Potential of Nettle Infusion to Protect Common Bean from Halo Blight Disease

Abstract

The use of plant-based preparations to replace chemical pesticides is a challenge for sustainable agriculture. Preparations from nettle (Urtica dioica L.) are good candidates, as the treatment of common bean plants (Phaseolus vulgaris L.) with aqueous suspensions of nettle reduced symptoms of halo blight disease caused by Pseudomonas syringae pv. phaseolicola (Pph). The aim of the present work was to test if nettle infusions (Ui) have similar effects and find out what activity is behind this: antimicrobial, promotion of the natural defenses of plants, and/or antioxidant. To achieve this, Pph growth was tested in the presence of infusions of nettle leaves collected in two different years (Ui₁₈ and Ui₂₂), and we found that it was only weakly inhibited at high concentrations of Ui₁₈. Interestingly, Ui₂₂ promoted bacteria growth at all concentrations. Second, we estimated the production of reactive oxygen species (ROS) in response to flagellin22 (flg22) in common bean leaf discs, since recognition of this bacterial peptide usually leads to ROS accumulation in tissues as a plant immune response. However, leaf discs that were preincubated in Ui showed no accumulation of ROS after flg22 treatment, suggesting that Ui can neutralize ROS production. Finally, in a Pph inoculation experiment of common bean plants grown in vitro, we observed that pretreatment of plants with Ui drastically reduced foliar oxidative damage and disease symptoms 6 h after inoculation. This effect was more noticeable for Ui₂₂, which was related to the higher antioxidant activity found in this extract in comparison with Ui₁₈. These results suggest that the protective properties of Ui are mainly due to the content of antioxidant bioactive compounds.

1. Introduction

A challenge for agriculture is to increase crop yields by controlling pathogenic micro-organisms that cause plant diseases and severe crop losses in production worldwide. Farmers use conventional pesticides to control phytopathogens, but these are pollutants, harmful to the environment and, in addition, the target organisms can quickly develop resistance to them [1]. Natural plant-based preparations (PBPs) are emerging as innovative and sustainable alternatives, due to their advantages in terms of safety, easy biodegradability, broad-spectrum efficacy, environmental friendliness, and low risk of resistance and toxicity [2]. From an economic and environmental point of view, it is desirable that the raw materials selected to obtain these preparations are residues from other manufacturing processes or wild species with no other industrial or agronomic use [3], such as nettle (Urtica dioica L.).

Nettle is a perennial herb widespread throughout temperate regions of Europe, Asia, North Africa, and North America, growing up to an altitude of 1800 m in moist soils, meadows, forests, and abandoned fields in dappled-shaded spots. Two universally recognized characteristics of this plant are the stinging effect of the trichomes on its stems and leaves, and its many uses in folk medicine and as a wild vegetable. Nowadays, nettle is considered a multipurpose plant with applications in the food/feed, medicinal, cosmetic, and fiber sectors [4,5,6]. Most of these applications are related to its rich nutritional profile and the presence of many bioactive compounds that lie behind the pharmacological, antimicrobial, and antioxidant activities attributed to this plant [7,8,9]. Agriculture also benefits from the properties of nettle, so that different aqueous nettle preparations are traditionally used as natural fertilizers and pesticides. In fact, Commission Implementing Regulation (UE) 2017/419 approved nettle as a basic substance allowed in organic farming for crop protection against insects, mites, and pathogenic fungi [10], but not against plant disease-causing bacteria, due to a lack of sufficient experimental results to support this activity. While the negative effect of nettle extracts on human pathogenic and food spoilage bacteria has been widely demonstrated, there are hardly any studies on phytopathogenic bacteria [11]. It is therefore interesting to investigate the antibacterial potential of nettle extracts, especially in view of the limited availability of chemical bactericidal agents and the legal restrictions to which their application is subject [12].

Plants have per se natural defense mechanisms against pathogenic bacteria. They perceive bacterial presence shortly after infection via transmembrane receptors that recognize highly conserved molecular patterns, such as flagellin22 (flg22), characteristic of the flagellum of bacteria such as Pseudomonas spp. [13]. Immediately after this recognition, plant cells produce a release of H₂O₂ and an influx of intracellular calcium, which activate a signal transduction pathway that involves, among others, certain plant hormones such as salicylic acid (SA), and finally triggers the expression of numerous plant defense genes [14]. This H₂O₂ production follows a two-phase kinetics, so that once the early and transient H₂O₂ generation occurs, a second, massive and prolonged H₂O₂ production, called oxidative burst, takes place after a few hours, with the aim of creating unfavorable conditions for pathogen growth [15]. However, this oxidative burst could also cause damage to plant tissues if cellular antioxidative defenses are not activated quickly and sufficiently.

