Selenium Nanoparticles (SeNPs) Inhibit the Growth and Proliferation of Reproductive Structures in Phytophthora capsici by Altering Cell Membrane Stability

Abstract

Selenium nanoparticles (SeNPs) are currently receiving attention for controlling plant pathogenic microorganisms, are expected to be especially effective against the genus Phytophthora, and show high anti-oomycete activity. SeNPs synthesized with plant extracts have shown low toxicity, high bioavailability, and mechanisms of action that alter cellular integrity and damage key components of phytopathogen metabolism, causing denaturation and cell death. The aim of this study was to evaluate the inhibitory activity of SeNPs on mycelial growth and the development of reproductive structures in Phytophthora capsici in vitro. Different concentrations of SeNPs (0 to 400 µg/mL) in culture media were used to analyze mycelial growth, sporangium formation, zoospores, and germination of the germ tube. To explain the changes in morphology and development of P. capsici, increased relative conductance and activation of glycerol synthesis were related to osmotic stress and damage to membrane permeability. In addition, SeNPs inhibited the production of exopolysaccharides (EPSs), which are compounds associated with pathogen virulence. A lower accumulation of its biomass evidences alterations in the oomycete growth. The percentage of inhibition of mycelial growth increased with higher SeNP concentrations and incubation time, reaching 100% growth inhibition at 300 and 400 µg/mL. A concentration-dependent reduction in the number of spores, sporangia, and zoospore germination was observed. Concentrations of 50 and 100 µg/mL of SeNPs reduced biomass production by 30%. The increase in glycerol levels indicated an osmoregulatory response to SeNP-induced stress. Also, the increase in electrical conductivity suggested plasma membrane damage, which supports the potential of SeNPs as antifungal agents by inducing cell disruption and structural damage in P. capsici. These results provide new knowledge on the in vitro mechanism of action of SeNPs against P. capsici and offer a new biological alternative for the control of diseases caused by oomycetes.

1. Introduction

Phytophthora capsici is a hemibiotrophic oomycete with heterothallic reproduction [1]; worldwide, it is considered a crop-destructive phytopathogen of vegetable, ornamental, and tropical crops of more than 15 plant families [2]. P. capsici is distributed in North America, most of South and Central America, and parts of Africa, Europe, Asia, and Australia [3], with temperatures of 25 to 28 °C and humidity conditions above 80% [4]. In its biotrophic form, its infection begins with the germination of sporangia and indirect germination from zoospores that attach to and enter epidermal cells or between host cells, reaching the cortex and colonizing the root. Once the hyphae enter the endodermis and vascular bundles, the pathogen becomes established, and necrotrophy forms [3]. Under field conditions, water contact with P. capsici favors its asexual reproduction by producing sporangia and flagellated motile zoospores [2]. Once zoospores come into contact, upon adherence with the plant, they encyst and germinate, forming germ tubes that enter the host directly with the help of effectors or through natural openings [5]. The hyphae grow within the tissue and form haustoria to obtain nutrients [6]. In the case of sexual reproduction, oospores develop inside the infested stems or fruits of host plants. In contrast, others remain in the soil and germinate when conditions are favorable for humidity and temperature, promoting germination [7]. Once introduced into crops, it spreads rapidly, making its eradication more difficult and causing yield losses of up to 100% depending on environmental conditions, ecotypes, and the amount of inoculum in the soil [3]. Due to its complex management, its biology and epidemiology have been studied [8], and different fungicides have been developed for its management and control [9]. However, the excessive use of these products has caused severe damage to human health and the ecosystem, in addition to the fact that the oomycete can develop mycobacterial resistance [10]. Therefore, there is a need to create new technologies that are more environmentally friendly, as well as integrated management plans to address their pathogenicity and resistance [4].

Recently, one of the most promising areas for researchers is the creation of biogenic nanoparticles as antifungal agents [11]. Ecological methods for synthesizing selenium nanoparticles (SeNPs) using plant extracts are simple, inexpensive, and environmentally friendly [12]. Compounds in plant extracts act as natural reductants and can function as capping agents, stabilizing the size of SeNPs and improving their biological properties [13]. For example, some phenolic compounds, such as gallic acid, thymol, tannins, flavonoids, and polyphenols, have been recognized to enhance the antifungal activity of SeNPs [14]. In addition, their high surface area-to-volume ratio and size allow them to cross the cell wall and cytoplasmic membrane of fungi easily [14].

