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. 2025 Oct 28;10(44):53278–53288. doi: 10.1021/acsomega.5c08101

Critical Role of Nanoparticle Size in Regulating Zinc Oxide’s Antimicrobial Performance Against Citrus Canker Pathogens

Wenhua Rao †, Qianying Li †,‡, Shang Gao †,‡, Minyu Li †,‡, Chao Shen †, Tao Lin †, Xiaohong Pan ‡, Binghai Lou §, Guocheng Fan †,*, Jinfeng Hu †,*
PMCID: PMC12612876  PMID: 41244412

Abstract

Nanozinc oxide (ZnO) is a known material for its high therapeutic efficacy against citrus canker. However, the influence of particle size on the antibacterial activity of nano-ZnO against Xanthomonas citri subsp. citri (Xcc), as well as the distinct bacterial response mechanisms to different nanoparticle sizes, remain poorly understood, limiting its broader application. In this study, differences in the antibacterial potentials of nano-ZnO particles with sizes of 30, 90, and 200 nm against Xcc were investigated. The results showed that the zeta potential of the three nano-ZnO variants displayed no significant differences. However, the antibacterial efficacy of nano-ZnO against Xcc was strongly dependent on particle size, with smaller particles exhibiting greater antibacterial activity. This is attributed to their increased capacity to damage bacterial cell membranes and DNA, which is linked to a higher generation of reactive oxygen species and greater release of Zn2+ within the bacterial cells. In addition, small-sized nano-ZnO exhibits superior efficacy in inhibiting biofilm formation, exopolysaccharide secretion, and xanthomonadin production in Xcc. Fourier transform infrared multivariate and two-dimensional correlation analyses revealed that macromolecular components in Xcc exhibited more pronounced responses to smaller-sized nano-ZnO particles, further supporting their superior antibacterial efficacy. In conclusion, this study provides valuable insights into the size-dependent antibacterial mechanisms of nanomaterials and offers a theoretical basis for the effective application of nano-ZnO in the control of citrus canker.

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1. Introduction

Citrus canker, a highly destructive disease in citrus cultivation, is primarily caused by Xanthomonas citri subsp. citri (Xcc) and significantly reduces both yield and quality. , Currently, control of citrus canker primarily relies on agents such as zinc thiazole and the Bordeaux mixture. However, the pathogen has developed resistance to these and several other chemical treatments. Therefore, identifying novel antibacterial agents and evaluating their variations in their efficacy against Xcc are of greater scientific importance.

With the continuous advancement of nanotechnology, nanopesticides, nanofertilizers, nanobiosensors, and nanoantibacterial agents, either as nanomaterials themselves or as delivery carriers, have found widespread application in both industrial and agricultural sectors. − In agriculture, nanometal and nanometal oxide materialsincluding nanosilver, nanocuprous oxide, and nanozinc oxide (nano-ZnO)are gaining attention for plant disease control (e.g., citrus canker). Their popularity stems from several advantages: easy synthesis, tunable size and morphology, ability to supply essential metal ions, and effectiveness in boosting plant resistance to pathogens. − Moreover, the multitarget mode of action of nanometal and nanometal oxide antibacterial agents makes it difficult for pathogens to develop resistance, , boosting their potential for effective plant disease management. , Therefore, further research on the antibacterial mechanisms of nanometal and nanometal oxide materials against plant pathogens, alongside evaluations of how intrinsic properties such as particle size, morphology, and surface charge affect their efficacy, is crucial for advancing the application of nanometal- and nanometal oxide-based agents and enhancing plant disease control strategies.

Particle size stands as a critical factor influencing the antibacterial efficacy of the nanomaterials. Generally, smaller nanomaterial particles exhibit enhanced antibacterial activity, which is a phenomenon driven by several mechanisms. First, reduced particle size facilitates easier penetration into bacterial cells, enabling more direct delivery of antibacterial effects. Second, smaller particles possess a larger specific surface area, which increases their contact area with bacterial cellsstrengthening their disruptive impact on bacterial cell membranes. Additionally, smaller metal/metal oxide particles demonstrate superior metal ion release efficiency compared with their larger counterparts. These released ions play a pivotal role in inducing reactive oxygen species (ROS) generation and damaging essential bacterial components (e.g., DNA and proteins). , However, when nanoparticles are reduced below a specific size threshold, their bactericidal efficacy may diminish. Therefore, evaluating the influence of particle size on antibacterial performance is critical when selecting nanomaterials for plant pathogen controlan effort that will guide the development of more effective plant disease management strategies.

