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Article

Biogenic Zinc Oxide Nanoparticles Protect Tomato Plants Against Pseudomonas syringae pv. tomato

by
Benedetta Orfei
,
Anna Scian
,
Daniele Del Buono
,
Michela Paglialunga
,
Ciro Tolisano
,
Dario Priolo
,
Chiaraluce Moretti
* and
Roberto Buonaurio
Department of Agricultural, Food and Environmental Sciences, University of Perugia, Borgo XX Giugno 74, 06121 Perugia, Italy
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 431; https://doi.org/10.3390/horticulturae11040431
Submission received: 4 March 2025 / Revised: 8 April 2025 / Accepted: 14 April 2025 / Published: 17 April 2025
(This article belongs to the Special Issue Advances in Sustainable Control of Bacterial Pathogens in Horticultural Crops)
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Abstract

:
The control of bacterial plant diseases is very challenging and often relies on the application of copper compounds, although the frequent emergence and spread of resistant bacterial strains compromise their efficacy. Additionally, copper-based compounds raise environmental and human health concerns, leading to their inclusion in the European Commission’s list of candidates for substitution. As a promising and sustainable alternative, we investigated the efficacy of biogenic zinc oxide nanoparticles (ZnO-NPs) in protecting tomato plants against Pseudomonas syringae pv. tomato (Pst), the causal agent of bacterial speck disease. ZnO-NPs exhibited significant in vitro antibacterial activity (EC95 = 17.0 ± 1.1 ppm) against the pathogen. Furthermore, when applied to the foliage of tomato plants at 100 ppm before or following Pst inoculation, they induced significant reductions in symptom severity and bacterial growth in planta, which were comparable to those shown by plants treated with acibenzolar-S-methyl, a plant defense inducer. Gene expression assessed by qPCR revealed the involvement of the systemic acquired resistance (SAR) pathway in tomato plants treated with ZnO-NPs before inoculation, suggesting that the observed protection could be due to a priming effect. Finally, infected plants showed oxidative stress, with higher H2O2 and malondialdehyde (MDA) contents. ZnO-NPs reverted this effect, containing the content of the above molecules, and stimulated the production of metabolites involved in dealing with oxidative perturbations (carotenoids and phenols), while unaffecting flavonoids and anthocyanins.

1. Introduction

Tomato (Solanum lycopersicum L.) is one of the most relevant horticultural crops worldwide and can be cultivated in a broad range of climates, both in the open field and in greenhouses. In 2021, tomato confirmed its enormous agricultural and economic relevance with a production of about 189 million tons grown on 5.16 Mha, ranking in second place among the most cultivated vegetables, right after potato [1]. Such an important production is owed to tomato nutritional value, since this crop is a natural source of retinol, ascorbic acid, phylloquinone (A, C and K1 vitamins, respectively), potassium and folate, among others. Moreover, this crop is one of the main dietary sources of lycopene, an antioxidant with several health benefits, including the ability to reduce the risk of heart disease and cancer [2,3]. However, due to the domestication and selection process, tomatoes have become susceptible to many diseases that are responsible for significant yield losses [4]. Among them, bacterial speck caused by Pseudomonas syringae pv. tomato (Pst) is renowned as one of the most impactful diseases [5,6,7]. The effective management of bacterial diseases strongly relies on chemical control, which, however, can often have a significant environmental impact. Furthermore, the emergence and acquisition of multi-drug resistance in bacteria, with the increasing restriction measures on copper-based products due to their impact on the environment, make the current measures of control inadequate [8,9,10]. In this context, nanotechnology focuses on developing nanostructured materials that could be employed in agriculture as an effective alternative to conventional products to control bacterial diseases [11,12,13]. Recent research shows the effectiveness of metal and metal oxide nanoscaled materials, which can control pests at already very low doses, such as nanopesticides [14,15,16].
Zinc (Zn) is an essential metal trace element that, in its cationic form, plays a multifaceted biochemical role, participating in a plethora of different processes. To cite a few examples, it is a cofactor for many enzymes, proteins and transcriptional factors. It plays a significant role in biological processes such as DNA and protein synthesis, chloroplast development and functionality, cell division, and signaling pathway regulation [17,18,19]. Despite being a heavy metal like copper, zinc has a lower impact on the environment than other cationic metals, and its scarcity or low bioavailability in the agricultural soil can even cause zinc deficiency, an issue considered more relevant than its toxicity [20,21,22].
Zinc oxide (ZnO) was enlisted as both a plant disease and a weed chemical control agent by the US Environment Protection Agency (US-EPA) in 1990 (US EPA/OPPTS, 1992) and recognized as a safe (GRAS) compound by the US Food and Drug Administration (FDA) [23,24]. For its biocompatibility and antimicrobial properties, in nanostructured form, zinc oxide nanoparticles (ZnO-NPs) can be considered a promising tool for disease management in agriculture [25]. Indeed, ZnO-NPs have been successfully tested as antimicrobial agents, showing noteworthy activity against several bacteria, fungi and viruses [26]. In particular, it has been demonstrated that ZnO-NPs can protect Nicotiana benthamiana and tomato plants against Tobacco mosaic virus (TMV) [27,28]. ZnO-NPs have also been shown to reduce the growth of and provoke hyphal distortion and cell wall damage in important phytopathogenic fungi such as Alternaria spp. and Botrytis cinerea [29,30,31], even in boscalid-resistant strains [32]. Other studies revealed the capacity of this nanomaterial to inhibit mycelial growth in conidia and mycotoxin production in Fusarium spp. and Colletotrichum spp. [33,34]. Concerning phytopathogenic bacteria, ZnO-NPs have been shown to protect plants, stimulating plant biomass production and inhibiting the growth of disease-causing agents such as Xanthomonas oryzae pv. oryzae in rice, Ralstonia solanacearum in tomato, Dickeya dadantii in potato and Pectobacterium betavasculorum in beetroot [30,35,36,37,38]. In tomato, previous studies assessed ZnO-NP efficacy in controlling the major phytopathogenic bacteria Ralstonia solanacearum, Pseudomonas syringae pv. tomato and Xanthomonas hortorum pv. gardneri [39,40,41,42,43]. Finally, the application of ZnO NPs to plants can also lead to a significant increase in the content of both enzymatic and non-enzymatic antioxidants, thus stimulating the activation of the plant defense antioxidant system, improving plant capacity to deal with reactive oxygen species (ROS) [44]. ZnO-NPs can modify the content of flavonoids and other phenolic compounds, which are often involved in plant protection against biotic and abiotic stresses. For instance, quantitative reverse-transcription (qRT) PCR revealed that ZnO-NPs increased the levels of total flavonoids and flavonols, inducing the expression of flavonol structural genes (GbF3H, GbF3′H and GbFLS) [45]. Although their mechanism of action is not yet fully understood, the effectiveness of ZnO-NPs against various plant pathogens makes them a promising alternative to conventional agricultural products, leading to the development of nanoformulations such as Zinkicide [46,47].
Nanomaterials and ZnO-NPs can be synthesized by applying chemical, physical, or biological processes [48]. However, conventional chemical and physical synthesis processes are generally energy-consuming and expensive and involve solvents and technologies that can have a relevant environmental impact. In contrast, the emerging biogenic synthesis strategies based on the use of biological extracts are cheaper and do not cause environmental hazards, allowing for the production of nanomaterials and fine-tuning their size and shape [49]. In line with the above, the biogenic synthesis of nanostructured materials focuses on using extracts from bacteria, fungi, yeasts, plants and algae, as they contain metabolites that can operate as reducing agents, ligands and capping agents. For this reason, the procedures to obtain bio-fabricated nanoparticles are considered safer and greener than conventional methods and have gained increasing interest as a sustainable alternative to the latter [50,51].
With this in mind, this research aimed to assess the ability of biogenic ZnO-NPs to protect tomato plants. More specifically, we assessed some physiological and biochemical traits in plants treated with biogenic ZnO-NPs and the activity of ZnO-NPs against the pathogen Pseudomonas syringae pv. tomato, the causal agent of bacterial speck in tomatoes. In particular, their action against this bacterium was assessed in vitro and in planta. Ultimately, our experiments investigated the most effective dosage for pathogen control, focusing on the cellular redox state and the content of some main antioxidant metabolite clusters.

