ZIF-93-Based Nanomaterials as pH-Responsive Drug Delivery Systems for Enhanced Antibacterial Efficacy of Kasugamycin in the Management of Pear Fire Blight

Abstract

Kasugamycin (KSM) is easily affected by photolysis, acid–base destruction, and oxidative decomposition in the natural environment, leading to its poor durability and low effective utilization rate, which affects its control effect on plant bacterial diseases. Nanomaterials modified with environment-responsive agents enable the control of the release of pesticides through intelligently responding to external stimuli, thereby improving efficacy and reducing environmental impact. In this study, a pH-responsive controlled release system was constructed using zeolitic imidazolate frameworks (ZIF-93) for the sustained and targeted delivery of KSM. The synthesized KSM@ZIF-93 exhibited a diameter of 63.93 ± 11.19 nm with a drug loading capacity of 20.0%. Under acidic conditions mimicking bacterial infection sites, the Schiff base bonds and coordination bonds in ZIF-93 dissociated, triggering the simultaneous release of KSM and Zn²⁺, achieving a synergistic antibacterial effect. Light stability experiments revealed a 34.81% reduction in UV-induced degradation of KSM when encapsulated in ZIF-93. In vitro antimicrobial assays demonstrated that KSM@ZIF-93 completely inhibited Erwinia amylovora at 200 mg/L and had better antibacterial activity and persistence than KSM and ZIF-93. The field experiment and safety evaluation showed that the control effect of KSM@ZIF-93 on pear fire blight at the concentration of 200 mg/L was (75.19 ± 3.63)% and had no toxic effect on pollen germination. This pH-responsive system not only enhances the stability and bioavailability of KSM but also provides a targeted and environmentally compatible strategy for managing bacterial infections during the flowering period of pear trees.

1. Introduction

The fire blight disease caused by Erwinia amylovora, a type of bacteria, is one of the most destructive diseases affecting plants in the Rosaceae family, such as pear and apple [1,2]. This disease is characterized by rapid spread, wide transmission routes, and severe damage [3]. The disease has spread to nearly 60 countries and regions. It was first discovered in Xinjiang’s Yili region in 2016 and quickly spread to most pear-producing areas of Xinjiang in recent years [4]. Pear fire blight can greatly reduce the yield and merchantability of pears in the current season by infecting the flower apparatus and killing fruit branches, and even cause the loss of the whole plant and orchard. Fire blight disease has been included in the “Class I crop diseases and pest list” by the Ministry of Agriculture and Rural Affairs of China in 2020 [5]. The flower apparatus is the most easily infected by E. amylovora [6]. Therefore, chemical control in the flowering period is the most effective measure to prevent the spread of pear fire blight [3,7]. As traditional bactericides such as agricultural streptomycin and bismerthiazol have been discontinued, the use of copper preparations, kasugamycin (KSM), hexythiazox, and so on has become the main pesticides for the control of pear fire blight in production. Copper preparations are prone to causing phytotoxicity, and spraying during the flowering period can easily reduce the fruit setting rate of pear trees [8,9,10]. KSM, as a biological pesticide, has a similar antibacterial effect on E. amylovora as agricultural streptomycin. It is often used in the prevention and control of pear blight [11,12]. KSM is easily affected by photolysis, acid–base destruction, and oxidative decomposition in the natural environment, leading to its poor durability and low effective utilization rate, which affects its control effect on plant bacterial diseases [13,14,15]. In order to solve these problems, researchers have designed a variety of slow-release formulations, such as kasuga-silica-conjugated nanospheres and KSM-pectin conjugate, to increase the stability of KSM and extend its effective time [16,17]. In addition, KSM preparations commonly used in the market are easy to cause drug damage when used during flowering because of the high toxicity of their adjuvants to pollen [18,19]. Therefore, it is necessary to design an environmentally responsive controlled release system without adjuvants, which can ensure the security and controlled release of KSM.