Among various metal and metal oxide nanomaterials, nano-ZnO is one of the most widely applied materials, exhibiting excellent safety for plants, animals, and the environment. , Nano-ZnO can provide essential Zn2+ nutrition to plants, promoting carbohydrate synthesis and chlorophyll production and enhancing stress resistance. Moreover, crops treated with nano-ZnO can contribute to human dietary Zn2+ intake, thus improving immune function and neural regulation. ,− As a semiconductor material, nano-ZnO also exhibits a remarkable ability to generate ROS under light exposure, contributing to its antimicrobial properties. On the other hand, the Zn2+ released upon interaction with pathogens can directly act on them, oxidizing and inactivating critical biomolecules. , Available evidence generally demonstrates that the antibacterial activity of nano-ZnO against human pathogenic bacterial pathogensincluding Escherichia coli and Staphylococcus aureusexhibits a positive correlation with decreasing particle size. , Currently, nano-ZnO has been applied in controlling bacterial plant diseases such as citrus canker and citrus Huanglongbing, , as well as viral diseases like tobacco mosaic virus. However, the size-dependent antibacterial effects of nano-ZnO, along with the differential responses of plant pathogen macromolecules to nanomaterial-induced stress, remain insufficiently understood. Further investigation into these mechanisms is essential to optimize the application and enhance the efficacy of nano-ZnO in controlling plant pathogens.

This study focused on investigating the influence of particle size on the antibacterial efficacy of nano-ZnO against Xcc. It specifically examined the mechanisms behind the size-dependent antibacterial effects of nano-ZnO, including bacterial cell membrane damage, DNA disruption, virulence factor suppression, Zn2+ release, and ROS induction. Furthermore, two-dimensional Fourier transform infrared correlation spectroscopy (2D-FTIR-COS) was employed to analyze the differential responses of macromolecules in Xcc to nano-ZnO of varying particle sizes (Scheme ). The biocompatibility of nano-ZnO was further validated by investigating its impact on the photosynthetic performance of citrus plants. The findings provide novel insights into the size-dependent antibacterial mechanisms of nanomaterials and offer theoretical guidance for the optimized application of nano-ZnO in controlling citrus canker.

1. Differential Responses of Xcc to Nano-ZnO with Varied Particle Sizes.

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2. Materials and Methods

2.1. Materials

Nano-ZnO (30, 90, and 200 nm), crystal violet, acetic acid, and spectroscopic-grade potassium bromide (KBr) were purchased from Macklin. Nanosilver (90 nm) was provided by XFNANO. Yeast extract and tryptone were obtained from Oxoid, while phosphate-buffered saline (PBS) dry powder and agar powder were sourced from Coolaber. All other reagents used in this study were of analytical grade.

2.2. Characterizations of Nano-ZnO

The particle size distribution of nano-ZnO in the dry state was characterized by using transmission electron microscopy (TEM, Tecnai G2 F30, FEI). The crystal structure was analyzed via powder X-ray diffraction (XRD, D8 Advance, Bruker). Zeta potential and hydrodynamic size in PBS were measured using a Zetasizer Nano Particle Analyzer (ZS90, Malvern). More details are shown in Text S1 (Supporting Information).

2.3. Antibacterial Ability of Nano-ZnO

2.3.1. Antibacterial Activity Test

The antibacterial efficacy of nano-ZnO against Xcc (wild-type strain obtained from Citrus sinensis (L.) Osbeck, 30–1) was evaluated according to previously described methods. Briefly, Xcc was cultured in Luria–Bertani (LB) medium at 28 °C until the optical density at 600 nm (OD600) reached 1.0. The bacterial cells were collected by centrifugation, washed three times with PBS, and resuspended in an equal volume of PBS. Three different sizes of nano-ZnO were dispersed in PBS using an ultrasonic cleaner (KQ3200DE, Kunshan) for 20 min to ensure homogeneous dispersion. The bacterial suspension was mixed with nano-ZnO dispersions at a 1:1 volume ratio, resulting in final nano-ZnO concentrations of 125, 250, 500, and 1000 mg/L. The mixtures were incubated in a shaker at 28 °C and 200 rpm for 16 h in the dark. After treatment, 5 μL aliquots were spotted onto solid LB plates and incubated at 28 °C for 48 h in an inverted position protected from light. The antibacterial activity of nano-ZnO was evaluated by observing bacterial growth inhibition on the plates.

