Design and research of new virulence factor inhibitors for plant bacterial disease control

2.1 Instruments and chemicals

All of the reagents were supplied by Bositai, Ltd. (Chongqing, China) and Titan Chemical Co., Ltd. (Shanghai, China). The melting point determination was performed by using the X-4B melting point instrument (Shanghai, China) without correction. The 500 NMR spectrometer (Bruker, Karlsruhe, Germany) was used to measure spectrum data, and the solvent employed was CDCl₃. High-resolution mass spectrometry (HRMS) was performed by using a Thermo Scientific Q Exactive (Thermo Scientific, Missouri, USA). An FEI Nova Nano 450 (FEI, USA) was used to observe the morphology of phytobacteria. The Leici DDSJ-3O8F conductivity meter (Shanghai, China) was used to measure relative conductivity.

2.2 General processes for synthesizing title compounds

Intermediates 1–5 were synthesized by previously reported methods (Feng et al., 2022; Hu et al., 2023; Xiang et al., 2020; Zhan et al., 2023). Target compounds were obtained through Intermediates 5 react with N-piperazine. The synthesis of Z1 was provided as an illustration. Intermediate 5 and K₂CO₃ were added sequentially to a 100 mL round-bottomed flask, stirring at 80 °C for 30 min, followed by the addition of N-piperazine and refluxing for 8–10 h. The reaction progress was monitored using thin layer chromatography (TLC) with a dichloromethane: MeOH = 10:1. Once the reaction was complete, the system was cooled to room temperature, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Z1 was separated and purified by column chromatography with dichloromethane: methanol = 50:1. Z2-Z22 were synthesized with reference to Z1. The detailed steps for the preparation of intermediates 1–5 were in the Supporting Information.

2.3 Biological activity test

2.4 The effect of Z6 on the growth rate of Xoo

Xoo growth curves were generated following previous work with slight modifications (Zeng et al., 2020). Initially, the suspension of Xoo bacteria cultured overnight was adjusted to 0.2 (OD_595nm) with sterile NB, then, Z6 (0, 1.5626, 3.125, 6.25, 12.5, 25 mg/L) were added to Xoo cell suspension in a 180 rpm shaker. The OD_595nm was recorded every 3 h over a period of 30 h. Each experiment consists of three parallel groups and the average absorbance value was calculated.

2.5 The effect of Z6 on the membrane permeability of Xoo cells

Following the previous literature (Wang et al., 2022). The suspension of Xoo cells cultured to the logarithmic phase was centrifuged at 5000 rpm for 10 min. The supernatant was discarded, and the remaining cells were washed with a 5 % glucose solution until the conductivity matched that of 5 % glucose. The concentrations of Z6 (0, 1.5626, 3.125, 6.25, 12.5, 25 mg/L) were combined with the bacterial solution and further incubated in a 180 rpm shaker. Conductivity measurements were taken every 3 h over a total of 18 h (L2). Additionally, different concentrations of Z6 were mixed with 5 % glucose to measure the conductivity (L1). A control group was subjected to boiling water for 5 min, and the resulting conductivity (L0) was measured. The relative conductivity (L’) was calculated by formula (L’ = L2-L1)/L0.

2.6 Effect of Z6 on Xoo extracellular polysaccharides (EPS)

The EPS content assay was following the previous literature. The impact of varying concentrations of Z6 (0, 1.5626, 3.125, 6.25, 12.5, 25 mg/L) on the EPS content of Xoo was investigated through the weighing method, 1 % DMSO serving as the negative control (Jiang et al., 2019).

2.7 Biofilm determination

The method was adapted from previous literature (Bae et al., 2018). 150 μL of Xoo bacterial suspension (OD_595nm = 0.2) was interacted with various concentrations of Z6 (0.0975, 0.195, 0.39, 0.78, 1.56 mg/L), further incubated at 28 °C and 180 rpm. After 72 h, the culture base was removed, and the biofilm was stained with crystal violet for 15 min. Subsequently, the crystal violet was removed, and the biofilm was washed three times with sterile water. 95 % ethanol was then added to dissolve the crystal violet after 4 h. The inhibition rate of the biofilm was evaluated by measuring the absorbance at OD_595nm. A negative control was prepared using Xoo bacterial liquid containing 1 % DMSO.