One of the main mechanisms of action of SeNPs is to produce reactive oxygen species (ROS) inside fungal cells, creating the rupture of the cell wall, altering its permeability, inhibiting the synthesis of different proteins and lipids, damaging their metabolic cycles, and reducing gene expression, leading to cell death by the loss of vital structures and processes [15]. For example, SeNPs have been shown to alter the biosynthesis of the ergosterol pathway in the cell wall of Candida albicans, leading to loss of rigidity, homogeneity, and integrity of the plasma membrane [16]. Other fungi, such as Aspergillus niger and Fusarium oxysporum, showed changes in their morphology and alterations in their structures due to the effect of SeNPs synthesized with Elaeagnus indica leaf extracts [17]. In another study, SeNPs inhibited radial growth and sporulation of Penicillium digitatum [18]. Cell wall and membrane morphology can change in response to electrostatic attraction due to impaired permeability and functional disorders of energy metabolism, resulting in cell death [19]. The antifungal activity of NPs also depends on the structural variability of the morphology of each species of phytopathogenic fungus [20].

In studies with other NPs, for example, silver NPs (AgNPs), growth inhibition of P. capsici was reported with doses of 250 µg/mL [21]. Likewise, up to 92.6% inhibition was observed with 100 ppm in the in vitro assay and 95% in the reduction of infection in chili bell pepper plants, with the application of 10 ppm AgNPs on this same phytopathogen [22]. A similar effect was reported with copper NPs (CuNPs), where 10 ppm had a 72% inhibitory effect on the growth of P. capsici on the seventh day of incubation [23]. Silicon dioxide nanoparticles (SiONPs), coated with Cu and suspended in carboxymethylcellulose at concentrations of 75 to 125 ppm, inhibited 93% of P. capsici infection on Piper nigrum plants [24].

SeNPs offer several advantages over other NPs, unlike AgNPs, which can produce cytotoxicity at high concentrations. In addition, SeNPs have high adsorption capacity because their surface charges can be conjugated with negatively or positively charged groups [25]. Thus, SeNPs show higher bioavailability, bioactivity, dispersibility, and lower toxicity. However, SeNPs can cause toxicity in a concentration-, size-, and method-dependent manner, mainly where high doses, larger sizes, and chemical synthesis can cause adverse effects in living organisms [26]. Meanwhile, SeNPs obtained through green synthesis are safer in attenuating the generation of ROS under in vitro exposure to ionizing radiation in Saccharomyces cerevisiae cells [27]. Although there are studies on SeNPs, mediated by plant extracts, against pathogenic fungi of agricultural importance, there are few or no studies on their antifungal effect in species of the genus Phytophthora, specifically in P. capsici. Therefore, the objective of this study was to determine the inhibitory activity of SeNPs synthesized from extracts of Calendula officinalis on mycelial growth, development of reproductive structures, and cell membrane damage in Phytophthora capsici under in vitro conditions. These findings contribute to understanding the antimicrobial activity of SeNPs, offering possible solutions for combating diseases in crops affected by this oomycete.

2. Materials and Methods

To evaluate the in vitro antimicrobial potential of SeNPs, experimental assays were considered during the vegetative and reproductive stage of P. capsici, including the effect on oomycete mycelial growth, as well as on pathogenicity parameters (number of sporangia and zoospores, zoospore germination, and germ tube elongation) that are determinants in the infection of plants by P. capsici [28]. Exopolysaccharide (EPS) content was included to identify the role of SeNPs in pathogen virulence [29]. As a possible mechanism of action of SeNPs, damage to cell membrane permeability and the release of intracellular contents is proposed, for which electrical conductivity and glycerol content were measured [30].