2.3.2. Time-Kill Assay

We conducted a time-kill assay to further evaluate the differences in antibacterial activity among nano-ZnO of varying sizes. The cultured Xcc was first diluted with LB medium to a bacterial concentration of 5 × 107 colony-forming units per milliliter (CFU/mL). This suspension was then mixed with ZnO-30, ZnO-90, ZnO-200, nanosilver (positive control), and ultrapure water separately, adjusting the material concentrations to the minimum inhibitory concentration (MIC) of ZnO-30 (125 mg/L). The mixtures were incubated at 28 °C with shaking at 200 rpm. At 0, 2, 4, 6, 8, and 24 h postincubation, 100 μL of the bacterial suspension was withdrawn, serially diluted 10-fold with PBS, and plated onto LB agar plates. After 48 h of incubation, the CFUs were counted.

2.4. Antibacterial Mechanisms of Nano-ZnO

2.4.1. DNA Damage

Following the procedure outlined in Section , treated Xcc cells were collected by centrifugation to obtain bacterial pellets. Genomic DNA was then extracted using a bacterial genomic DNA extraction kit (Tiangen, China). DNA damage was assessed through gel electrophoresis analysis.

2.4.2. Ultrastructure

Bacterial cells treated with nano-ZnO of three different particle sizes were collected and fixed overnight at 4 °C in 2.5% glutaraldehyde. Following a standard dehydration protocol and gold sputtering, morphological changes in the Xcc cell membranes were examined using a scanning electron microscope (SEM, TM-1000, Hitachi).

2.4.3. LIVE/DEAD Assays

Considering that membrane permeability changes during bacterial cell death, SYTO 9 and propidium iodide (PI) dyes (Invitrogen/Thermo Fisher Scientific) were employed to assess Xcc membrane permeability and bacterial viability. After treatment with nano-ZnO, bacterial cells were washed twice with PBS and then incubated with an SYTO 9/PI dye mixture (1:1 ratio, concentrations of 0.005 and 0.03 mM, respectively) at 28 °C in the dark for 15 min. Following three additional washes with PBS, the fluorescence signals of SYTO 9 (excitation/emission: 488/520 nm) and PI (excitation/emission: 488/630 nm) were observed using confocal laser scanning microscopy (CLSM 900, Zeiss). Based on the statistical analysis using ImageJ, the green fluorescence intensity (SYTO 9) and red fluorescence intensity (PI) of the samples were measured, and the SYTO 9/PI ratios were calculated.

2.4.4. ROS Monitoring

Following the method described in Section , treated Xcc cells were collected by centrifugation and incubated with a 10 μM DCFH-DA fluorescent probe at 28 °C for 30 min in the dark. After washing three times with PBS, intracellular ROS levels were quantified using CLSM with excitation and emission wavelengths of 488 and 525 nm, respectively.

2.4.5. Determination of Biofilm Formation, Exopolysaccharide Secretion, and Xanthomonadin Production in Xcc

The impact of nano-ZnO on the formation of Xcc biofilms was investigated via a crystal violet staining assay. The phenol-sulfuric acid method was utilized to quantify bacterial extracellular polysaccharide (EPS) production by Xcc following nano-ZnO exposure. Additionally, xanthomonadin was isolated from nano-ZnO-treated Xcc cells using absolute ethanol, and its content was determined by measuring OD441 with a Varioskan LUX microplate reader (Thermo Fisher Scientific). Detailed experimental protocols are provided in Text S2 (Supporting Information).

2.5. Zinc Ion Release

Nano-ZnO PBS dispersions (10 mL, 2000 mg/L) were placed in 3.5 kDa dialysis bags and then immersed in 100 mL of ultrapure water with rapid stirring. After equilibration for 16 h, 5 mL aliquots were sampled from the external dialysate. The collected samples were subjected to nitric acid digestion, and the concentration of Zn2+ was subsequently analyzed via inductively coupled plasma optical emission spectrometry (ICP-OES; Thermo Fisher Scientific).