2.8 Scanning electron microscopy (SEM)

SEM was utilized to analyze the biofilm morphology of Z6, with the experimental methods being derived from previous literature (Zheng et al., 2024).

2.9 Determination of extracellular enzyme activity

The experiment was conducted following the method described in previous literature (He et al., 2010). Amylase and cellulase were selected as representative enzymes to study the impact of Z6 on extracellular enzymes.

2.10 Chlorophyll content determination

The levels of total chlorophyll (Ct), chlorophyll a (Ca), and chlorophyll b (Cb) in rice leaves were measured following established protocols (Wu et al., 2021). Each treatment was replicated three times.

2.11 Determination of defense enzyme activity

Defense enzyme activity was determined using POD, SOD and CAT content detection kits from Beijing Solebao Technology Co., Ltd (Lu et al., 2023). The rice leaves were evenly sprayed with Z6 (200 mg/L) until the liquid dripped, followed by inoculation with Xoo 24 h later. Samples were collected on 1, 3, 5, and 7 d post inoculation and stored at −80 °C. The measurement of POD, SOD and CAT content followed the kit's instructions, with each treatment being replicated 3 times.

2.12 Hypersensitivity (HR) analysis

HR assays were measured by using a previous study with slight modification (Jin et al., 2020). After Xoo cells (OD_595nm = 0.1) were incubated with varying concentrations of Z6 (6.25, 12.5 mg/L) for 2 h, 100 μL of treated-Xoo cells were infiltrated with N. benthamiana leaves by using the osmotic pressure method in the greenhouse for 24 h. Subsequently, the leaves were photographed under natural light, ultraviolet and staining with trypan blue solution for 48 h. A bacterial solution with OD_595nm = 0.1 served as the positive control, while tween water containing 1 % DMSO was used as the negative control.

2.13 The effect of Z6 on rice seed germination

Mature rice seeds were carefully chosen and subjected to alcohol disinfection before being divided into three groups (Liu et al., 2019; Rombolà et al., 2015; Zhou et al., 2024). Each group was then placed in a petri dish containing varying concentrations of Z6 (0, 200, and 500 mg/L) and incubated at 25 °C. The growth of rice seeds was observed on the 2nd, 4th, and 6th d of incubation. Additionally, the same concentrations of Z6 were sprayed on mature rice leaves, and the growth of rice was monitored after 7 d. Each experiment consisted of three parallel groups.

3.1 Chemistry

A total of 22 compounds were synthesized following the procedure outlined in Scheme 1. The structures of all compounds were confirmed using NMR and HRMS, with comprehensive spectral data provided in the Supporting Information.

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Scheme 1

Synthetic route of the Z1-Z22.

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3.2 Bioactivity test

TC was regarded as the control drug and the study evaluated the inhibitory effect of the target compound against 8 phytopathogenic bacteria through the turbidimetric method. Table 1 indicated that incorporating piperazine fragments onto chalcone can notably enhance the compound's antibacterial activity. Specifically, most of title compounds exhibited significant antibacterial activity against Xoo and Xac, the introduction of electron-withdrawing or electron-donating groups could further enhance their antibacterial activity, with inhibition rates of most compounds are 100.0 %. Additionally, Z5 (94.4 %) and Z6 (90.3 %) displayed superior inhibitory effects on Ac compared to TC (67.8 %), Z6 (99.5 %) and Z16 (98.7 %) showed better inhibitory effects on Xf than TC (15.5 %).