2.1. SeNPs Preparation and Characterization

To obtain the SeNPs, the green synthesis followed the methodology described by Hernández-Díaz et al. [12] with slight modifications. A 10 mM sodium selenite (Na₂SeO₃) solution was prepared, and 10 mL was taken and placed into a 20 mL clear glass vial. Ascorbic acid (AsAc) was made at 40 mM. Methanolic extract of Calendula officinalis flowers (COF extract) was used at a 100 mg/mL concentration. The vial with Na₂SeO₃ was placed on a heating rack at 40 °C, and stirring was set to 950 rpm. Subsequently, 3.5 mL of AsAc was added, pouring 500 μL every 5 min. The COF extract was added in 20 μL increments every 2 min until a final volume of 120 μL was reached. The incorporation of AsAc and COF extract was alternated and aggregated depending on the time intervals. At the end of the approximately 35 min process, the vial was removed from the heat and agitation, covered with aluminum foil to protect it from light, and kept at 4 °C for 24 h to conclude the reaction. The resulting SeNPs were purified by filtration using 0.22 µm membranes.

The peak maximum absorbance was determined using the NanoDrop OneC micro-UV–vis spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). Dynamic light scattering (DLS) analysis was performed to obtain average hydrodynamic diameter, polydispersity (PdI), and zeta potential [31].

2.2. Molecular Identification of Oomycete Strain and Phylogenetic Analysis

DNA extraction was performed using the CTAB 2% method with five-day-old P. capsici growth cultures. The internal transcribed spacer region (ITS) of ribosomal DNA was amplified by primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATATGC-3′) [32]. The amplification reaction was performed using the GoTaq^® Green Master Mix, 2X kit (PROMEGA, Madison, WI, USA). The conditions for the PCR reactions were as follows: (1) an initial denaturation at 94 °C for 5 min; (2) 25 cycles at 94 °C for 30 s, 64 °C for 1 min, and 72 °C for 1 min; (3) a final extension at 72 °C for 10 min. PCR products were visualized by electrophoresis on 1.5% agarose gels and then observed in ultraviolet light with a photodocumenter (Universal Hood II, Bio-Rad, Hercules, CA, USA) in Quantify One software ver. 4.6.9. (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Once the amplified product bands were verified, the PCR samples were purified with the Wizard^® SV Gel and PCR Clean-Up System kit (PROMEGA, Madison, WI, USA), following the supplier’s instructions. The final amplified and purified PCR products were sequenced at the Laboratorio Nacional de Biotecnología Agrícola, Medica y Ambiental (LANBAMA/IPICyT) in San Luis Potosí, Mexico.

The sequences obtained were assembled with the Chromas Pro v1.5 program (Technelysium Pty Ltd., Tewantin, QLD, Australia). The taxonomic assignment was performed using the GenBank database’s BLAST algorithm. The phylogenetic tree was constructed using the neighbor-joining algorithm with 1000 replicates in MEGA version 11 (https://megasoftware.net/, accessed on 21 December 2024) (The Biodesign Institute, Tempe, AZ, USA), using the reported sequences of the Phytophthora species described in the clade. Pythium ultimum (JN695789.1) was used as an outgroup.

2.3. Growth Conditions of P. capsici in V8 Culture Medium with SeNPs

The strain molecularly identified as P. capsici from the strain collection at the Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ, Zapopan, Mexico) was used. The strain was reseeded with a sterile handle in Petri dishes in V8 solid culture medium (160 mL filtered V8 juice, 0.3 g CaCO₃, and 15 g agar per liter of medium) [33]. Then, it was placed in incubation under dark conditions at 25 °C until colonial growth was observed.

For the SeNPs assays, P. capsici was first seeded in a solid V8 medium and incubated for five days under the same conditions as previously described. Then, 10 mL of sterile distilled water was added to each plate with mycelial growth, and a triangular handle was used to dislodge the mycelium until a cloudy suspension was obtained gently. One mL of the suspension was taken, added to an Erlenmeyer flask containing 80 mL of V8 liquid medium (160 mL filtered V8 juice, 0.3 g CaCO₃, and 840 mL distilled water), and placed in a shaking incubator (LSI-3016R, LabTech, Daihan, Republic of Korea) at 150 rpm and 25 °C for two days. Previously filtered (0.22 µm syringe membranes) SeNPs were added under axenic conditions at final concentrations of 12.5, 50, 100, 200, 300, or 400 μg/mL and incubated for 7 days. Distilled water was used as a control (fungal growth without SeNPs), and the antifungal drug cycloheximide (400 µg/mL) was used as a positive control.