2.6. 2D-FTIR-COS

Following the procedure described in Section , Xcc suspensions were mixed with nano-ZnO at a 1:1 (v/v) ratio to yield final concentrations of 62.5, 125, 250, 500, 1000, and 2000 mg/L. The mixtures were incubated at 28 °C while being shaken in the dark for 16 h. After incubation, the bacterial cells were collected by centrifugation, and the resulting pellets were lyophilized for 24 h. For FTIR analysis, 1 mg of the lyophilized sample was uniformly mixed with 200 mg of KBr and analyzed using an FTIR spectrometer (T10, Persee).

All FTIR spectra were preprocessed using the Savitzky–Golay algorithm to perform baseline correction and smoothing. Spectral data from the 1800–800 cm–1 fingerprint region were extracted for subsequent two-dimensional correlation spectroscopy (2D-COS) analysis using 2D-shige software (Shigeaki Morita, Kwansei-Gakuin University). The sequence of the functional group changes in Xcc macromolecules in response to different nano-ZnO particle sizes was determined by applying Noda’s rules to interpret synchronous and asynchronous cross-peaks. ,

2.7. Biocompatibility of Nano-ZnO in Citrus Plants

Fresh leaves from lemon seedlings were excised and placed on 0.5% agar plates to maintain turgor and prevent desiccation. Two treatment solutions were then applied: PBS (negative control) and nano-ZnO suspension (1000 mg/mL), with each solution directly pipetted onto the abaxial leaf surface at a volume of 10 μL. The plates were subsequently incubated in a controlled environment chamber (14 h light/10 h dark photoperiod with 65% relative humidity at 25 °C). The citrus leaves were observed using a large multispectral fluorescence imaging system (PlantExplorer; PhenoVation B.V., Wageningen, Netherlands) 20 d later.

2.8. Statistical Analysis

All experiments were performed in triplicate, and data are presented as the mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA) with SPSS version 22.0.

3. Results and Discussion

3.1. Characterizations of Nano-ZnO

The physicochemical properties of nano-ZnO of varying sizes were characterized, including dry-state particle size, zeta potential, and hydrodynamic size in PBS. TEM analysis revealed that ZnO-30 particles primarily exhibited a spherical morphology, whereas ZnO-90 and ZnO-200 displayed a mixed morphology characterized by both spherical and rod-shaped features (Figure a–c). XRD analysis confirmed that all three nano-ZnO variants were indexed to the wurtzite crystal structure of ZnO (JCPDS No. 36-1451), validating their structural identity (Figure d–f). Upon dispersion in PBS, all nano-ZnO samples displayed negative surface charges (−20.3, −20.3, and −23.8 mV, respectively) and tended to aggregate (Figure g,h). The particle sizes of nano-ZnO in PBS displayed negligible differences, which is likely attributed to the aggregation behavior of these materials in PBS. Although the samples varied in dimensional variations (30, 90, and 200 nm, the average size obtained from the TEM results), they shared comparable surface properties and crystalline structures. Overall, the nano-ZnO we selected exhibits negligible variability in surface properties yet displays significant size disparities, which is particularly critical for subsequent size-mediated antimicrobial testing.

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Characterization of nano-ZnO. (a–c) TEM images of ZnO-30, ZnO-90, and ZnO-200, respectively; (d–f) XRD patterns of ZnO-30, ZnO-90, and ZnO-200, showing characteristic diffraction peaks consistent with the ZnO phase (wurzite, JCPDS No. 36-1451); (g) Zeta potential measurements of nano-ZnO dispersions in PBS; and (h) hydrodynamic size distribution of nano-ZnO in PBS.