In recent years, stimulus-responsive pesticide delivery systems based on nanocarriers have attracted widespread attention due to their ability to control pesticide release, improve target activity, and minimize the adverse effects on the environment [20,21,22,23]. Metal–organic frameworks (MOFs), composed of metal nodes and organic linkers through coordination bonds, have attractive development prospects in modern materials research because of open unsaturated metal sites [24,25,26]. They have been widely used in gas storage, separation, catalysis, and drug delivery. Among the nanocarriers studied, zeolitic imidazolate frameworks (ZIFs) nanocarriers as an excellent carrier for high drug loading environmentally responsive pesticide control systems have many unique advantages, such as large pore size capacity, huge specific surface area, controllable size distribution, easy-to-adjust skeleton structure, good thermal and chemical stability, good biocompatibility and surface modification [27,28,29]. ZIF-93 is a kind of ZIF material; it is mainly a coordination polymer formed by the coordination reaction of Zn²⁺ and 4-methylimidazole-5-formaldehyde [30,31]. ZIF-93 is mostly stable under normal physiological conditions, while under acidic conditions, the protonation reaction of N atom on 4-methylimidazole-5-carboxaldehyde destroys the coordination bond between Zn²⁺ and N atoms, resulting in the collapse and dissolution of the three-dimensional internal void structure, releasing Zn²⁺ and the drug loaded inside, realizing the synergistic antibacterial effect against bacteria. This feature can be used to construct a pH-intelligent responsive controlled release system [31,32,33]. In addition, the highly reactive-free aldehyde group of ZIF-93 could be covalently conjugated with the amino group in the drug molecule to form a Schiff base bond through the Schiff base reaction. The resulting Schiff base bonds could be rapidly degraded under acidic conditions, enabling the active ingredient to be released on demand [34].

In this study, a pH-intelligent responsive controlled release system (KSM@ZIF-93) was constructed by covalently coupling the aldehyde group of ZIF-93 with the amino group of KSM through a Schiff base reaction, which could effectively reduce the unintentional leakage and photodegradation of KSM. The change in pH value of the infected site of the pathogen would destroy the coordination bond and Schiff base bond on the nanomaterials, and release Zn²⁺ and KSM, which not only makes the nano-pesticide have the characteristics of slow release and targeting, but also forms a double killing effect on the pathogen. The characterization, the light stability, the release kinetics, the biological activity, and pollen germination safety of the prepared KSM@ZIF-93 were fully evaluated. This paper aims to construct a pH-intelligent responsive controlled release system (KSM@ZIF-93).

2. Materials and Methods

2.1. Materials

Pear pollen was purchased from Hebei Jiamingliang Pollen Co., Ltd. (Shijiazhuang, China). Kasugamycin (KSM, purity 98%) was purchased from Shanghai Yi en Chemical Technology Co., Ltd. (Shanghai, China). Phosphoric acid, 4-methylimidazole-5-formaldehyde, potassium bromide, and acetonitrile were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Zinc nitrate hexahydrate and Tris-HCl buffer were purchased from Chengdu Huaxia Reagent Co., Ltd. (Chengdu, China). Sodium dodecyl sulfate (SDS) was purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). PBS buffer solution was purchased from Xiamen An Yongbo Technology Co., Ltd. (Xiamen, China). Sucrose was purchased from Tianjin Xinbote Chemical Co., Ltd. (Tianjin, China). Boric acid was purchased from Tianjin Beilian Fine Chemicals Development Co., Ltd. (Tianjin, China). Kasugamycin aqueous solution (4%) was purchased from Shanxi McCro Biological Technology Co., Ltd. (Yuncheng, China). The ultrapure water was made by the observation station of Tarim University (Alar, China). The strain of pear fire blight (E. amylovora) was obtained from plant pathology Laboratory of the College of Agronomy, Tarim University, (Alar, China).

2.2. Synthesis of KSM@ZIF-93

2.2.1. Synthesis of ZIF-93

The preparation of ZIF-93 was referred to the method reported by Jin et al. [35]. A total of 0.882 g Zn(NO₃)₂ 6H₂O was first dissolved in 60 mL methanol and quickly added to 60 mL methanol solution containing 2.61 g 4-methylimidazole-5-formaldehyde. The mixture was stirred at room temperature for 20 min and then centrifuged at 13,000 r/min for 15 min to collect precipitate, and washed with ethanol three times.

