reaching 87.7%. Taken together, these results suggested that N30 and N42 could restrict the colonization and infection of phytopathogens in plants by concurrently reducing EPS synthesis and biofilm formation.
Figure 5.
Effects of N30 and N42 on biofilm formation and EPS production in Xoo and Xac. (A–C) Biofilm formation in Xoo treated with N30, Xoo treated with N42, and Xac treated with N42, respectively. Biofilm biomass was quantified by crystal violet staining and measured at OD570(bar graphs); the inhibition ratios were shown as line graphs. (D–F) EPS production in Xoo treated with N30, Xoo treated with N42 and Xac treated with N42, respectively. EPS concentrations (mg L−1) are shown as bar graphs and inhibition ratios as line graphs. Different lowercase letters indicate statistically significant differences between means at P ≤ 0.05. Error bars represent the SD of three independent biological replicates.
3.3.4. Effects of N30 and N42 on extracellular enzymes
Extracellular enzymes, as important virulence factors, contribute to bacterial pathogenicity by degrading key structural components in plants. 42 This study determined the effects of N30 and N42 against the extracellular enzyme activities of phytopathogens to investigate their regulatory roles on the key virulence factors of the pathogens, and the results are presented in Fig. 6. At the concentrations of 10, 100 and 200 μg mL−1, the effect magnitudes of N42 on extracellular cellulase in Xoo were 14.9%, 31.1% and 48.6%, respectively, whereas those for amylase were 23.5%, 48.6% and 66.4%. The effect magnitudes of N42 were superior to those of N30 (cellulase: 13.9%, 22.6%, 35.7%; amylase: 18.0%, 33.6%, 58.2%). These findings provided supportive evidence for understanding the higher biological activity of N42 from an enzymological perspective. Furthermore, N42 also exerted certain effects on the extracellular enzyme activities of Xac. At the aforementioned concentrations, the effect magnitudes of N42 on Xac extracellular cellulase were 18.7%, 36.3% and 85.2%, respectively, and those on amylase were 8.0%, 25.2% and 43.5%. Obviously, N42 exerted a stronger effect on Xac‐derived cellulase than on amylase at the same concentration, suggesting that N42 exhibited enzyme‐specific selectivity toward extracellular enzymes of phytopathogens. These results indicated that N30 and N42 reduced the extracellular cellulase and amylase activities of the tested phytopathogens in a concentration‐dependent manner. It should be noted that relatively high concentrations (100 and 200 μg mL−1) also markedly affect bacterial growth and viability according to the growth curve results. Therefore, the decreased extracellular enzyme activities observed at higher concentrations may be partially attributed to suppressed bacterial proliferation, rather than purely specific inhibition of extracellular virulence enzymes alone.
Figure 6.
Effects of N30 and N42 on extracellular enzyme activities of Xoo and Xac. (A,C,E) Cellulase activities; (B,D,F) amylase activities. Treatments included N30 and N42 at concentrations of 0, 10, 100 and 200 μg mL−1. The bar graphs show the average diameter of inhibition zones (mm), and the corresponding inhibition ratios are presented as line graphs. Different lowercase letters indicate statistically significant differences between means at P ≤ 0.05. Error bars represent the SD of three independent biological replicates.
3.3.5. Effects of N30 and N42 on the swarming motility of Phytopathogens
Swarming motility, a key virulence factor in phytopathogenic bacteria, enables bacterial movement on plant surfaces to target infection sites and establish colonization; co‐regulated with quorum sensing, it also promotes biofilm formation, enhances stress resistance, accelerates tissue penetration and exacerbates disease symptoms. 43 , 44 This study systematically observed and quantitatively analyzed the swarming motility of Xoo treated with N30 and N42, as well as Xac treated with N42 (Fig. 7), to explore the effects of these compounds on the swarming motility of phytopathogens. The results demonstrated that both phytopathogens displayed marked collective migration outward from the inoculation site on semi‐solid medium, forming bacterial rings with an extensive spreading range and large diameter in the control group. By contrast, the colony‐spreading capacity of the phytopathogens was subject to concentration‐dependent inhibition on semi‐solid medium supplemented with N30 or N42. These findings suggest that N30 and N42 can effectively attenuate the swarming motility of phytopathogens.
Figure 7.
Effects of N30 and N42 on swarming motility of Xoo and Xac. (A,C,E) Representative swarming plates treated with different concentrations of N30 or N42; (B,D,F) Quantitative analysis. For Xoo, N30 and N42 were applied at 0, 0.78, 1.56, 3.13, 6.25 and 12.5 μg mL−1. For Xac, N42 was applied at 0, 1.56, 3.13, 6.25, 12.5 and 25 μg mL−1. Bar graphs show average swarming zone diameters (mm); line graphs show inhibition ratios. Different lowercase letters indicate significant differences (P ≤ 0.05). Error bars represent the SD of three independent biological replicates.