3.2. Antibacterial Ability of Nano-ZnO

The direct antibacterial efficacy of nano-ZnO against Xcc was evaluated. Results demonstrated that nano-ZnO (30, 90, and 200 nm) exhibited concentration-dependent antibacterial activity, with higher concentrations leading to greater inhibition (Figure ). At an Xcc abundance of 108 CFU/mL, the minimum bactericidal concentrations (MBCs) of ZnO-30, ZnO-90, and ZnO-200 against Xcc were 1000 mg/L, >1000 mg/L, and >1000 mg/L, respectively. When the Xcc abundance decreased to 107 CFU/mL, the MBCs of these three nano-ZnO materials were reduced to 500, 1000, and 1000 mg/L, respectively. Further decreasing the Xcc abundance to 106 CFU/mL resulted in even lower MBCs: 125, 125, and 500 mg/L for ZnO-30, ZnO-90, and ZnO-200, respectively. In summary, the antibacterial potency followed the order ZnO-30 > ZnO-90 > ZnO-200, suggesting that smaller nanoparticles within this size range exhibit enhanced antimicrobial activitya trend consistent with previous reports on nanomaterial–bacteria interactions. ,,

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Antibacterial activity of nano-ZnO against Xcc (the numerical values 125–1000 shown in the figure denote the final concentrations of nano-ZnO in mixtures with Xcc.).

3.3. Time-Kill Assay

To further evaluate how nano-ZnO particle size influences its antibacterial activity against Xcc, we conducted time-kill assays. As shown in Figure , the negative control group displayed a rapid upward trend. In contrast, the nano-ZnO treatment group first increased at 2 h and then decreased moderately between 2 and 8 h before entering another upward phase. Specifically, the final LOG10 CFU/mL values were 7.74 (ZnO-30), 7.92 (ZnO-90), 7.95 (ZnO-200), and 8.08 (negative control). These results suggest that nano-ZnO effectively inhibits Xcc, with smaller particles showing stronger inhibition. For the nanosilver treatment (positive control), Xcc levels decreased over the first 4 h and then increased at a steady rate. Its final LOG10 CFU/mL value was 7.73, which is comparable to that observed in the ZnO-30 group. These findings align with the results in Figure and previous studies. ,,

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Time-kill curve of Xcc induced by nano-ZnO and nanosilver (125 mg/L).

3.4. DNA Damage, Ultrastructural Alterations, and Virulence Factor Suppression in Xcc

DNA damage and cell membrane rupture are key mechanisms underlying bacterial death induced by nanomaterials. Therefore, the damage to Xcc genomic DNA and cell membranes under nano-ZnO exposure was analyzed. As shown in Figure a, compared to the control group, the DNA band intensity decreased in all nano-ZnO-treated groups, with band intensity weakening as the nano-ZnO particle size decreased, indicating higher levels of DNA damage. Meanwhile, the ultrastructural changes on the Xcc surface were further examined by using SEM (Figure b). The control cells displayed a plump and intact morphology, whereas the membrane rupture and gaps (indicated by red arrows) were particularly evident in groups treated with smaller-sized nano-ZnO. These structural disruptions and decrease in DNA content of Xcc may be attributed to the direct contact of nano-ZnO with bacterial cells, ROS generation, and the release of Zn2+ (This hypothesis was further presented and discussed in the subsequent results). , Additionally, we investigated the antibacterial mechanism of nano-ZnO against Xcc by assessing its virulence factors (Figure c–e). At equivalent concentrations, small nano-ZnO exhibited the most potent inhibitory effects on Xcc biofilm formation, EPS secretion, and xanthomonadin production. Given that these virulence factors are critical pathogenic determinants of Xcc, ,, small-sized nano-ZnO may thus demonstrate greater potential for controlling citrus canker.

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Effects of nano-ZnO on DNA integrity (a), cellular ultrastructure (b), biofilm formation (c), EPS secretion (d), and xanthomonadin production (e) of Xcc.

3.5. LIVE/DEAD Assays

The response of Xcc to nano-ZnO particles of different sizes was further confirmed by using the LIVE/DEAD kit (Figure ). The nucleic acid dye PI penetrates only the membranes of dead bacteria, emitting red fluorescence under CLSM, whereas SYTO 9 can enter both viable and dead cells, emitting green fluorescence. After treatment with the three nano-ZnO materials, Xcc all exhibited red PI fluorescent signals, and the STYO-9/PI ratios of all groups significantly decreased as the nano-ZnO concentration decreased (Figure b–d, P < 0.05). Additionally, at the same nano-ZnO treatment concentration, the STYO-9/PI ratios of Xcc in the ZnO-30, ZnO-90, and ZnO-200 treatment groups showed a significant downward trend (Figure e, P < 0.01), consistent with the results presented in Figure . These findings indicate that smaller nano-ZnO induces significantly greater damage to Xcc cells compared to larger particles, consistent with previous observations.