2. Materials and Methods

2.1. Duckweed Growth Condition and Biogenic Zinc Oxide Nanoparticle (ZnO-NP) Synthesis

The biogenic ZnO-NPs were synthesized using a previously published procedure [48]. In particular, the synthesis was carried out using a hydroalcoholic extract of the aquatic plant duckweed (Lemna minor L.). In greater detail, certain classes of metabolites in the extract can operate as capping and modulating agents during biogenic synthesis, allowing for nanoparticle formation. The suitability of the plant extract was previously assessed through profiling with ultra-high-pressure liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-ESI/QTOF-MS Agilent Technologies 6550iFunnel, Santa Clara, CA, USA). Such determination revealed the presence of phenols and their derivatives, phenolic acids and flavanols, which may play a key capping and modulating role during biogenic synthesis [48]. Finally, the biogenic ZnO-NPs showed a spherical shape and dimensions in the 10–20 nm range [52].

2.2. Bacterial Growth and Inoculum Preparation

In the present study, Pseudomonas syringae pv. tomato (Pst) strain DAPP-PG 215 [53,54] was used. This strain was obtained from the phytopathogenic bacterial collection of the Plant Protection Section of the Department of Agricultural, Food and Environmental Sciences, University of Perugia (Italy), where it was stored in vials containing 15% glycerol at −80 °C. After revival on nutrient agar medium (NA; Thermo Fisher Scientific, Dreieich, Germany) at 27 ± 2 °C for 48 h, strain purity was verified before usage by plating a single colony in plates containing NA enriched with 50 g L−1 of sucrose (NAS). Afterward, to prepare the inoculum, Pst DAPP-PG 215 was grown on NA plates for 24 h at 27 ± 1 °C, and the bacterial culture was suspended in sterile deionized water until reaching the concentration of 108 cells mL−1, corresponding to an optical density (OD) of 0.06 at 660 nm.

2.3. Seed Germination and Seedling Growth

Seeds of tomato (Solanum lycopersicum L.; cv. Rio Grande) were sown in modular-tray substrate (Klasmann-Deilmann, GmbH; Geeste, Germany) and incubated in a growth chamber programmed at 22 ± 2 °C under a 12 h light/12 h dark cycle with 70 ± 90% RH and 240 µE m−2 s−1 light illumination. Every 2 days, both seedbed and seedlings were watered with 50 mL of water. At the 2nd to 3rd true-leaf stage, the plants were transplanted into 9 × 9 × 12.5 cm pots (one plant per pot) containing the modular-tray substrate (140 g per pot).