2.2.2. Synthesis of KSM-Linked ZIF-93 (KSM@ZIF-93)

The KSM was covalently linked to the ZIF-93 by Schiff base reaction. Firstly, 100 mg KSM was dissolved in 3 mL Tris-HCl buffer (1.5 M, pH 8.5) at room temperature. Then, 50 mg of prepared ZIF-93 was dispersed in 3 mL Tris-HCl buffer (1.5 M, pH 8.5) with an ultrasonic cleaner. The solution was added dropwise to a flask containing KSM solution and stirred continuously at room temperature in dark for 24 h. Finally, the products were collected by centrifugation and cleaned with ultra-pure water three times to remove unreacted KSM molecules. The precipitate was dried in a drying oven before further processing.

2.3. Characterization of KSM@ZIF-93

The morphology of the obtained sample was characterized by a ZEISS Sigma500 scanning electron microscope (SEM) (Oberkochen, Germany). The crystal structural analyses of products were carried out by using a Rigaku UltimaIV X-ray diffractometer (XRD) (Akishima, Japan) with a Cu Kα incident source at a scanning rate of 10°/min with a 2θ range from 5° to 90°. The chemical functional groups of ZIF-93, KSM, and KSM@ZIF-93 were determined by a Brooke-TENSOR II Fourier Transform Spectrophotometer (FTIR) in transmission mode using the KBr pellet technique. Particle size and zeta potential of ZIF-93 and KSM@ZIF-93 particles were measured by DLS with a Malvern-Zetasizer Nano ZS. The content of KSM in KSM@ZIF-93 was estimated by a TG209F1 thermogravimetric analyzer (TGA) under a nitrogen atmosphere with continuous heating from ambient temperature to 600 °C at a heating rate of 10 °C/min.

The content of KSM in KSM@ZIF-93 was determined by high-performance liquid chromatography (HPLC) system with ultraviolet detector (ALLIANCE E2695, Waters Corporation, Milford, CT, USA). The chromatographic column of the system was Kromasil ODS C18 (250 mm × 4.6 mm, 5 μm; Dikma, Lake Forest, CA, USA). The column temperature was room temperature. The UV detection wavelength was 210 nm. The mobile phase composition was acetonitrile: 0.1% phosphoric acid + 0.1% SDS aqueous solution (0.1% phosphoric acid: SDS = 70:30) = 40:60. The flow rate was maintained at 1 mL/min, and the injection volume was 20 μL. The mobile phase was ultrasonically degassed for 15 min before use, and the chromatographic system was pre-balanced with the mobile phase for 30 min before analysis. All mobile phases and samples used for HPLC analysis were filtered through a 0.45 μm membrane filter.

2.4. Light Stability of KSM@ZIF-93

In order to evaluate the photostability of KSM and KSM@ZIF-93 under ultraviolet light irradiation, a certain amount of sample was added to a closed dialysis bag (MW: 3500 Da), then immersed in 50 mL PBS buffer solution (pH 7) and exposed to a 30 W ultraviolet lamp with a distance of 20 cm. Within a predetermined time interval, 1 mL of the solution containing KSM Stir at room temperature in the dark for 24 h were taken from the liquid outside the dialysis bag of each sample and supplemented with the same volume of PBS buffer solution to measure the KSM content of the sample at 210 nm by HPLC system. The samples incubated in the dark were set as the control group, and all tests were repeated three times. The degradation efficiency of KSM in the sample was calculated by the following equation:

$$\mathrm{D}\mathrm{e}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y}(\mathrm{\%})={\displaystyle \frac{A_0-A_t}{A_0}}\times 100$$

In the equation, A_t is the concentration of KSM at irradiation time t, A₀ is the initial concentration of KSM.

2.5. Controlled Release Kinetics

The release characteristics of KSM from KSM@ZIF-93 under different pH values (5, 6, 7) were studied by dialysis bag method. The corresponding pH buffer solution (PBS) was prepared. A 2 mL amount of KSM@ZIF-93 dispersion (25 mg/mL) was placed in a sealed dialysis bag. Then, the bag was submerged in 50 mL buffer solution at the corresponding pH. The solution was wrapped in tinfoil to avoid light and placed on a constant temperature shaker at 25 °C. At a predetermined time interval, 0.5 mL solution was taken from the liquid outside the dialysis bag of each sample, and the same volume of PBS buffer was supplemented. The content of KSM at 210 nm was analyzed by HPLC system. All tests were repeated three times.