EC₅₀ values were determined for select compounds as shown in Table 2 and Fig. S1. The results indicate that Z21 exhibits significant inhibitory activity against Xac, with an EC₅₀ value of 0.83 mg/L, outperforming TC (114.60 mg/L); Z6 (0.29 mg/L) shows a notably higher inhibitory effect against Xoo compared to TC (91.00 mg/L); Z5 (46.20 mg/L) demonstrates a superior inhibitory effect on Ac in comparison to TC (56.25 mg/L); and Z16 (95.79 mg/L) displays a better inhibitory effect on Xf than TC (211.30 mg/L). From Table 1 and Table 2, there is a common feature of the high activity compounds that inhibit Xac, Xf, and Ac: R₁ being an electron-withdrawing group. Moreover, when the R₁ group was an electron-withdrawing group, the activity of anti-Xoo was better than when R₁ was an electron-donating group. Z6 (n = 3, R₁ = 4-Br, R₂ = -Isopropyl, EC₅₀ = 0.29 mg/L) > Z14 (n = 4, R₁ = 4-CH₃, R₂ = -Isopropyl, EC₅₀ = 4.27 mg/L); Z4 (n = 3, R₁ = 4-Br, R₂ = -Methyl, EC₅₀ = 0.50 mg/L) > Z14 (n = 3, R₁ = 4-CH₃, R₂ = -Methyl, EC₅₀ = 2.29 mg/L).

The EC₅₀ value of Z6 was 0.29 mg/L, which outperforms TC (EC₅₀ = 91.00 mg/L), while Z16 has an EC₅₀ value of 0.32 mg/L, similar to Z6. Following the result of EC₅₀ values, the two compounds were mixed in a specific ratio (v/v) by the method of combination and synergism, with each group being tested three times (Liang et al., 2021). The results showed that the best inhibition effect was observed when Z6:Z16 = 4:1 (98.3 %). The specific experimental results were presented in Table S2 and Fig. S2. Z6:Z16 = 4:1 (98.3 %) > Z6:Z16 = 2:1 (97.8 %) > Z6:Z16 = 1:1 (94.0 %) > Z6: Z16 = 1:0 (89.8 %) > Z6:Z16 = 0:1 (87.9 %) > Z6:Z16 = 1:2 (84.4 %) > Z6:Z16 = 1:4 (69.4 %). Analysis revealed that as the proportion of Z6 increases, the inhibition rate also increases. Consequently, Z6 was chosen as the focus for subsequent in vivo activity testing and mechanism research.

3.3 Effect of Z6 on growth curves of Xoo cells

The experimental results depicted in Fig. 2a, demonstrate that Z6 significantly inhibits the growth of Xoo at concentrations of 25 and 12.5 mg/L, in comparison to the control group, the inhibitory effect on Xoo growth diminishes slightly at concentrations of 6.25 and 3.125 mg/L, the inhibitory effect on Xoo growth at 1.5625 mg/L was comparable to that of the control group, indicating a concentration-dependent inhibitory effect of Z6 on Xoo.

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Fig. 2

The effect of Z6 on Xoo cells.

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3.4 Determination of cell membrane permeability of Xoo by Z6

The permeability of cell membranes can be assessed through relative conductivity. Fig. 2b illustrates that Z6 enhances Xoo cell membrane permeability in a dose-dependent manner. Xoo cells were treated with varying concentrations of Z6 (0, 1.5625, 3.125, 6.25, 12.5, and 25 mg/L) for 18 h resulted in a gradual increase in cell conductivity and membrane permeability. This indicates that following the interaction between Xoo and Z6, the cell membrane is compromised, leading to the release of intracellular substances and an increase in relative conductivity.

3.5 Analysis of EPS and biofilm content

EPS serves as the primary substrate for biofilm formation and also plays a role in aiding bacteria to evade plant defense mechanisms. The penetration of antibiotics into plants can be impacted by EPS, consequently contributing to enhanced drug resistance (Kakkar et al., 2015). Thus, the impact of Z6 on EPS secretion was explored (Fig. 2c), it demonstrated that Z6 diminishes EPS content in a dose dependent manner, aligning with the observations from the growth curve analysis.

Furthermore, the inhibitory effect of Z6 on biofilm growth at various concentrations was quantitatively assessed using crystal violet staining by measuring the value of optical density (OD_595nm), as shown in Fig. 2d. When Xoo were treated with Z6 (1.56, 0.78, 0.39, 0.195, 0.0975 mg/L), the inhibition rates were 84.7, 58.7, 28.4, 12.7, 4.8 %, respectively. These results collectively indicateed that Z6 exhibits a significant inhibitory effect on biofilm formation and EPS production reduction.