2.4. ZnO-NPs’ Activity In Vitro Antimicrobial Assay

The effect of increasing concentrations of ZnO-NPs on the in vitro growth of Pst DAPP-PG 215 was evaluated in 96-well ELISA microplates, measuring changes in OD630 every hour, from 0 to 24 h at 27 °C, using a Thermo Scientific MultiSkan EX microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) set to gently shake the microplate every min for 5 s. Each well was filled with 160 µL of King’s B broth (KB) [55], 20 µL of a Pst suspension (108 cells mL−1) prepared as reported above and increasing concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ppm) of ZnO-NPs resuspended in 100 µL of sterile ultrapure H2O; the final volume per well was 200 µL. Eight replicates per each ZnO-NP concentration were included in each of three experiments. The data obtained were used to elaborate growth curves and calculate the effective concentrations (ECs).

2.5. Effect of ZnO-NPs in Protecting Tomato Plants Against Pst

Once reached the 5th true-leaf stage, to verify the efficacy of both a preventive and a curative treatment with ZnO-NPs against Pst DAPP-PG 215, the tomato plants were foliar spray-treated at two different time points. For the preventive treatment, the plants were treated two days before inoculation with Pst, while for the curative treatment, the plants were sprayed five days after inoculation. Two different concentrations of ZnO-NPs were chosen for a first experiment, in which the treatments were arranged as follows: control, ZnO-NP 50 ppm preventive treatment, ZnO-NP 100 ppm preventive treatment, ZnO-NP 50 ppm curative treatment, and ZnO-NP 100 ppm curative treatment. We included 4 replicates for each treatment inoculation, which was performed by adaxially and abaxially leaf-spraying the plants with a Pst suspension (108 cells mL−1) using an airbrush (Airstar 200/hp 1.5/24 L). The control plants were treated with H2O and, after 2 days, challenged with Pst (108 cells mL−1). After inoculation, the plants were covered with plastic bags for the first 2 days. The plants were then transferred to a growth chamber conditioned at 22 ± 2 °C under a 12 h light/12 h dark cycle with 70 ± 90% RH and 240 µE m−2 s−1 light illumination. Disease symptoms were recorded 14 days post-inoculation (dpi), in the detached leaves (from the third to the eighth true leaf) of each plant, by evaluating the percentage (%) of necrotic area using the Horsfall and Barratt scale [56]. Based on the results of this first evaluation, a second experiment was carried out including the following treatments: control, ZnO-NP 100 ppm preventive treatment, ZnO-NP 100 ppm curative treatment, and acibenzolar-S-methyl (ASM) (benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester) [2.1 × 104 mol L−1 (0.0441 g L−1)] treatment. This ASM dose is reported in the literature as non-phytotoxic to tomato seedlings and plants and protective against root-knot nematodes [57] and Pst [58]. Moreover, it is similar to the dose that activates defense responses in tomato plants [59]. The replicates and treatment time points were the same as those in the previous experiment, but ASM was applied two days before inoculation only. Inoculation with Pst DAPP-PG 215 (108 cfu mL−1) was performed two days after the preventive treatments for all the tomato plants, which were then transferred to the growth chamber. Inoculation, plant incubation and symptom assessment were conducted in the same manner as in the first experiment.

2.6. ZnO-NP Impact on Bacterial Growth in Planta

The effect of the ZnO-NP and ASM (positive control) treatments on Pst growth in planta were determined 3, 6 and 9 dpi. From 3 plants per treatment, three leaf disks (15 mm diameter) from the 3rd, 4th and 5th true leaf (where the disease symptoms were more evident) were ground in a mortar with sterile deionized water. The leaf homogenates were diluted 10-fold, and 100 µL of each dilution was streaked onto NA plates. After 48 h of incubation at 27 ± 1 °C, the Pst colonies were counted.

2.7. Plant Defense Gene Expression in ZnO-NP-Treated Plants

Tomato leaves were collected and snap-frozen in liquid nitrogen at 24 and 48 h post-treatment (hpt) from plants treated with water (negative control), ZnO-NPs, or ASM, and at 0, 24, and 96 h post-inoculation (hpi) from plants treated and then inoculated after two days with Pst. Total RNA was isolated from frozen, homogenized leaf tissue using the PureLink™ RNA Mini Kit (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). The DNase treatments were carried out using TURBO™ DNase (2 U μL−1) (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). RNA quantification and quality were assessed using a spectrophotometer (Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis; Thermo Fisher Scientific, Dreieich, Germany) and agarose gel electrophoresis, respectively. First-strand cDNA synthesis was performed starting from 1000 ng of total RNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer’s instructions, in a final volume of 20 μL. The cDNA products were then diluted 1:20 in RNase-free water for use in real-time qPCR. Gene-specific fragments were amplified by quantitative reverse transcriptase–polymerase chain reaction (RT-qPCR) using 5 μL of diluted cDNA, 10 μL of SsoFast EvaGreen Supermix (Bio-Rad Laboratories Inc., Hercules, CA, USA) and 0.4 μM solutions of each specific primer in a 20 μL reaction volume. The amplification steps were incubation at 98 °C for 2 min, followed by 40 cycles at 98 °C for 3 s, annealing for 30 s, incubation at 95 °C for 10 s, cooling at 65 °C for 1 min, and finally incubation at 95 °C with a 0.2 °C increase every 10 s, measuring fluorescence. Primer sequences for the pathogenesis-related protein 1b1 (PR1B1) and β-1,3-glucanase (GluB) genes, annealing temperatures and targets of the analyzed genes are provided in Supplementary Table S1. The tomato actin and elongation factor 1-α (EF1-α) genes were used as reference housekeeping genes for normalization. To monitor potential primer dimers and non-specific amplification products, a dissociation curve was included at the end of the qPCR program. Relative gene expression was quantified using the threshold cycles (Cq) according to the Pfaffl method [60], with modifications by Vandesompele et al. [61] to account for two reference genes.