2.6. Antimicrobial Assays In Vitro

The minimum inhibitory concentration (MIC) was determined by medium dilution method [36]. A 10 mL amount of LB liquid medium containing different concentrations of KSM, ZIF-93, and KSM@ZIF-93 (6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L were, respectively, poured into sterilized 50 mL centrifuge tubes, and then 200 µL of activated E. amylovora bacteria solution (about 1 × 10⁸ CFU·mL⁻¹) was added. The LB liquid medium without drug was used as a blank control group. The culture solutions were cultured at 28 °C and 220 r/min for 18 h, and 200 μL of each shaking culture solution was coated on LB solid medium. The LB solid medium was cultured in a constant temperature incubator at 28 °C for 24 h, and the MIC of each treatment was determined according to no visible colony growth. Each treatment was repeated three times.

2.7. Field Experiment

The suspensions of KSM, ZIF-93, and KSM@ZIF-93 with concentrations of 400 mg/L, 200 mg/L, 100 mg/L, and 50 mg/L were prepared as test agents. The sterile water treatment was used as blank control group. A fine needle was used to pierce 10 fine holes in the center of the leaves of living pear trees with the same growth, and then E. amylovora bacteria solution (about 1 × 10⁸ CFU·mL⁻¹) was sprayed on the leaves of the punctures, and after 24 h, different concentrations of test agents were sprayed on the leaves. Each treatment was tested on 10 living pear leaves with the same growth, and three replicates were set up. The disease spots were observed and recorded 10 days later. The method of Paprstein et al. [37] was referred to and improved in the formulation of the disease grading criteria for the live leaves inoculated by pathogenic bacteria of pear fire blight (Table 1). At the same time, the disease index and control effect were calculated according to the following formula.

$$\mathrm{D}\mathrm{i}\mathrm{s}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e} \mathrm{i}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{x}={\displaystyle \frac{\sum (B_1\times B_2)}{B_3\times B_4}}\times 100$$

$$\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{e}\mathrm{f}\mathrm{f}\mathrm{e}\mathrm{c}\mathrm{t}(\mathrm{\%})={\displaystyle \frac{C_1-C_2}{C_1}}$$

In the equation, B₁ is the disease level representative value, B₂ is the number of diseased leaves at all levels, B₃ is the total number of leaves surveyed, B₄ is the highest disease level representative value, C₁ is the disease index of control group, and C₂ is the disease index of treatment group.

Table 1. Grading standards of pear fire blight.

Progression of Disease	Symptom
0	Leaves without diseased spots.
1	The area of leaf diseased spots accounted for 1–5% of the inoculated leaf area.
3	The area of leaf diseased spots accounted for 6–15% of the inoculated leaf area.
5	The area of leaf diseased spots accounted for 16–30% of the inoculated leaf area.
7	The area of leaf diseased spots accounted for 31–50% of the inoculated leaf area.
9	The area of leaf diseased spots accounted for more than 50% of the inoculated leaf area.

Table 1. Grading standards of pear fire blight.

Progression of Disease	Symptom
0	Leaves without diseased spots.
1	The area of leaf diseased spots accounted for 1–5% of the inoculated leaf area.
3	The area of leaf diseased spots accounted for 6–15% of the inoculated leaf area.
5	The area of leaf diseased spots accounted for 16–30% of the inoculated leaf area.
7	The area of leaf diseased spots accounted for 31–50% of the inoculated leaf area.
9	The area of leaf diseased spots accounted for more than 50% of the inoculated leaf area.

2.8. Safety Evaluation

The infection of pear fire blight mainly occurs in the flowering period. Therefore, the flowering period is a key period for the prevention and control of pear fire blight. Pollen germination is extremely sensitive to pesticides, so phytotoxicity analysis can be carried out by monitoring pollen germination rate. The inhibitory effects of KSM aqueous solution (4%) and KSM@ZIF-93 on pollen germination rate were evaluated by simulated field spraying experiments. 60 g sucrose and 60 mg boric acid were dissolved in 600 mL ultrapure water and stirred and dissolved to form basic culture solution. 100 mL basic culture solution was taken in a conical flask and frozen at 4 °C for later use. 7.5 g amount of agar powder was added in the remaining 500 mL basic culture solution to fully dissolve and sterilize at high temperature. Therefore, the germination medium was prepared. 0.5 g amount of pollen was weighed and put into the prepared 100 mL basic culture medium, shaken at 150 rpm for 30 min, and a pollen suspension about 0.5% was obtained. In the simulated field spraying experiment, the fungicides containing different concentrations of KSM, ZIF-93, KSM aqueous solution (4%), and KSM@ZIF-93 (100 mg/L, 200 mg/L, and 400 mg/L) were sprayed on the surface of the sterilized germination medium and dried naturally for 2 h. Then, 0.8 mL of the diluted pollen solution was immediately applied to the solid germination medium, and the pollen germination rate was recorded after 12 h of dark incubation in a constant temperature incubator at 25 °C [38]. The sterile water was used as the blank control group. Five-point sampling mode was used to observe and record the test results on the microscope. All treatments were repeated three times.