3.3.6. Effects of N30 and N42 on the activities of defense enzymes in rice
In order to explore the potential effects of N30 and N42 on host plant responses, this study assayed the defense enzymes (SOD, CAT and POD) activity dynamics in rice plants (Fig. 8). The results showed that both N30 and N42 modulated the activities of defense enzymes in rice plants after inoculation. Regarding SOD activity, both the N30 and N42 treatment groups exhibited similar trends of change, with their activity peaking on day 3 after inoculation. Among them, the N42 treatment group reached the highest peak of 191.9 U g−1, exceeding the levels observed in the N30 group (163.9 U g−1) and all control groups TC (155.3 U g−1), ZT (137.2 U g−1) and CK (159.0 U g−1). As for CAT activity, the N30 treatment group peaked on 5 dpi with an activity of 751.8 U g−1, whereas the N42 treatment group demonstrated a continuous increase from day 1 to day 7 after inocution, reaching a maximum of 883.0 U g−1. Both values were significantly higher than those in all control groups TC (336.4 and 257.4 U g−1), ZT (504.6 and 412.8 U g−1) and CK (259.9 and 224.3 U g−1). In the case of POD activity, both the N30 and N42 treatment groups also peaked on day 3 after inoculation, with activities of 57 036 and 62 001 U g−1, respectively. Notably, the POD activity in the N42 treatment group was significantly higher than that in the N30 treatment group and all control groups TC (52 397 U g−1), ZT (40 670 U g−1) and CK (47 791 U g−1). These results showed that N30 and N42 effectively modulated the activities of rice defense enzymes SOD, CAT and POD upon pathogen infection, suggesting a potential correlation with host defense response regulation.
Figure 8.
Effects of N30 and N42 on the activities of defense enzymes in rice. (A) Catalase (CAT, U g−1) activities; (B) Superoxide dismutase (SOD, U g−1) activities; (C) Peroxidase (POD, U g−1) activities. Enzyme activities were measured at day 1, 3, 5 and 7 after inoculation with N30, N42 at 200 μg mL−1, and after control treatments (CK: 0 μg mL−1, TC and ZT: 200 μg mL−1). The radial bar graphs show enzyme activity values at each time point. Different lowercase letters indicate statistically significant differences between treatments at P ≤ 0.05. Error bars represent the SD of three independent biological replicates.
4. CONCLUSIONS
A series of novel piperidine‐functionalized formononetin derivatives were designed and synthesized, and their antibacterial activities, antibacterial modes and related phenotypes were systematically evaluated. The results showed that some derivatives exhibited significant in vitro inhibitory activities against Xoo and Xac. Among them, N30 and N42 demonstrated potent antibacterial activities, with EC50 values significantly lower than those of the control agents ZT and TC. In vivo pot experiments further confirmed the favorable curative and protective effects of N30 and N42 against plant diseases. Furthermore, the antibacterial modes and related phenotypes under N30 and N42 treatment were investigated from multiple perspectives. On the one hand, SEM and biochemical analyses showed that N30 and N42 could cause bacterial cell‐envelope damage, suppress biofilm formation, reduce EPS, weaken swarming motility and reduce the activities of extracellular virulence‐related enzymes. These changes were accompanied by reducing bacterial growth and infection‐related behaviors. On the other hand, N30 and N42 enhanced the activities of key defense enzymes (SOD, POD, CAT) in rice, which may contribute to better physiological performance of host plants under pathogen stress. Together, these observations suggest a potential combined effect of direct actions on phytopathogens and modulatory influences on plant physiology.
Overall, this study identified two piperidine‐functionalized formononetin derivatives, N30 and N42, which exhibited significant antibacterial activities in vitro and in vivo. The multilevel phenotypic changes induced by these two compounds provide a scientific basis and experimental foundation for the further exploration of their antibacterial properties, as well as the elucidation of their molecular targets and modes‐of‐action. These results also demonstrate the potential of formononetin derivatives in the research and development of novel green antibacterial agents, thereby offering valuable references for the green control of plant bacterial diseases.
CONFLICT OF INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Supporting information
Data S1. Supporting Information.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the National Key Research and Development Program of China (no. 2024YFE0214300), and the Natural Science Foundation of Guizhou Province (grant nos QKHJC‐MS [2026]240 and QKHJC‐MS [2026]388).
Contributor Information
Shuang Feng, Email: feng_shuang1989@sina.com.
Wei Xue, Email: wxue@gzu.edu.cn.
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supplementary material of this article.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data S1. Supporting Information.
Data Availability Statement
The data that supports the findings of this study are available in the supplementary material of this article.