Despite the battery of mechanisms that plants have to defend themselves, there are crops susceptible to pathogens, such as the Riñón variety of common bean (Phaseolus vulgaris L.) that is unable to prevent the halo blight disease caused by Pseudomonas syringae pv. phaseolicola (Pph), a hemi-biotrophic gamma-proteobacteria [16]. The disease provokes yield losses of up to 45%, with the main symptoms being general chlorosis, stunting, and distortion of growth [17]. A work from our group revealed that Riñón variety plants can perceive the bacteria and initiate defense responses, but the expression of defense-related genes, the activation of an effective antioxidant system, and the production of an SA peak are limited, which would explain its susceptibility to Pph [18]. In other work, we showed that one way to protect these plants is by priming plant defenses for a future Pph infection with 2,6-dichloroisonicotinic acid (INA), a synthetic derivative mimicking the resistance-inducing activity of SA [19]. However, treatment with PBPs is preferable to chemicals such as INA, for the reasons outlined above and because PBPs may have certain substances with antimicrobial activity and others with the potential to stimulate the plant immune system, given their complex composition. Thus, they would protect crops against pathogens through both direct and indirect pathways [20].

With all this in mind, the next step in our research was to test the ability of four PBPs to protect bean plants against Pph. As a result of this study, De la Rubia et al. [3] found that the most effective was an aqueous suspension of powdered nettle leaves. Nettle suspension inhibited the Pph growth at concentrations ranging from 0.4 to 10 mg/mL, and its application to plants stimulated the expression of six genes involved in defense, mainly PR1 (pathogenesis-related 1). Therefore, given the apparent antibacterial and defense-stimulating activities of nettle suspension, it is conceivable that it could be used to diminish the impact of halo blight disease on common bean plants, and even to protect other crops. However, field application of nettle extracts in the form of suspensions may not be practical or feasible, as the presence of suspended particles may prevent uniform distribution of the product, which is likely to lead to variable results between plants. In addition, the particles could clog nozzles, pipes, etc. A solution to these possible problems would be to use clarified nettle infusions instead of suspensions.

Nettle and its extracts have another property that can be exploited to protect crops against phytopathogens: their antioxidant activity, due in part to the presence of reducing compounds. These could attenuate the oxidative activity of ROS produced in plants under biotic and abiotic stress and reduce the oxidative damage to tissues. In fact, Urtica dioica has been found to exhibit higher antioxidant activity than species of the same genus and other plant genera [21,22], and even than some of the standard compounds used in the assays to measure it [23,24].

In view of the above, we proposed that nettle infusions could have an agronomic application as a natural PBP capable of protecting Pph-susceptible common bean varieties against halo blight disease. To confirm this, it was first necessary to study whether nettle infusions have bactericidal properties, can promote defense responses in plants, as was the case with suspensions [3], and/or have some other activity that indirectly protects plants. Thus, the aims of the present work were (1) to assess the antibacterial activity of nettle leaf infusions (Ui) against Pph; (2) to evaluate whether Ui extracts can induce a primary defense response such as H₂O₂ accumulation in common bean (cv. Riñón) tissues; (3) to know if the treatment of the plants with Ui really increases their resistance to Pph; and (4) to check whether any of these capacities of Ui are related to its antioxidant activity. We tested infusions obtained from fresh nettle leaves collected in 2022 (U₂₂), and from leaves harvested in 2018, dried, and preserved (U₁₈) to date, as the latter were the same used by De la Rubia et al. [3] to obtain the suspension.

2. Materials and Methods

This section presents the experiments carried out in the study and the methodology used, as well as the treatments applied to the data obtained.

2.1. Nettle Sampling and Preparation of Nettle Infusions

Nettle (Urtica dioica L.) was harvested from a field in León (Spain, coordinates: 42° 38′ 04.8″ N; 5° 31.5′ 50.5″ W) during the spring season in 2022. The isolated leaves were frozen at −80 °C, lyophilized, and pulverized. The nettle powder was stored under vacuum and dark conditions at room temperature until use. In this work, we also used the same material as in De la Rubia et al. [3] as a reference. The later consisted of nettle leaves collected in 2018 from another León location (coordinates: 42° 43′ 47.5″ N; 5° 50′ 29.1″ W), which were dried at 60 °C until at constant mass and pulverized.