2.9. Data Processing

The experimental data were analyzed by SPSS 26.0 statistical analysis software (SPSS, Chicago, IL, USA). The data were analyzed by Duncan’s multiple range test (p < 0.05) and expressed as the mean ± standard error of the mean for all the experiments. Origin 2022 and Excel 2016 were used to plot the graphs. All treatments were replicated three times.

3. Results

3.1. Preparation and Characterization of KSM@ZIF-93

The preparation mechanism and acid-triggered release mechanism of KSM@ZIF-93 are shown in Figure 1A. The zeolitic imidazolate framework material (ZIF-93) was synthesized by the coordination reaction of Zn²⁺ and N atom on 4-methylimidazole-5-carbaldehyde by the stirring synthesis method at room temperature. Schiff base bond was formed by covalent coupling of the aldehyde group on ZIF-93 and the amino group of KSM, so that KSM was successfully grafted onto the surface of ZIF-93 nanoparticles to prepare KSM@ZIF-93.

The morphologies and size of the synthesized ZIF-93 and KSM@ZIF-93 were measured by SEM and DLS, and the results are shown in Figure 2. The results showed that the synthesized ZIF-93 had good dispersibility and spheroid morphology with a diameter of around (59.39 ± 8.72) nm. When the KSM was grafted on the ZIF-93, the morphology of KSM@ZIF-93 was similar to that of ZIF-93, and a diameter of around (63.93 ± 11.19) nm, indicating that the zeolite imidazole framework was not destroyed after Schiff base reaction, and ZIF-93 has good stability. In order to qualitatively verify the formation process of KSM@ZIF-93, ZIF-93 and KSM@ZIF-93 were, respectively, tested for zeta potential, and the results are shown in Figure 3A. The zeta potential of ZIF-93 was 13.6 mV. When the KSM was grafted on the ZIF-93, the zeta potential of KSM@ZIF-93 decreased (−11.4 mV), which may be due to the neutralization of negatively charged carboxylate ions on the KSM molecules with the positive ions on the surface of ZIF-93. The results indicated that KSM was successfully grafted onto ZIF-93, and KSM@ZIF-93 was synthesized.

The crystal structure of ZIF-93 before and after grafting KSM was characterized by XRD analysis, and the results are shown in Figure 3B. The simulated XRD pattern of ZIF-93 showed eight characteristic diffraction peaks at 6.229°, 7.631°, 9.856°, 12.476°, 13.236°, 13.956°, 15.924°, and 18.764°, which belong to the crystalline planes of (200), (211), (310), (400), (411), (420), (510), and (600), respectively. According to the analysis of X-ray characteristic peak data, the main characteristic peaks of the prepared ZIF-93 have a good correlation with the simulated characteristic peak of ZIF-93, which proves that Zn²⁺ and 4-methylimidazole-5-formaldehyde were successfully coordinated. By comparing ZIF-93 with KSM@ZIF-93, KSM@ZIF-93 obtained by grafting KSM had similar characteristic peaks to ZIF-93, indicating that loading KSM by Schiff base reaction did not change the metal skeleton integrity of ZIF-93, and ZIF-93 had good stability. Similar results have been reported by Vasconcelos et al. [39] and Liang et al. [40].