3.6 Effect of Z6 on the morphology of Xoo cells

SEM was utilized for visualization to further observe the impact of Z6 on Xoo cells, as shown in Fig. 3, in the control group, Xoo cells appeared uniform and plump. However, after Xoo were interacted with Z6 about 9 h, some cells exhibited shrinkage and ruptured cell membranes. This effect was more pronounced at 100 mg/L, with increased cell rupture and shrinkage significantly, showing contraction, collapse and deformation. SEM analysis clearly indicates that Z6 has the potential to damage Xoo cells.

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Fig. 3

SEM images for Xoo after incubated in diferent concentrations of Z6.

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3.7 Analysis of extracellular enzyme content

In addition to secreting virulence factors like EPS, plant bacteria also release amylase and cellulase, such as amylase, cellulase, pectinase, etc. These enzymes play a crucial role in breaking down extracellular macromolecules to acquire nutrients, supporting bacterial growth, and aiding in the infection of plants.

The impact of Z6 on Xoo extracellular amylase and cellulase was assessed at concentrations of 6.25, 12.5, and 25 mg/L. In the experiment of evaluating the impact of Z6 on amylase with KI/I₂, the average diameter of the hydrolysis circle in the control group (1 % DMSO) was 17 cm (Fig. 4). When different concentrations of Z6 were interacted with Xoo cells, the corresponding diameters of the hydrolysis circles were 14, 12 and 10 cm. These findings suggest that Z6 possesses the ability to inhibit Xoo amylase activity.

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Fig. 4

The impacts of Z6 on Xoo extracellular enzymes.

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Similarly, After Xoo cells were treated with Z6 at 0.0, 6.25, 12.5 and 25 mg/L, the cells were cultured in cellulose dishes for three days, stained with 0.1 % Congo red for about 15 min, and washed with 1 mol/L NaCl for three times for decolorization. It can be observed that the hydrolysis of cellulase appears. The diameter of hydrolytic rings was 17 cm, 15 cm, 13 cm, 10 cm, respectively. The results showed that Z6 could inhibit the activity of Xoo cellulase.

3.8 Chlorophyll content determination

The process of photosynthesis was intricately linked to the chlorophyll content of plants. The chlorophyll content of rice leaves was examined following treatment with CK, Xoo, Z6, and Z6 + Xoo on 1, 3, 5, 7 d (Fig. 5). Among the susceptible group, the Ct content was Z6 + Xoo > Xoo, and Z6 + Xoo shows an upward trend followed by a downward trend, peaking on the 3rd day (Fig. 5c). In the healthy group, the Ct content was highest in Z6 compared to CK, with Z6 consistently maintaining the highest Ct content throughout the 1–7 d period. The distributions of Ca and Cb followed a similar pattern to Ct (Fig. 5a, b). These findings suggested that Z6 supplementation leads to an increase in chlorophyll content in rice, ultimately stimulating photosynthesis and bolstering plant disease resistance.

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Fig. 5

Analysis of chlorophyll content and the activities of defensive enzyme in rice leaves of Z6.

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3.9 Determination of defense enzyme activity

In plants, antioxidant enzymes such as POD, SOD and CAT play a crucial role in protecting cells from damage caused by reactive oxygen species and in preventing or reducing the formation of hydroxyl radicals. Normally, there exists a delicate balance between the generation and removal of free radicals within plant cells (Yi et al., 2022). However, under unfavorable conditions, this balance was disturbed, leading to the accumulation of free radicals that can inflict damage on the cell (Hatami and Ghorbanpour 2024).