Infusions (Ui₂₂ and Ui₁₈) were obtained by mixing the nettle powder in water at a concentration of 100 mg/mL and autoclaving (121 °C for 15 min) the mixture. After centrifugation (4000× g, 5 min), the supernatant was collected and filtered (Millex-GP, 3.3 cm, 0.22 µm Ø, Millipore Merck Science, Madrid, Spain).

2.2. Bioassays of Nettle Infusions against Pseudomonas syringae pv. Phaseolicola

Pseudomonas syringae van Hall 1902 (Pph), the P. syringae pv. phaseolicola 1448A from “Colección Española de Cultivos Tipo” (CECT321, Valencia, Spain) was grown on liquid King’s B (KB) medium for two days at 30 °C and 220 rpm. Afterwards, a Pph solution was prepared removing the KB medium by centrifugation at 4800× g for 10 min and resuspending the pellet in a volume of 0.9% (w/v) NaCl to obtain a final concentration of 10⁸ colony-forming-units (CFU)/mL, as described by De la Rubia et al. [18]. The medium for bioassays was Tryptic Soy Agar medium (TSA, BD DifcoTM, Waltham, MA, USA) with glucose 5 g/L. After autoclaving this medium, water (control) or the infusions (final concentrations of 1, 4, 10, and 20 mg/mL) were added, and the media were dosed into Petri dishes (25 mL/dish). Once the media had jellified, a single well was made in the middle of each Petri dish to place 20 µL of the bacteria solution previously prepared. Dishes were incubated at 30 °C for seven days and the area of Pph growth halos was quantified using ImageJ (v. 1.53e) software [25]. Bacterial growth was calculated as the percentage of the halo area measured in the presence of Ui relative to the halo area in its absence.

2.3. Detection of ROS Released from Common Bean Leaf Discs in Response to flagellin22

In this study, we employed plants grown in vitro of Phaseolus vulgaris L. variety Riñón (Protected Geographical Identification of Bean of La Bañeza-León, Spain), common bean hereafter. Seeds were sterilized with 70% ethanol (v/v) for 30 s followed by 0.4% NaClO (w/v) for 20 min [3]. After washing with sterile water, the seeds were sown in a hydrated peat: perlite mixture (Blumenerde universal substrate, Gramoflor, Vechta, Germany) contained in closed and autoclaved 980 mL glass containers (PhytoTech Labs, Lenexa, KS, USA). Cultures were maintained at 25 ± 2 °C, 16 h light photoperiod, and 45 µmol/m²s light intensity at least for 14 days, when plants were at V1 stage (two cotyledonary leaves expanded, but without true leaves developed).

The ROS production was determined in leaf discs from common bean plants grown in vitro up to V1 stage using the luminol assay as described by De la Rubia et al. [19]. Discs were excised from the cotyledonary leaves of seven plants, placed in the wells of a white plate with 200 µL of sterile water, 20 mg/mL Ui₁₈, or 20 mg/mL Ui₂₂, and incubated overnight at room temperature. The solution was then removed, and 100 µL of a solution containing 20 µM luminol L-012 (Wako Chemicals, Richmond, VA, USA) and 100 µg/mL peroxidase from horseradish typo VI.A (Sigma Aldrich, Madrid, Spain, P6782) was added and incubated for 30 min more in darkness [26]. This was followed by the addition of 100 µL of water to the negative control or 100 µL of 2 µM flg22. The luminescence emitted by the luminol due to ROS production was measured as relative luminescent units (RLUs) in a Varioskan LUX (Thermo Scientific, Waltham, MA, USA) microplate reader for 120 min.