The structures of KSM, ZIF-93, and KSM@ZIF-93 were characterized by FTIR spectra, and the results are shown in Figure 3C. In the KSM molecule, the peak at 1693 cm⁻¹ was attributed to the C=O stretching vibration absorption peak of the carbonyl group, the peak at 1660 cm⁻¹ and 1622 cm⁻¹ were assigned to the bending vibration of amine group (N-H), and the double peaks at 3489 cm⁻¹ and 3381 cm⁻¹ were the stretching vibration absorption peak of amine group (N-H), indicating that there were primary amino groups in the molecule [41,42]. In the FTIR spectra of ZIF-93, the peaks at 1666 cm⁻¹ and 1633 cm⁻¹ were related to the stretching vibration of the aldehyde group (C=O) in the 4-methylimidazole-5-formaldehyde ligand, and the peak at 1543 cm⁻¹ was the imidazole ring [43,44,45]. After the Schiff base reaction between KSM and ZIF-93, the peaks at 3489 cm⁻¹ and 3381 cm⁻¹ in KSM shifted and became significantly smaller, indicating that many primary amines in KSM disappeared and KSM was successfully grafted onto ZIF-93. The absorption peaks of the aldehyde group (2787 cm⁻¹ and 2737 cm⁻¹) in ZIF-93 were weakened, which indirectly indicated that the Schiff base reaction occurred. In addition, the characteristic band (C=O) at 1633 cm⁻¹ in ZIF-93 and the characteristic band at 1622 cm⁻¹ in KSM disappeared, indicating that the Schiff base reaction occurred. The amine group in the KSM molecule reacted with the aldehyde group of ZIF-93 to form a C=N bond, and KSM was successfully grafted onto ZIF-93.

The loading capacity and thermal stability of KSM@ZIF-93 were studied by thermogravimetric analysis (TGA), and the results are shown in Figure 3D. The original weight loss in the range of 32–178 °C might be caused by the removal of free and bound water molecules from the samples. TGA curve of KSM shows that the weight loss between 178 and 252 °C may be due to the decomposition and volatilization of KSM [46]. The TGA curve of ZIF-93 shows that the 43.3% weight loss between 178 and 800 °C was mainly due to the breaking or decomposition of the coordination bond in ZIF-93. After functionalization with KSM, KSM@ZIF-93 loses more weight (63.3%) than ZIF-93 at 178–800 °C, which may be caused by the cleavage of the Schiff base bond and the decomposition of the ZIF-93 skeleton. According to the TGA result analysis, the load ratio of KSM in KSM@ZIF-93 was 20.0%.

3.2. Release Kinetics

The KSM-triggered release behavior in KSM@ZIF-93 was shown in Figure 1B. When E. amylovora infects pear trees, the pH value of the infected site would change, and the change in pH value would destroy the Schiff base bonds on KSM@ZIF-93 and realize the on-demand release of KSM, indicating that KSM@ZIF-93 has good pH response ability under acidic conditions.

Figure 4 shows the release curves of KSM from KSM@ZIF-93 at different pH values (5, 6, and 7). The results showed that KSM@ZIF-93 had different pH response properties, and the release of KSM molecules shows an increase in a typical pH-dependent manner. After incubation in PBS buffer solution with pH 7 for 96 h, the cumulative release of KSM in KSM@ZIF-93 was very small, about 7.45%. By contrast, the release amount of KSM reached up to 42.63% at pH 6 and 64.68% at pH 5 within a period of 96 h, respectively, indicating that KSM@ZIF-93 could exist stably under neutral conditions and gradually release under acidic conditions. In summary, the rate of KSM release from KSM@ZIF-93 increases gradually with the increase in acidic strength, which may be due to the accelerated acid-responsive imine bond breaking in KSM@ZIF-93, which not only achieves the on-demand release of KSM@ZIF-93 but also improves the pharmacokinetics and drug utilization rate.

3.3. Light Stability of KSM@ZIF-93

In order to evaluate the effect of ultraviolet radiation on the stability of KSM@ZIF-93, the degradation rate of KSM was determined by HPLC, and the results are shown in Figure 5. KSM and KSM@ZIF-93 showed different degrees of degradation under ultraviolet radiation, while the degradation rates of KSM and KSM@ZIF-93 under non-radiation conditions were relatively low and could be ignored. The KSM dispersed in aqueous solution was rapidly decomposed under ultraviolet radiation. After 48 h of irradiation, the degradation rate of KSM was as high as 90.23%, indicating that KSM was sensitive to ultraviolet radiation and easy to be degraded. The degradation rate of KSM@ZIF-93 formed by covalent coupling of KSM with ZIF-93 through Schiff base reaction was 55.42%, which had a good protective effect. The results showed that KSM@ZIF-93 could reduce the degradation of KSM by ultraviolet radiation and enhance the photostability of KSM, which provided the possibility to prolong the efficacy of KSM in the natural environment.