2.8. H2O2, MDA and Antioxidant Determination

The hydrogen peroxide (H2O2) content in fresh leaves was determined by extracting 0.5 g of leaves with 5 mL of 0.1% (w/v) trichloroacetic acid (TCA). The suspension was then centrifuged at 6000 rpm for 10 min, and 0.5 mL of the supernatant was used for H₂O₂ quantification following the method described by Velikova et al. [62]. The malondialdehyde (MDA) content was assessed using the extract obtained as described above. In particular, 1 mL of the extract was incubated at 95 °C for 20 min with 2.0 mL of 20% (w/v) trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. After cooling, the MDA content was assessed spectrophotometrically [59]. Carotenoids and anthocyanin were determined in the leaves by extracting 0.5 g of leaves with 5 mL of methanol. According to Lichtenthaler and Buschmann [63], the methanolic suspension was then filtered and analyzed spectrophotometrically.
The total phenolic compounds (TPCs) were determined using 0.5 mL of the same methanolic extract as above, adding 7 mL of distilled water and 0.5 mL of Folin–Ciocalteu’s reagent. Two mL of 2% sodium carbonate (w/v) was added after 5 min and left to react for 2 h. The TPC content was determined according to Paradiković et al. [64] and expressed as gallic acid equivalent (GAE) per gram of fresh weight. Finally, the total flavonoid content (TFC) was assessed. To this end, 0.5 g of fresh plant tissue was extracted with methanol and centrifuged for 10 min at 6000 rpm. To one mL of supernatant, we added in sequence, at five-minute intervals, 0.3 mL of 5% NaNO2, 0.3 mL of 10% AlCl3 and 2 mL of 1 M NaOH. Subsequently, the volume was brought to 10 mL with H2O. The quantification was carried out spectrophotometrically, and the TFC was expressed as mg of catechin equivalents (CEs) per gram of fresh weight.

2.9. Statistical Analysis

The data obtained from the Pst in vitro growth experiments were analyzed using the non-linear regression dose–response model proposed by Streibig et al. [65] incorporated in BIOASSAY97 Excel-macro [66], setting the equation parameters as in Orfei et al. [67]. The resulting Pst in planta growth data were separately submitted to one-way (treatment) analysis of variance (ANOVA), with significant differences compared using Tukey’s multiple range test (p < 0.05), via Excel-macro DSAASTAT [68]. The disease severity results assessed via visual observation were compared using Kruskal–Wallis and Dunn’s post hoc tests (p < 0.05) using R software (version 4.4.0; April 2024) [69].

3. Results and Discussion

3.1. ZnO-NPs’ Activity in Vitro Antimicrobial Assay

Among all the ten ZnO-NP concentrations investigated in vitro, eight (ranging from 30 to 100 ppm) totally suppressed bacterial growth from the first measurement (one hour of incubation; Figure 1). Therefore, it was possible to conclude that 30 ppm corresponded to the minimum inhibitory concentration (MIC) value. A dose–response curve was then obtained from the data to calculate the effective concentrations EC10 (2.43 ± 0.46 ppm), EC20 (6.43 ± 0.49 ppm), EC90 (17 ± 1.08 ppm) and EC95 (23.7 ± 2.37 ppm), which evidenced bacterium growth reduction compared to the results obtained for the untreated control. The EC values confirmed a growth-inhibiting activity according to those observed by other studies for ZnO-NPs against various pathogens [26]. Considering the genus Pseudomonas, similar in vitro tests showed a significant reduction in the development of a hazardous human pathogen associated with a broad and severe spectrum of infections such as Pseudomonas aeruginosa [70,71,72], even though the concentrations found to be effective were significantly higher than those selected for our work. However, reductions in the growth of Pseudomonas spp. comparable to those highlighted by our experiments have been recently observed [73]. By contrast, for Pseudomonas syringae pv. tomato, agar disc diffusion assays have previously demonstrated a substantial reduction in bacterial growth on plates, at concentrations of both 100 and 300 ppm [40,41].

3.2. Effect of ZnO-NPs in Protecting Tomato Plants Against Pst

The 50 ppm concentration of ZnO-NPs proved ineffective in protecting tomato plants, regardless of whether it was applied two days before inoculation with Pst or five days after. In contrast, the 100 ppm concentration significantly reduced the disease symptoms in the preventive treatment (Figure 2a). Consequently, in the next experiment, the effectiveness of the 100 ppm ZnO-NP preventive treatment was compared to that of the plant defense activator acibenzolar-S-methyl (benzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester, ASM), both applied two days before inoculation. Fourteen days after inoculation, the symptoms following the 100 ppm ZnO-NP treatment were significantly reduced compared to those in the untreated control (−50%, Figure 2b). These results are consistent with observations reported by Elsharkawy et al. [41], where both 100 ppm ZnO-NP treatments applied one day before and one day after inoculation significantly reduced the disease symptoms.