The activities of POD, SOD and CAT in rice leaves were analyzed, which treated with CK, Xoo, Z6, and Z6 + Xoo on 1, 3, 5, and 7 d, as depicted in Fig. 5d–f. In terms of POD activity, the content of Z6 + Xoo showed a gradual increase from 1 to 5 d, peaking on the 5th day at a significantly higher level than the group of Xoo (Fig. 5d); In terms of SOD activity, the SOD content after Z6 + Xoo treatment showed a trend of first increasing and then decreasing. The samples of Z6 + Xoo reached their peak on the 5th day, which was significantly higher than the activity Xoo treatment (Fig. 5e); CAT activity peaked on the fifth day after Z6 + Xoo treatment, which was significantly higher than the activity induced by Xoo treatment (Fig. 5f). These results indicate that Z6 enhances plant POD, SOD and CAT activities, thereby boosting plant defense capabilities.

3.10 HR analysis

Plant HR is the process by which cells in and around the infection site undergo programmed cell death to halt the spread of pathogens within the plant. N. benthamiana has the ability to detect effector molecules released by the rice BLB, leading to a HR (Liu et al., 2020). In this study, N. benthamiana was utilized to assess the capacity of Z6 to induce HR in tobacco leaves following pretreatment with Xoo bacteria. The results depicted in Fig. 6, indicate that in the Xoo control group, tobacco leaves exhibited significant wilt lesions, while no noticeable changes were observed in the 1 % DMSO blank group. However, the leaf lesions decreased gradually when Xoo was pretreated with Z6 at 6.25 and 12.5 mg/L. BIO-RAD and trypan blue staining were used to visualize necrotic lesions, and the results showed that Z6 effectively hinders the production of effector molecules by Xoo, thereby impeding further infection in plants.

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Fig. 6

HR of Z6 pretreated Xoo on N. benthamiana.

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3.11 In vivo antibacterial activity of Z6

The research aims to identify a potential antibacterial agent for managing rice BLB. Rice was inoculated with Xoo bacteria by cutting leaf method. After 14 d, the disease index and control effect were calculated by measuring the length of rice leaf spots. The experimental results are shown in the Fig. 7 and Table 3. Z6 demonstrated notable curative (47.9 %) and protective activity (51.5 %) against Xoo at a concentration of 200 mg/L, surpassing TC (30.5, 47.9 %).

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Fig. 7

Curative and protective activities of 200 mg/L Z6 against rice BLB under greenhouse conditions.

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3.12 Qualitative analysis of the effect of Z21 on citrus bacterial canker disease

Green lemons were specifically chosen for a study testing the efficacy of Z21 in controlling citrus bacterial canker disease. The result was shown as Fig. 8. Lemons treated with Xac displayed yellow, and a substantial portion of the lemons was infected and decomposed; lemons injected with water showed no discernible changes and the lesion area of lemons treated with Z21 and Xac was smaller than the control drug TC. Thus, qualitative analysis experiments indicateed that the promising antibacterial properties of Z21 in protective and curative activities against citrus bacterial canker disease.

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Fig. 8

Curative and protective activity of Z21 against citrus bacterial canker disease.

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3.13 Effect of Z6 on rice seed germination

The experimental results are presented in Fig. 9a. On the second day, rice seeds at concentrations of 500, 200, and 0 mg/L all germinated. Subsequently, on the fourth and sixth days, seeds at these concentrations exhibited normal growth. Thus, the experiment revealed that Z6 at the concentrations of 200 and 500 mg/L did not have a significant impact on rice seed germination.

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Fig. 9

Toxicity experiment of Z6.

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3.14 Toxicity experiments

The phytotoxicity test demonstrated that rice treated with 200 and 500 mg/L of Z6 exhibited normal growth with healthy leaves (Fig. 9b). Similarly, the same concentrations of Z21 were sprayed with lemon leaves, which showed normal growth and healthy leaves after 7 d (Fig. S3). This indicated that Z6 and Z21 didn’t exhibit toxicological activity against rice and lemon at concentrations of 200 and 500 mg/L. Furthermore, the physicochemical properties and potential toxic effects of Z6 were also assessed using the online prediction tool ADMETlab 2.0. The results indicated no human hepatotoxicity (H-HT), AMES toxicity, eye irritation, or corrosiveness for Z6 (Table S3, Fig. S4). These findings suggested that Z6 holds promise as a protective and curative agent against bacterial diseases in plants.