2.4. Pph Inoculation of Plants and 3,3′-Diaminobendicin (DAB) Staining for Checking Oxidative Damage—Measurement of Non-Enzymatic Antioxidant Activity of the Nettle Infusions

Common bean plants grown in vitro for 14 days (V1 stage), and a 10⁸ CFU/mL Pph solution obtained as described (Section 2.2) but resuspended in water with 0.1% (v/v) Tween-20 [18] were used in an experiment whose design is shown in Figure 1. Upper and lower side of cotyledonary leaves of plants were sprayed with 2 mL of 20 mg/mL Ui₁₈, 20 mg/mL Ui₂₂, or sterile water (control) per leaf. Seven days after this treatment, when the plants were 21 days old, half of them were sprayed with 2 mL of the Pph solution or with 2 mL of water in the case of the control plants. After 6 h, uninfected (control, Ui₁₈, and Ui₂₂) and infected (control+, Ui₁₈+, and Ui₂₂+) plants were observed and photographed, to describe the phenotypic symptoms of halo blight disease.

To check the oxidative damage caused by Pph infection, the H₂O₂ production in leaves was stained with DAB, following the method described in Daudi and O’Brien [27] and modified by De la Rubia et al. [28]. Briefly, after the leaves had been incubated in the DAB solution, the chlorophyll was removed by immersing them in a bleaching solution and boiling. Leaves were air-dried for 5 min and photographed. The H₂O₂ accumulation as a marker of oxidative damage was estimated as the intensity of the brown color of each leaf due to DAB oxidation, using Adobe Photoshop Create (v. 23.0.0) software.

Antioxidant activity of Ui₂₂ and Ui₁₈ was estimated by the Ferric Reducing Antioxidant Power (FRAP) method [29] modified to be performed in 96-well plates. The FRAP reagent was prepared using 10 mM of TPTZ-Fe³⁺ (2,4,6-tri-(2-pyridyl)-s-triazine-Fe³⁺) solution in 40 mM HCl, 20 mM FeCl₃x6H₂O, and sodium acetate buffer (300 mM, pH 3.6) (1:1:10 v/v/v). Aliquots (10 μL) of Ui₂₂ and Ui₁₈ were added to the FRAP reagent (300 μL) and incubated for 15 min at room temperature. The absorbance of the reaction product was measured at 593 nm in a Multi-Detection Varioskan LUX (Thermo Scientific) microplate reader with FRAP reagent as blank. Several dilutions of the infusions were assayed, and the results were calculated by interpolating in a calibration curve made with Trolox (0.1 to 5 mM), which was used as a standard reducing agent. The results, which were expressed as mM equivalents of Trolox, allowed us to determine the total concentration of non-enzymatic antioxidants involved in the reducing capacity of the infusions [30].

2.5. Statistical Analysis and Data Representation

All results are expressed as the means ± standard error (SE) of three independent replicates, except for the ROS release experiment (Section 2.3), in which data represent means ± SE for n equal to seven. Statistical analyses were performed using SPSS software (v. 26.0.0.1). Data normality was first checked with a Kolmogorov–Smirnov test. ANOVA and Tukey post hoc tests were applied to assess differences between more than two means, and a t-Student test was used to compare pairs of means. The means were significantly different considering p < 0.05. The results were represented using Excel Office 2019, 2403 version, and GraphPad Prisma 8.0.1 (2.4.4, La Jolla, CA, USA).

3. Results

The following are the results of tests carried out to find out whether Ui extracts have any protective effect on plants against Pph infection and, if so, what type of Ui activity is responsible for this possible effect: bactericidal/bacteriostatic, induction of plant defense responses, and/or antioxidant.

3.1. Effect of the Nettle Infusions on Pph In Vitro Growth

To understand the potential of Ui₁₈ and Ui₂₂ to inhibit Pph growth, in vitro bioassays were performed on media containing different concentrations of both tested extracts. Figure 2 shows the percentage of bacterial growth with respect to the control. The Ui₁₈ concentrations between 4 and 20 mg/mL decreased the relative growth of Pph, although 50% inhibition was not achieved. On the contrary, the addition of Ui₂₂ to the culture medium stimulated the growth of Pph, mainly at 20 mg/mL. Therefore, Ui₁₈ may have some bacteriostatic effect on Pph, but Ui₂₂ had the opposite effect, since it increased bacterial growth.

3.2. Nettle Infusion Putatively Neutralised the ROS Release Triggered by flg22 in Leaf Discs

Another way in which nettle infusions might protect common bean against Pph is by promoting the general plant defense response. To test this, ROS production in leaf discs in response to the bacterial-derived peptide flg22 (which mimics Pph inoculation) was monitored using a peroxidase/luminol-based assay. As was expected, in our experiment, the addition of flg22 produced a large ROS peak in the leaf discs preincubated in water (positive control), whereas no signal was detected in the absence of flg22 (Mock condition) (Figure 3). Similarly, no ROS production was observed when adding water to leaf discs preincubated in Ui₁₈ or Ui₂₂, but neither when flg22 was added. In the latter case, it is possible that the discs produced ROS and that reducing compounds present in the infusions neutralized them, thus masking the luminescent signal.