3.3. ZnO-NPs’ Impact on Bacterial Growth in Planta

In tomato plants, ZnO-NPs and ASM significantly reduced Pst DAPP-PG 215 growth when compared to the control, starting from the first time point of investigation at 3 dpi (Figure 3). However, at the last point examined (9 dpi), bacterial growth in the ASM-treated plants was not different from that in the control, whereas the ZnO-NP-treated plants were still capable of contrasting the pathogen’s development. ZnO-NPs’ ability to reduce bacterial populations in planta aligns with the findings of Shantharaj et al. [47], where the application of doses of 250 ppm and 500/100 ppm significantly mitigated the development of Xylella fastidiosa in the xylem vessels of tobacco and blueberry, respectively. The loss of efficacy found for ASM after 9 days from inoculation is consistent with its estimated temporal range of induced resistance in tomato, which is reported to range between 3 and 7 days [59,74].

3.4. Plant Defense Gene Expression in ZnO-NP-Treated Plants

Preventive treatment before inoculation resulted in a significant increase in the expression of the PR1B1 marker gene only in the case of ASM administration and exclusively 24 hpt (3.3 Log2fold compared to 0.9 Log2fold and 1.7 Log2fold for control and ZnO-NP-treated plants, respectively; see Figure 4). PR1B1 induction is generally associated with the salicylic acid (SA) pathway. However, by 48 h post-treatment (hpt), PR1B1 expression in ASM-treated plants was found to be comparable to that exhibited by the control and ZnO-NP-treated plants. After inoculation with the bacteria, PR1B1 expression increased similarly in all plants at one hour, regardless of the treatment. Despite this, gene expression was significantly higher in both ASM- and ZnO-NP-treated plants compared to the untreated control at 24 hpi (7.5 Log2fold for ZnO-NPs, 6.5 Log2fold for ASM and 4.9 Log2fold for the control). At 96 hpi, PR1B1 expression remained significantly high in the control and ZnO-NP-treated plants (8.1 and 7.9 Log2fold, respectively), while it substantially decreased in ASM-treated plants (3.7 Log2fold).
In contrast, the expression of the gluB gene, which encodes β-1,3-glucanase and is associated with the jasmonic acid (JA) and ethylene (ET) pathways, did not significantly differ from that in the control across the treatments or time points examined (Figure 5). These findings suggest that ZnO-NPs cannot induce a defense response before inoculation, as ASM does. Instead, the expression of the JA pathway was maximally activated only after inoculation and remained elevated for at least 96 h. Based on these results, we hypothesize that ZnO-NPs may exert a priming effect, enabling a rapid increase in SA pathway expression following the pathogen challenge, relative to the control. The induction of the SA pathway by zinc and zinc nanoparticles has been reported in other studies [41,75]. In particular, the ability of ZnO-NPs to prime seed has been observed in various host plants and can help plants counteract the hampering effects due to biotic and abiotic stresses [76,77,78,79]. Unlike the findings of Elsharkawy et al. [41], our experiments did not evidence any ZnO-NP effect in inducing defensive responses in tomato plants before inoculation.

3.5. Oxidative Stress and Antioxidants

The next experiments focused on assessing the oxidative cellular status and the content of some antioxidants in plants treated with the most effective ZnO-NP concentration (100 ppm). In general, reactive oxygen species (ROS), under physiological conditions, modulate and regulate many biological processes through a network of intricate signaling pathways [80]. However, in situations of abiotic or biotic stress, plants can activate antioxidant responses with the scope of containing the oxidative impairment. In the same way, substances applied to plants, including some metal oxide nanomaterials, can increase the content of antioxidants, thus allowing plants to better deal with oxidative perturbations [81].
Pathogen infection can alter the cellular redox balance, prompting the accumulation of ROS, such as H2O2, which can hamper membrane stability, degrade proteins and damage DNA, among other negative impacts on cells [80]. The content of MDA, a lipid oxidation marker, reflects eventual damage due to membranes. Our experiments showed that samples infected with Pst exhibited at 96 h after the treatment (48 h after the inoculation) a significant increase in H2O2 levels (Table 1). This was accompanied by a higher level of MDA, thus confirming the oxidative impairment imposed by Pst. In contrast, at 96 h after treatment (48 h after inoculation), the infected plants treated with the nanomaterial did not show higher contents of H2O2 and MDA compared to the control samples. This result highlights the beneficial effect prompted by ZnO-NPs, mitigating the oxidative perturbations associated with the infection (Table 1).
As these results indicated the capacity of ZnO-NPs to modulate the cellular redox status, some clusters of antioxidant metabolites were assessed in the plants subjected to the different treatments (Table 2). Carotenoids are light-harvesting pigments with antioxidant–protective functions, operating as photoprotectors by removing ROS from the chloroplast when the plant is subject to biotic and abiotic stressors [82]. Accordingly, these biomolecules are classified among the most effective chemicals scavenging ROS in plants. Carotenoids can contain ROS when pathogens exploit them to establish infections. Our results showed a generally higher carotenoid content in ZnO-NP-treated plants at 72 and 96 h after treatment (24 and 48 h after inoculation) compared to the controls (Table 2).
In line with our findings, ZnO-NPs improved the carotenoid content in carrot plants infected with Pectobacterium carotovorum, Xanthomonas campestris pv. carotae, Meloidogyne javanica, Alternaria dauci and Fusarium solani [83]. Differently, the anthocyanin content was generally not affected by the ZnO-NP treatments, despite their antioxidant role and capacity of mediating defense signaling pathways related to the SA and JA pathways [84]. Finally, the content of TPCs and the TFC were investigated to obtain any evidence of their involvement in contrasting Pst infection. These secondary metabolites are pivotal for plant defense against pathogens [85]. In particular, phenolics and flavonoids can directly act as antimicrobials or be involved in the plant immune responses. They can accumulate in response to the oxidative stress determined by pathogen infection and act as ROS scavengers [86].
Regarding the TPCs and TFC, the general enhancement or maintenance of TPCs following pathogen’s infection further points out the direct involvement of ZnO-NPs in strengthening plant defenses. In particular, the TPC levels increased in plants treated with ZnO-NPs but not infected compared to the control samples at 72 and 96 h after treatment (24 and 48 h after inoculation). In contrast, in infected samples not treated with ZnO-NPs, the TPC level decreased 96 h after treatment (48 h after the inoculation). On the contrary, when plants treated with ZnO-NPs were infected with Pst, the TPC content increased 72 h after treatment (24 h after inoculation) or did not change at the last experimental point. The changes observed in the TPC content align with other studies that showed that ZnO-NPs could act as a priming agent, thus prompting the activation of defensive pathways directly related to this important family of secondary metabolites [36].