3.4. Antimicrobial Assays In Vitro

In order to evaluate the antibacterial activity of KSM aqueous solution, ZIF-93 and KSM@ZIF-93 against E. amylovora, MIC tests were performed. As shown in Figure 6, the density of E. amylovora colonies was affected by KSM aqueous solution, ZIF-93, and KSM@ZIF-93 in a typical dose-dependent manner. The growth of E. amylovora was completely inhibited by ZIF-93, and KSM aqueous solutions were 400 mg/L and 200 mg/L, respectively, while the inhibitory activity of KSM@ZIF-93 achieved the same efficacy as 100 mg/L. The MIC of KSM@ZIF-93 was lower than that of KSM aqueous solution and ZIF-93, indicating that KSM@ZIF-93 had better antibacterial properties. The difference in antibacterial activity between KSM@ZIF-93 and KSM aqueous solutions could be explained by the fact that, under acidic conditions of pathogenic bacteria infection, the Schiff bond of KSM@ZIF-93 was broken, and KSM and ZIF-93 were released at the same time. The Schiff base bond cleavage facilitates the rapid release of KSM and zinc ions in ZIF-93-KSM, which can achieve a synergistic antibacterial effect. Several studies have also reported that zinc ions released from zinc-based metal–organic frameworks inhibited the growth of pathogens through disrupting the cell membrane of the microorganism and achieved synergistic treatment with antibiotics [29,40]. Therefore, ZIF-93 would be a desirable carrier that can evidently enhance the antimicrobial activity of the loaded active ingredients.

3.5. Field Experiment

Table 2. Control effect of KSM@ZIF-93 on pear fire blight.

Treatment Group	Effective Component Concentration (mg/L)	Disease Index	Control Effect (%)
Blank control	0	83.67 ± 1.27a	-
ZIF-93	50	71.10 ± 2.20b	15.03 ± 1.64h
	100	53.33 ± 2.25d	36.27 ± 1.92f
	200	35.93 ± 1.33f	57.06 ± 0.94d
	400	23.30 ± 1.10h	72.16 ± 0.97b
KSM	50	61.43 ± 3.42c	26.56 ± 4.41g
	100	42.20 ± 2.20e	49.55 ± 2.72e
	200	30.37 ± 1.68g	63.70 ± 1.96c
	400	21.10 ± 1.91h	74.80 ± 1.91b
KSM@ZIF-93	50	58.53 ± 3.36c	30.03 ± 3.92g
	100	32.20 ± 1.10g	61.52 ± 0.86c
	200	20.73 ± 2.77h	75.19 ± 3.63b
	400	15.57 ± 2.25i	81.37 ± 2.95a

Note: Values marked with different letters are significantly different according to Duncan’s multiple range test (p < 0.05).

shows the control effects of different concentrations of KSM, ZIF-93, and KSM@ZIF-93 on E. amylovora. Compared with the control group, after 10 days of treatment, the disease index of all treatment groups was significantly different, indicating that all concentrations of the three agents had a control effect on E. amylovora infecting pear leaves, and the infection area was reduced in a concentration-dependent manner. In addition, compared with the control group, the control effect of KSM@ZIF-93 (50, 100, 200, and 400 mg/L) was better than that of ZIF-93 and KSM at the corresponding concentrations after 10 days of treatment. When the application concentration was 200 mg/L, the control effect of KSM@ZIF-93 on pear fire blight was (75.19 ± 3.63)%, which was significantly higher than that of ZIF-93 [(57.06 ± 0.94)%] and KSM [(63.70 ± 1.96)%]. At the same time, the control effect of KSM@ZIF-93 at 200 mg/L was similar to that of ZIF-93 and KSM at 400 mg/L, indicating that KSM@ZIF-93 had a synergistic effect.