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STYO-9/PI measurement. (a) CLSM images of Xcc after exposure to nano-ZnO. (b–e) Fluorescence measurement of CLSM images. Different letters indicate significant difference in fluorescence intensity among different treatments (uppercase letters: P < 0.01, lowercase letters: P < 0.05).

3.6. ROS Monitoring

ROS, including oxygen-containing free radicals and peroxides, are closely linked to cellular oxygen metabolism and serve as key effector molecules in nanomaterial-induced bacterial death. ,, In this study, DCFH-DA fluorescent staining revealed distinct green fluorescence signals in the high-concentration ZnO-30 treatment groups, with signal intensity gradually decreasing as the concentration was lowered (Figure ). Following treatment with the three nano-ZnO materials, Xcc cells exhibited DCFH-DA fluorescent signals. Notably, the fluorescent signal intensity of all groups decreased significantly with decreasing nano-ZnO concentration (Figure b–d, P < 0.05). Additionally, at equivalent nano-ZnO treatment concentrations, the fluorescent signal intensity of Xcc in the ZnO-30, ZnO-90, and ZnO-200 treatment groups displayed a marked downward trend (Figure e, P < 0.01), consistent with the findings in Figures and . These findings are consistent with previous results and further confirm that smaller nano-ZnO particles have a stronger ROS-inducing effect against Xcc.

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ROS measurement. (a) Fluorescence of ROS in Xcc induced by nano-ZnO exposure. (b–e) Fluorescence measurement of CLSM images. Different letters indicate significant difference in fluorescence intensity among different treatments (uppercase letters: P < 0.01, lowercase letters: P < 0.05).

3.7. Zinc Ion Release

Zn2+ release from nano-ZnO was further analyzed using ICP-OES. The results showed that, in the absence of Xcc cells, smaller nano-ZnO particles released significantly higher amounts of Zn2+ in PBS (Figure ), likely due to their larger specific surface area accelerating Zn2+ dissolution from the particle surface. , Based on the above findings, we conclude that the superior anti-Xcc efficacy of smaller-sized nano-ZnO is attributable to its enhanced capacity for Zn2+ release and potent ROS-inducing ability.

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Zn2+ release efficiency of different-sized nano-ZnO particles in PBS (different letters indicate significant differences between treatments, P < 0.05).

3.8. 2D-FTIR-COS

2D-FTIR-COS is a powerful technique for investigating the dynamic responses of organic substances to external stimuli, as it improves spectral resolution by expanding overlapping peaks into a second dimension, therefore simplifying the interpretation of one-dimensional spectra. , Based on synchronous and asynchronous 2D-FTIR-COS maps (Figure ) derived from the raw FTIR spectra of bacteria exposed to varying nano-ZnO concentrations (Figure S1), the preferential response sequence of bacterial biomacromolecules to nano-ZnO is summarized in Table , with detailed spectral assignments provided in Tables S1–S3.

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Synchronous and asynchronous 2D-FTIR-COS maps generated from the FTIR spectra of Xcc with increasing nano-ZnO concentrations.

1. Response Sequences of Xcc Biomacromolecules to Nano-ZnO.

nanomaterials Xcc
ZnO-200 amide I of α-helical structures of proteins > Amide II band of proteins > C–H deformation of > CH2 in lipid proteins > PO asymmetric stretching of phosphodiesters in phospholipids > C–O–C dominated by ring vibrations in various polysaccharides > PO symmetric stretching in DNA, RNA, and phospholipids
ZnO-90 C–O–C dominated by ring vibrations in various polysaccharides > Amide I of α-helical structures of proteins > Amide II band of proteins > PO asymmetric stretching of phosphodiesters in phospholipids
ZnO-30 C–O–C dominated by ring vibrations in various polysaccharides > PO symmetric stretching in DNA, RNA, and phospholipids > Amide I of α-helical structures of proteins > Amide II band of proteins > PO asymmetric stretching of phosphodiesters in phospholipids
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The spectra revealed characteristic bands corresponding to amide I, amide II, C–H deformation, C–O–H stretching, phospholipid P–O in nucleic acids, C–O, PO symmetric stretching, and polysaccharide C–O–C vibrations. The cross-peak signs revealed the sequential binding affinity between these bands and nano-ZnO. Overall, carbohydrate and protein components in Xcc cells responded preferentially to nano-ZnO exposure, indicating strong interactions with both cell wall components and membrane proteins. This suggests that cell wall disruption is the primary mechanism of bacterial inactivation. In the ZnO-30 treatment group, shifts in nucleic acid PO bands following polysaccharide alterations indicate the immediate targeting of nucleic acids after membrane penetration (Table ). This leads to increased membrane and DNA damage, contributing to superior antibacterial efficacy consistent with previous findings. Therefore, the prioritized response of intracellular nucleic acids to ZnO-30 likely explains its stronger bactericidal effects.