3.3. Nettle Infusion Reduced Oxidative Damage Caused by Pph Inoculation on Common Bean

In view of the above result (3.2.), we hypothesized that Ui₁₈ and Ui₂₂ could protect the common bean plants through their antioxidant properties. To assess this hypothesis, we performed a bacterial inoculation experiment using plants grown in vitro. In addition, analyses related to the oxidative state of the infected leaves and the antioxidant capacity of the infusions were conducted. The results are shown in Figure 4 and Figure 5, and in

Table 1. Antioxidant activity of the nettle infusions (Ui₁₈ and Ui₂₂) at several concentrations.

Nettle Extract	1 mg/mL	10 mg/mL	20 mg/mL
Ui18	0.12 ± 0.04 a	1.06 ± 0.28 a	1.88 ± 0.21 a
Ui22	0.23 ± 0.06 a	1.75 ± 0.42 a	3.03 ± 0.59 b

Data are mean ± SE of three experimental replicates. Different letters imply significant differences for each concentration according to t-Student test (p < 0.05).

.

In general terms, all plants that were sprayed seven days previously with Ui showed a more vigorous appearance just before inoculation with Pph than control plants. The morphological analysis 6 h post-inoculation indicated that infected plants suffered leaf epinasty, whereas non-inoculated plants did not (Figure 4). However, this symptom was less pronounced when infected plants were pretreated with the infusions, particularly Ui₂₂. Moreover, Pph-inoculated leaves revealed the presence of chlorotic and/or necrotic tissue areas in control plants, whereas the leaves previously sprayed with the infusions, especially with Ui₂₂, remained almost green and normal, despite Pph inoculation (Figure 4).

Results of tissue oxidation, assessed by DAB staining of accumulated H₂O₂, showed that leaves from uninfected common bean plants (control, Ui₁₈ and Ui₂₂) were barely stained (Figure 5A), because they were not exposed to Pph, whereas the brown color was very intense and distributed over the entire leaves in control plants at 6 h post Pph inoculation (control+). However, the relative area and the intensity of the brown color in leaves from plants pretreated with Ui₁₈ (Ui₁₈+) seemed to be somewhat less than in control leaves. Leaves of plants pretreated with Ui₂₂ (Ui₂₂+) showed a very similar staining pattern to uninoculated leaves. These observations were supported by data obtained when quantifying the mean intensity of the brown color (Figure 5B), which was significantly higher in leaves from infected control plants than in the rest, according to a one-way ANOVA test (Table S1). This finding confirms the oxidative damage caused by Pph in unprotected plants. On the other hand, inoculation did not lead to significant differences in color intensity between plants previously treated or not with Ui₁₈ and Ui₂₂. However, when using a t-Student test to compare pairs of means, a significantly higher value was observed in the leaves of infected plants with respect to uninfected plants when the pretreatment was Ui₁₈, but no difference was found when the pretreatment was Ui₂₂.

Therefore, these results pointed out that previous treatment of common bean plants with nettle infusions reduced the symptoms of halo blight disease, as well as the oxidative damage caused by Pph infection. In addition, Ui₂₂ seemed to provide more protection than Ui_18. The antioxidant activity of the infusions, measured as mM-equivalents of Trolox by FRAP method, was also higher in U₂₂ than in U₁₈ (3.03 and 1.88 for 20 mg/mL, respectively). As expected, this activity decreased as the infusions were diluted, although it remained double or almost double at U₂₂ compared to at U₁₈ (Table 1). Thus, the data were consistent with a possible positive relationship between the concentration of reducing compounds in the infusions and their ability to protect plants.

In response to the proposed objectives and in view of the results obtained, we can state that the pretreatment of common bean plants with Ui extracts reduced the symptoms caused by Pph infection, and that this protective capacity seems to be related to the antioxidant activity of the infusions. However, the results of the bioassays do not allow to shed light on a possible direct effect of Ui on Pph growth, as they were inconsistent and extract-dependent. Finally, the incubation of leaf discs with the infusions did not enhance H₂O₂ production induced by flg22, but rather the presence of reducing agents in the infusions masked this primary tissue response.