4. Conclusions

Zinc, one of the first elements discovered and present in the earth’s crust, is pivotal in all living organisms [87]. In plants, zinc is notably and critically involved in many physiological processes such as cell multiplication, water and nutrient uptake, phytohormone activities and plant responses to biotic and abiotic stresses [88]. In addition, zinc possesses antimicrobial properties against a wide range of pathogens [89]. Consequently, nanoscaled zinc oxide is among the nanomaterials most extensively studied for plant protection against pathogens in recent years [90].
With this work, we aimed to investigate the bioactivity of biogenic zinc oxide nanoparticles (ZnO-NPs) synthetized using a Lemna minor extract. In particular, the nano-biofabricated material was tested against a common and challenging tomato pathogen, Pseudomonas syringae pv. tomato (Pst), a causative agent of bacterial speck disease. The in vitro experiment confirmed the antimicrobial activity of ZnO-NPs, as it was possible to observe a complete inhibition of Pst growth already at 30 ppm. Furthermore, the biogenic material significantly reduced the severity of tomato bacterial speck symptoms, when applied preventively (2 days before) or after (5 days later) pathogen inoculation. Regarding the in planta growth of Pst, ZnO-NPs and ASM were observed to reduce pathogen development significantly, but in the case of the resistance inducer, after 9 days, the bacterium was found at a concentration similar to that in the control. The gene expression analysis in tomato plants treated with ZnO-NPs showed a significant increase in the expression of defense genes compared to the control, following inoculation with Pst. Finally, the infected tomato plants evidenced the insurgence of oxidative stress; however, the ZnO-NP treatment counteracted this effect. The antioxidative protection of ZnO-NPs was further corroborated by the fact that this nanoscaled material stimulated the biosynthesis of metabolites involved in dealing with oxidative imbalances.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11040431/s1, Table S1: List of the primers used in this study for gene expression analysis via qPCR [91,92,93].

Author Contributions

Conceptualization, B.O., D.D.B., C.T., C.M. and R.B.; methodology, B.O., D.D.B., C.M. and R.B.; software, B.O., A.S. and M.P.; validation, B.O. and A.S.; formal analysis, B.O., D.D.B., C.M. and R.B.; investigation, B.O., A.S., M.P., D.D.B., C.T. and D.P.; resources, D.D.B., C.M. and R.B.; data curation, B.O. and A.S.; writing—original draft preparation, B.O. and A.S.; writing—review and editing, D.D.B., M.P., C.T., D.P., C.M. and R.B.; visualization, B.O., A.S., M.P. and C.M.; supervision, B.O., D.D.B., C.M. and R.B.; project administration, C.M. and R.B.; funding acquisition, D.D.B., C.M. and R.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Università degli Studi di Perugia, project VITALITY National Innovation Ecosystem grant ECS00000041—NextGenerationEU under the Italian Ministry of University and Research (MUR).