3.6. Safety Evaluation

As shown in Figure 7, the phytotoxicity of KSM@ZIF-93 was evaluated by the effect of different agents on pollen germination rate. Compared with the sterile water control, KSM, ZIF-93, KSM aqueous solution (4%), and KSM@ZIF-93 had no significant difference in pollen germination rate when the concentration was 200 mg/L, indicating that there was no toxic effect on pollen germination at this concentration. However, with the increase in the concentration of the drug, each drug showed different toxic effects. Compared with the blank control, KSM and ZIF-93 had no significant difference in pollen germination in the experimental concentration range, indicating that KSM and ZIF-93 had no toxic effect on pollen germination in the experimental concentration range. Compared with the blank control, the effects of KSM aqueous solution (4%) and KSM@ZIF-93 on pollen germination at the concentration of 400 mg/L were significantly different, and the pollen germination rates were reduced by 5.54% and 5.74%, respectively, indicating that the concentration had toxic effects on pollen germination. Compared with KSM aqueous solution, there was no significant difference in the effects of KSM@ZIF-93 on pollen germination at the concentration of 400 mg/L, indicating that the effects of the two reagents on pollen germination were similar at this concentration. In summary, KSM@ZIF-93 at 200 mg/L had no effect on pollen germination, which was consistent with the KSM aqueous solution (4%) commonly used in the flowering period, indicating that KSM@ZIF-93 could be applied to the prevention and control of pear fire blight in the flowering period within the concentration range of 200 mg/L, which was of great significance for the prevention and control of pear fire disease in the flowering period.

4. Discussion

KSM@ZIF-93 was prepared in this paper by coupling KSM with aldehyde-functionalized ZIF-93 nanoparticles through a Schiff base reaction. The crystal structure analysis of ZIF-93 before and after grafting with KSM shows that loading KSM by Schiff base reaction did not change the metal skeleton integrity of ZIF-93, and ZIF-93 had good stability. Similar results have been reported by Vasconcelos et al. [39] and Liang et al. Through the study of the light-shielding properties of KSM@ZIF-93, it was found that KSM@ZIF-93 has good photostability. This is consistent with the viewpoint reported by Liang et al. [40]. Due to the neutralization of negatively charged carboxylate ions on the KSM molecules with the positive ions on the surface of ZIF-93, the zeta potential of KSM@ZIF-93 is obviously lower than ZIF-93. Therefore, the KSM@ZIF-93 synthesized in this experiment retains the nanoscale size of ZIF-93 and solves the problem of easy photolysis of KSM.

The KSM-release behavior in KSM@ZIF-93 indicated that the release amount of KSM reached up to 42.63% at pH 6, and 64.68% at pH 5 within a period of 96 h, respectively. When pathogenic bacteria infect plants, the pH value of the infected site decreases. Under the acidic conditions of pathogenic infection, the Schiff base bond cleavage facilitates the rapid release of KSM and zinc ions in ZIF-93-KSM, which can achieve a synergistic antibacterial effect. Several studies have also reported that Zn²⁺ released from zinc-based metal–organic frameworks inhibited the growth of pathogens and achieved synergistic treatment with antibiotic bactericides [29,40]. The field efficacy results of this experiment also confirmed this view.

5. Conclusions

The change in pH at the infected site of the pathogen could gradually decompose the Schiff base bonds between ZIF-93 and KSM, and dissolve the metal–organic skeleton, realizing the fixed-point release of KSM and Zn²⁺ by KSM@ZIF-93, and achieving a synergistic antibacterial effect. In this study, KSM@ZIF-93 was synthetized by conjugating ZIF-93 and KSM by Schiff base reaction. The results show that KSM@ZIF-93 had a complete metal skeleton structure, good dispersibility and stability, the particle size was about (63.93 ± 11.19) nm, and the load ratio of KSM in KSM@ZIF-93 was estimated to be 20.0%. KSM@ZIF-93 could reduce the degradation of KSM by ultraviolet radiation and enhance the photostability of KSM. Meanwhile, KSM@ZIF-93 had excellent pH response performance and could simultaneously release KSM and Zn²⁺. The biological activity tests showed that KSM@ZIF-93 had a synergistic antibacterial effect and prolonged pharmacological duration on E. amylovora. More importantly, KSM@ZIF-93 had no effect on pollen germination in the concentration range of 200 mg/L, and good plant safety was of great significance for the prevention and control of pear fire blight at the pear flowering stage. Therefore, the construction of KSM@ZIF-93 with ideal antibacterial activity and plant safety provides a new method to solve the problem of pear fire blight, the prevention and control during flowering, and has great potential in the field of slow-release pesticides.