4. Discussion

Nettle (Urtica dioica L.) is traditionally considered as a weed in agriculture, but farmers have also used aqueous extracts of nettle, previously macerated for hours or days, as organic fertilizers to promote crop growth [31,32]. However, there is less evidence about the potential of nettle-based preparations for foliar application to protect plants against phytopathogens. In this sense, the studies performed in our laboratory by De la Rubia et al. [3] demonstrated that pretreatment with 1 mg/mL nettle aqueous suspension reduced the phenotypic symptoms of halo blight disease in plants of common bean inoculated with Pph and grown in vitro. The authors linked the protective effect of nettle suspension to its antimicrobial activity and to its ability to induce natural defenses in plants. Given that nettle infusions could have advantages over suspensions for agronomic proposals, it would be interesting to know if and how they could also protect common bean against Pph.

It has been previously shown that nettle leaf water extracts inhibited the growth of Gram-positive and Gram-negative phytopathogenic bacteria [11]. However, several works have shown that these extracts provoked only a slight reduction or no effect on bacteria growth of several pathogenic Pseudomonas species, such as Pseudomonas syringae pv. tomato or Pseudomonas aeruginosa [12,33,34]. In the present work, we found that the ability of two nettle infusions to inhibit Pph growth was different, so that Ui₂₂ stimulated bacteria growth, whereas Ui₁₈ showed limited bacteriostatic activity (Figure 2). Infusions were made with plants from two different locations and collected in two different years; therefore, we expected that the composition of each should be different, as it depended on factors such as harvest year, plant location, habitat, and climatic conditions [35,36]. Moreover, the drying method of the nettle leaves, which was lyophilization for Ui₂₂ and heating for Ui₁₈, could have conditioned the composition of the infusion, as shown by Shonte et al. [37]. Thus, the result was not surprising, given that the antimicrobial activity of U. dioica extracts has usually been related to their content of biologically active compounds [24,38,39].

The ability of Ui₂₂ to stimulate Pph growth could easily be explained by the high antioxidant activity detected in the Ui₂₂ extracts in comparison with Ui₁₈ (Table 1). In addition, nettle leaves and leaf water extracts are rich in nutrients such as amino acids, flavonoids, and vitamins, and ions such as calcium, magnesium, and zinc [8], therefore being a good media for bacterial growth in the absence of antimicrobial compounds.

Regarding Ui₁₈, it showed considerably less ability to inhibit Pph growth than the nettle water suspension [3], although both were prepared from the same nettle leaves collected in 2018. It was expected that the high pressure and temperature applied to obtain the infusion would improve the antimicrobial activity in the aqueous extracts, since several authors have shown that these conditions increase the extraction efficiency of the bioactive compounds [40,41]. Moreover, Garofulić et al. [42] even related this finding to the activity of the nettle extracts against Pseudomonas fragi and Campylobacter jejuni. Our results could be attributed, among other factors, to the loss of phytochemicals that probably occurred in nettle material during the four years of storage.

We also aimed to elucidate whether nettle infusions have the potential to activate the immune system of common bean plants, and thus increase the plant responsiveness to future pathogen attacks, a process known as immune priming [43]. To this end, we tested ROS production triggered by flg22, a mimetic peptide of Pph infection, in foliar discs previously incubated with 20 mg/mL of Ui₁₈ and Ui₂₂. This method is commonly used to reveal whether pathogen- or host-derived ligands can trigger early immune hallmarks [44], such as the H₂O₂ generation that occurs during the first hour of Pph infection in common bean [18]. Moreover, in a very similar experiment conducted with INA, De la Rubia et al. [19] found that this structural analogue of SA reinforced the ROS production in response to flg22 in common bean discs, consistently with the priming concept. In the present work, the signal due to luminol peroxidation by H₂O₂ released from the cells was observed in control discs, but not in those preincubated with infusions (Figure 3). This result may indicate either, that the infusions prevented ROS production, or that the pretreated discs released H₂O₂, but this was neutralized by the ROS-reducing compounds present in the infusions. The latter possibility is more plausible considering the high antioxidant activity attributed to U. dioica and its extracts in many studies [9], and references therein. In fact, both nettle infusions showed this activity (Table 1), which can easily explain the reduction in ROS production after flg22 treatment. However, these results only highlight the antioxidant capacity of nettle extracts, and a more detailed study of their ability to promote plant immune responses should be performed.