Data Availability Statement

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

We thank Luca Bonciarelli and Maurizio Orfei for technical assistance.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Horticulturae 11 00431 g001
Figure 1. (A) In vitro growth curves of Pseudomonas syringae pv. tomato (Pst) DAPP-PG 215 in the presence of ZnO-NPs (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ppm); each value is the mean from 8 replicates. (B) Dose–response curve calculated at 24 h after the ZnO-NP treatments. The effective concentrations are indicated in the plot.
Figure 1. (A) In vitro growth curves of Pseudomonas syringae pv. tomato (Pst) DAPP-PG 215 in the presence of ZnO-NPs (0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ppm); each value is the mean from 8 replicates. (B) Dose–response curve calculated at 24 h after the ZnO-NP treatments. The effective concentrations are indicated in the plot.
Horticulturae 11 00431 g001
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Figure 2. (a) Effect of zinc oxide nanoparticle (ZnO-NP) treatment 2 days before inoculation (preventive) and 5 days after inoculation (curative), at different concentrations in both cases (50 ppm, 100 ppm) in comparison with control treatment (H2O), expressed as bacterial speck disease severity according to Horsfall–Barratt (HB) scale at 14 days post-inoculation (dpi). Data were analyzed with Kruskal–Wallis test (p-value = 0.022), followed by a post-hoc Dunn’s multiple comparison test (p-value with Benjamin–Hochberg adjustment for control and 100 ppm ZnO-NP preventive treatments = 0.034). (b) Effect of zinc oxide nanoparticle (ZnO-NPs) and acibenzolar-S-methyl (ASM) treatments in comparison with control treatment (H2O) expressed as bacterial speck disease severity according to Horsfall–Barratt (HB) scale at 14 days post-inoculation (dpi). Both treatments were carried out 2 days before inoculation. Data were analyzed with Kruskal–Wallis test (p-value = 0.022), followed by a post-hoc Dunn’s multiple comparison test. Different letters reveal statistically significant differences based on Dunn’s multiple comparison test (p < 0.05; p-value with Benjamin–Hochberg adjustment for control and 100 ppm ZnO-NP preventive treatments = 0.034).
Figure 2. (a) Effect of zinc oxide nanoparticle (ZnO-NP) treatment 2 days before inoculation (preventive) and 5 days after inoculation (curative), at different concentrations in both cases (50 ppm, 100 ppm) in comparison with control treatment (H2O), expressed as bacterial speck disease severity according to Horsfall–Barratt (HB) scale at 14 days post-inoculation (dpi). Data were analyzed with Kruskal–Wallis test (p-value = 0.022), followed by a post-hoc Dunn’s multiple comparison test (p-value with Benjamin–Hochberg adjustment for control and 100 ppm ZnO-NP preventive treatments = 0.034). (b) Effect of zinc oxide nanoparticle (ZnO-NPs) and acibenzolar-S-methyl (ASM) treatments in comparison with control treatment (H2O) expressed as bacterial speck disease severity according to Horsfall–Barratt (HB) scale at 14 days post-inoculation (dpi). Both treatments were carried out 2 days before inoculation. Data were analyzed with Kruskal–Wallis test (p-value = 0.022), followed by a post-hoc Dunn’s multiple comparison test. Different letters reveal statistically significant differences based on Dunn’s multiple comparison test (p < 0.05; p-value with Benjamin–Hochberg adjustment for control and 100 ppm ZnO-NP preventive treatments = 0.034).
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Figure 3. Pseudomonas syringae pv. tomato growth in planta at 3, 6 and 9 days post-inoculation (dpi) in tomato plants pre-treated (2 days before inoculation) with ZnO-NPs 100 ppm and acibenzolar-S-methyl (ASM) in comparison to control (H2O), expressed as log10 colony-forming unit (cfu) per cm2 of leaf. Data are means ± SE of 3 replicates. Columns with different letters are significantly different (p ≤ 0.05) according to Tukey’s HSD test.
Figure 3. Pseudomonas syringae pv. tomato growth in planta at 3, 6 and 9 days post-inoculation (dpi) in tomato plants pre-treated (2 days before inoculation) with ZnO-NPs 100 ppm and acibenzolar-S-methyl (ASM) in comparison to control (H2O), expressed as log10 colony-forming unit (cfu) per cm2 of leaf. Data are means ± SE of 3 replicates. Columns with different letters are significantly different (p ≤ 0.05) according to Tukey’s HSD test.
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Figure 4. Relative gene expression of PR1b1 in tomato (cv. Rio Grande) leaves at 24 and 48 h post-treatment (hpt) with ZnO-NPs 100 ppm and acibenzolar-S-methyl (ASM) at 0, 24 and 96 h post-inoculation (hpi; treatment applied two days before inoculation) in comparison to control (H2O). Data are means of 9 replicates (3 biological, 3 technical) ± SE. Columns with different letters are significantly different (p ≤ 0.01) according to Tukey’s HSD test.
Figure 4. Relative gene expression of PR1b1 in tomato (cv. Rio Grande) leaves at 24 and 48 h post-treatment (hpt) with ZnO-NPs 100 ppm and acibenzolar-S-methyl (ASM) at 0, 24 and 96 h post-inoculation (hpi; treatment applied two days before inoculation) in comparison to control (H2O). Data are means of 9 replicates (3 biological, 3 technical) ± SE. Columns with different letters are significantly different (p ≤ 0.01) according to Tukey’s HSD test.
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Figure 5. Relative gene expression of gluB in tomato (cv. Rio Grande) leaves at 24 and 48 h post-treatment (hpt) with ZnO-NPs 100 ppm and acibenzolar-S-methyl (ASM) at 0, 24 and 96 h post-inoculation (hpi; treatment applied two days before inoculation) in comparison to control (H2O water, foliar). Data are means of 9 replicates (3 biological, 3 technical) ± SE. Columns with different letters are significantly different (p ≤ 0.01) according to Tukey’s HSD test.
Figure 5. Relative gene expression of gluB in tomato (cv. Rio Grande) leaves at 24 and 48 h post-treatment (hpt) with ZnO-NPs 100 ppm and acibenzolar-S-methyl (ASM) at 0, 24 and 96 h post-inoculation (hpi; treatment applied two days before inoculation) in comparison to control (H2O water, foliar). Data are means of 9 replicates (3 biological, 3 technical) ± SE. Columns with different letters are significantly different (p ≤ 0.01) according to Tukey’s HSD test.
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Table 1. Cellular redox state expressed as H2O2 and MDA contents found in tomato plant samples that were untreated and not inoculated (control), treated with ZnO nanoparticles but not inoculated (ZnO-NPs), untreated but inoculated with the pathogen (Pst), and both treated with ZnO nanoparticles and then inoculated with Pst (ZnO-NPs+Pst), collected after 72 and 96 h from the treatment date.
Table 1. Cellular redox state expressed as H2O2 and MDA contents found in tomato plant samples that were untreated and not inoculated (control), treated with ZnO nanoparticles but not inoculated (ZnO-NPs), untreated but inoculated with the pathogen (Pst), and both treated with ZnO nanoparticles and then inoculated with Pst (ZnO-NPs+Pst), collected after 72 and 96 h from the treatment date.
TimeTreatmentH2O2
(nmol g−1 FW)		MDA
(nmol−1 FW)
72 hControl32.5 ± 7.5 a8.11 ± 1.11 a
ZnO-NPs29.6 ± 4.3 a7.32 ± 1.38 a
ZnO-NPs+Pst32.0 ± 1.9 a7.40 ± 0.36 a
Pst37.4 ± 3.0 a8.70 ± 0.40 a
96 hControl32.3 ± 4.0 b7.35 ± 0.16 b
ZnO-NPs28.9 ± 3.4 b6.81 ± 0.27 b
ZnO-NPs+Pst32.9 ± 4.5 b7.30 ± 0.98 b
Pst41.6 ± 3.5 a9.02 ± 0.75 a
Different letters reveal statistically significant differences based on Duncan’s multiple comparison test (p < 0.05).
Table 2. Contents of carotenoids, anthocyanin and total phenolic compounds (TPCs) and total flavonoid content (TFC) in tomato plant samples that were untreated and not inoculated (control), treated with ZnO nanoparticles but not inoculated (ZnO-NPs), untreated but inoculated with the pathogen (Pst), and both treated with ZnO nanoparticles and then inoculated with Pst (ZnO-NPs+Pst), collected after 72 and 96 h from the treatment date.
Table 2. Contents of carotenoids, anthocyanin and total phenolic compounds (TPCs) and total flavonoid content (TFC) in tomato plant samples that were untreated and not inoculated (control), treated with ZnO nanoparticles but not inoculated (ZnO-NPs), untreated but inoculated with the pathogen (Pst), and both treated with ZnO nanoparticles and then inoculated with Pst (ZnO-NPs+Pst), collected after 72 and 96 h from the treatment date.
TimeTreatmentCarotenoids
(mg g−1 FW)		Anthocyanin
(µg g−1 FW)		TPC
(mg GAE g−1 FW)		TFC
(mg CE g−1 FW)
72 hControl 0.030 ± 0.005 b0.93 ± 0.10 a0.24 ± 0.02 bc1.44 ± 0.02 a
ZnO-NPs0.040 ± 0.002 a0.71 ± 0.07 a0.28 ± 0.01 a1.23 ± 0.20 ab
ZnO-NPs+Pst0.044 ± 0.002 a0.90 ± 0.08 a0.26 ± 0.01 ab1.04 ± 0.15 b
Only Pst0.030 ± 0.001 b0.81 ± 0.18 a0.22 ± 0.03 c1.03 ± 0.20 b
96 hControl 0.030 ± 0.001 b0.84 ± 0.20 a0.25 ± 0.03 b1.09 ± 0.04 ab
ZnO-NPs0.032 ± 0.003 b0.83 ± 0.02 b0.30 ± 0.01 a1.18 ± 0.08 a
ZnO-NPs+Pst0.059 ± 0.005 a1.01 ± 0.25 a0.25 ± 0.01 b1.14 ± 0.11 ab
Only Pst0.040 ± 0.013 a0.89 ± 0.13 ab0.19 ± 0.01 c1.00 ± 0.04 b
Different letters reveal statistically significant differences based on Duncan’s multiple comparison test (p < 0.05).
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MDPI and ACS Style