3.9. Biocompatibility of Nano-ZnO in Citrus Plants

We further evaluated the safety of nano-ZnO for citrus plants using a spot application method. After 20 days of treatment, no mesophyll tissue necrosis was observed at the application sites of nano-ZnO treatments across three particle sizes, and there were no differences in photosynthetic indices between these nano-ZnO treatments and the control group (Figure ). Collectively, these results indicate that nano-ZnO exhibits strong biocompatibility with citrus plants, laying a foundation for its subsequent applications.

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Safety evaluation of nano-ZnO to citrus (Fv/fm for maximum photosynthetic efficiency; Chlorophyll for all chlorophylls; Ari for anthocyanin reflectance index; and Ndvi for normalized vegetation index).

4. Conclusions

In summary, the findings demonstrate that the antibacterial efficacy of nano-ZnO against Xcc is size-dependent, with smaller particles exhibiting superior antimicrobial activity. This enhanced efficacy is mainly due to their increased Zn2+ release and ROS generation, thus inducing: (1) significant biomacromolecular responses in bacterial membranes; (2) marked structural membrane disruption; (3) notable DNA damage in the pathogen; and (4) suppression of virulence factors. Thus, smaller-sized nano-ZnO particles (ZnO-30) may demonstrate significant advantages in controlling citrus canker. Given that within a specific particle size range the antibacterial activity of nano-ZnO against Xcc increases as particle size decreases, future research should focus on two key priorities. First, refined synthesis protocols are advanced to produce smaller antimicrobial nanoparticles with enhanced properties. Second, optimizing existing agrochemical agentsincluding ZnO, copper hydroxide, and silver nanoparticlesto ensure that the cost of reducing their particle size remains lower than the economic benefits gained from improved control of plant diseases. Meanwhile, it is critical to carry out thorough assessments of the biosafety of these modified materials, thus ultimately enhancing the sustainability and efficacy of plant disease management strategies. In summary, this study offers novel insights into the size-dependent antimicrobial effects of nanomaterials and establishes a theoretical framework for optimizing the use of nano-ZnO in citrus canker management.

Supplementary Material

ao5c08101_si_001.pdf (209KB, pdf)

Acknowledgments

This work was supported by the Exploration Project of Fujian Academy of Agricultural Sciences, China (ZYTS202413), the Basic Research Special Foundation of Public Research Institutes of Fujian Province, China (2023R1022002), the Natural Science Foundation of Fujian Province, China (2023J05062), and the “5511” Collaborative Innovation Project of High-quality Agricultural Development and Surpassment in Fujian Province (XTCXGC2021011, XTCXGC2021017). The authors would like to thank MJEditor (www.mjeditor.com) for its linguistic assistance during the preparation of this manuscript.

Data used is available throughout the manuscript text.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c08101.

  • Characterization of Nano-ZnO and the determination of Xcc’s virulence factors; FTIR spectra of Xcc treated with various concentrations of nano-ZnO; and 2D-FTIR-COS result on the assignment and sign of each cross-peak in Xcc exposed to nano-ZnO synchronous and asynchronous (in the brackets) map of bacteria with increasing ZnO concentrations (PDF)

Wenhua Rao obtained the project, conceived the idea, performed experiments, wrote the manuscript, and revised the manuscript. Qianying Li and Shang Gao performed experiments, wrote the manuscript, and revised the manuscript. Minyu Li, Chao Shen, and Tao Lin performed experiments and analyzed the data. Xiaohong Pan and Binghai Lou helped to revise the article and provided suggestions. Guocheng Fan and Jinfeng Hu obtained the project, supervised the project, and contributed significantly to the analysis.

The authors declare no competing financial interest.

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