Finally, to assess the capacity of the infusions to protect common bean plants and, if so, to relate it to their antioxidant activity, we carried out a bacterial inoculation experiment in plants grown in vitro previously treated with Ui₁₈ and Ui₂₂. In very similar trials, it was found that both INA and aqueous nettle suspensions reduced the symptoms of halo blight disease [3,19], which were analyzed seven days after inoculation. Much earlier, after only six hours, we had already observed the characteristic symptoms of Pph infection in untreated plants: the appearance of chlorosis and lesions on leaves, mainly affecting the foliar margins (Figure 4). These symptoms probably resulted from the oxidative damage caused by the accumulation of H₂O₂ produced by the plants as a defensive response or as part of the hypersensitive response upon the presence of the pathogen [45,46]. In line with this, DAB staining revealed higher H₂O₂ levels in inoculated leaves than in non-inoculated ones (Figure 5A). Nowogórska and Patykowski [47] and De la Rubia et al. [18] also measured significantly higher concentrations of H₂O₂ after 6 h of Pph infiltration in plants of a resistant variety of common bean (cv. Korona) and in leaf discs of the susceptible variety Riñón, respectively. Moreover, an earlier H₂O₂ peak that occurred in leaf discs during the first hour post-infiltration was accompanied by increased lipid peroxidation, indicating that the tissues had undergone oxidative damage [18].

In our experiment, the infected plants also showed leaf epinasty, a process promoted by the gaseous phytohormone ethylene, which regulates plant responses to biotic and abiotic stresses [48]. It is conceivable that common bean plants synthesized ethylene in response to the stress caused by Pph infection [49,50] and that its accumulation in the closed containers led to epinasty.

Pretreatment of common bean plants with nettle infusions apparently had no detrimental effects in plants and greatly alleviated the symptoms of halo blight disease, even leaf epinasty (Figure 4). In the same way, the accumulation of H₂O₂ in the leaves of the infected plants previously sprayed with infusions was statistically equivalent to uninfected plants (Figure 5B). These findings were more noteworthy for Ui₂₂, which also had a higher antioxidant capacity than Ui₁₈ (Table 1). Thus, taking all these results together, we can conclude that the infusions protected the plants from pathogen spread and symptoms due to their antioxidant properties. Many of the biological activities and uses of nettle and its extracts have been linked to its antioxidant activity, which in turn is related to its content of phenolic compounds, especially phenolic acids and flavonoids [7,8,9]. In our opinion, the former might be more involved in the protective potential of nettle infusions than the latter, since the stability and antioxidant activity of flavonoids are compromised when they are subject to temperatures above 75–100 °C [51,52]. On the contrary, the heating of the infusion process itself could modify the cell wall structure and/or break down some covalent bound of phenolic acids to insoluble polymers, releasing these compounds and increasing their concentration in aqueous preparation, as Lou et al. [40] proposed.

5. Conclusions

A nettle infusion, obtained by autoclaving aqueous suspensions of powdered leaves, protected common bean plants against the phytopathogen Pph, as the foliar application of the infusion to plants grown in vitro reduced the disease symptoms and the oxidative damage caused by the bacteria, as early as 6 h after the infection. Moreover, the protective potential of two different infusions (Ui₁₈ and Ui₂₂) seemed to be positively related to their non-enzymatic antioxidant activity. Since only Ui₁₈ had some ability to inhibit bacterial growth, while U₂₂ promoted it, and as the infusions did not increase Pph-triggered tissue ROS production, but rather the opposite, the protection could be related to the presence of reducing compounds capable of neutralizing ROS. In addition, and despite the differences in antioxidant activity, Ui₁₈ and Ui₂₂ showed an excellent effect in protecting common bean against halo blight, which contrasted with their opposite effects on Pph growth. It is likely that the composition of the two extracts was different, so that Ui₁₈ may have contained some compounds with antimicrobial activity that were not present in Ui₂₂. To answer this question, the chemical characterization of nettle infusions should be addressed in the future, with the aim of finding specific bioactive compounds. Agronomic studies will also be needed to formulate a final product based on Ui and to verify its effectiveness in the field. In any case, this study opens the door to the use of nettle infusions as natural pesticides, replacing traditional products that are unhealthy for humans and animals, as well as environmentally unsustainable.