Orfei, B.; Scian, A.; Del Buono, D.; Paglialunga, M.; Tolisano, C.; Priolo, D.; Moretti, C.; Buonaurio, R. Biogenic Zinc Oxide Nanoparticles Protect Tomato Plants Against Pseudomonas syringae pv. tomato. Horticulturae 2025, 11, 431. https://doi.org/10.3390/horticulturae11040431

AMA Style

Orfei B, Scian A, Del Buono D, Paglialunga M, Tolisano C, Priolo D, Moretti C, Buonaurio R. Biogenic Zinc Oxide Nanoparticles Protect Tomato Plants Against Pseudomonas syringae pv. tomato. Horticulturae. 2025; 11(4):431. https://doi.org/10.3390/horticulturae11040431

Chicago/Turabian Style

Orfei, Benedetta, Anna Scian, Daniele Del Buono, Michela Paglialunga, Ciro Tolisano, Dario Priolo, Chiaraluce Moretti, and Roberto Buonaurio. 2025. "Biogenic Zinc Oxide Nanoparticles Protect Tomato Plants Against Pseudomonas syringae pv. tomato" Horticulturae 11, no. 4: 431. https://doi.org/10.3390/horticulturae11040431

APA Style

Orfei, B., Scian, A., Del Buono, D., Paglialunga, M., Tolisano, C., Priolo, D., Moretti, C., & Buonaurio, R. (2025). Biogenic Zinc Oxide Nanoparticles Protect Tomato Plants Against Pseudomonas syringae pv. tomato. Horticulturae, 11(4), 431. https://doi.org/10.3390/horticulturae11040431

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