2.4. SeNPs Effect on Mycelial Growth

To evaluate the in vitro effect of SeNPs, assays were performed using the mycelial growth inhibition method for the phytopathogen, using the poisoned media technique, following the methodology of Lazcano-Ramírez et al. [34]. Petri dishes were prepared with sterilized V8 medium, and before solidification, different volumes of SeNPs or Na₂SeO₃ were added to a final concentration of 12.5, 50, 100, 200, 300, and 400 μg/mL in a 50 mL volume of V8 medium. A commercial antifungal control cycloheximide (CHX) (400 μg/mL) and absolute control (without SeNPs) were included. Two independent assays were performed, with four replicates each (one Petri dish consisted of one replicate). A five-day-old oomycete explant was placed in the center of each Petri dish, according to the inoculum conditions described in Section 2.3. The inoculated plates were incubated at room temperature and in darkness. The diameter of each colony, per SeNP concentration, was measured every day for seven days. The percentage of inhibition was determined according to the equation reported by Joshi et al. [35].

(1)$\%\mathrm{growth}\mathrm{inhibition}={\displaystyle \frac{\mathrm{Growth}\mathrm{in}\mathrm{the}\mathrm{control}-\mathrm{Growth}\mathrm{in}\mathrm{the}\mathrm{treatment}}{\mathrm{Growth}\mathrm{in}\mathrm{the}\mathrm{control}}}\times 100$

2.5. Sporangium Production and Zoospore Release Under SeNP Treatments

To induce the formation of P. capsici sporangia, 5 mm explants of the oomycete were seeded on Petri dishes in solid V8 medium, incubated at 25 °C for 5 days under dark conditions, and then transferred under continuous light for another 5 days. For sporangia harvesting, 10 mL of sterile distilled water was added to each plate, and a triangular spreader was used to rub the surface of the medium and gently wash the mycelium until it became cloudy. To carry out zoospore release, the freshly prepared plates were transferred to 4 °C for 30 min and then placed at room temperature under continuous light [36].

To determine the effect of SeNPs on sporangium formation and zoospore production, the concentrations that presented an inhibitory effect more significant than 50% on mycelial growth were selected (Section 2.3). V8 liquid culture medium was prepared with SeNPs at final concentrations of 0, 100, 200, 300, and 400 μg/mL [37]. Then, sporangia and zoospores were adjusted to a concentration of 1.0 × 10⁻⁴ and cultured in an equal volume of V8 medium and the different concentrations of SeNPs (1:1, v/v) in 2 mL tubes. The suspensions were mixed with an equal volume of distilled water for the control. All samples were homogenized manually. From each treatment, 100 µL was taken, and the total number of spores and sporangia was recorded in a Newbauer chamber and optical microscope (BH2-RFCA, Olympus, OM SYSTEM, Hachioji-City, Japan) at 24, 48, 72, 96, and 120 h. The total number of spores and sporangia were recorded at 24, 48, 72, 96, and 120 h. The count was repeated eight times per day. Counting was repeated eight times for each concentration. For the percentage of inhibition of spores and sporangia, the equation from Koka et al. [38] was used.

(2)$\%\mathrm{Spore}\mathrm{germination}\mathrm{inhibition}={\displaystyle \frac{\mathrm{Gc}-\mathrm{Gt}}{\mathrm{Gc}}}\times 100$

Gc and Gt represent the total number of spores or sporangia in the control and treatments.

2.6. Effect of SeNPs on Germ Tube Growth

Water agar culture medium was prepared at a concentration of 2% (w/v). SeNPs were added at final concentrations of 0, 100, 200, 300, and 400 μg/mL, similar to that mentioned in Section 2.5. Then, in the resulting medium, zoospores were added and cultured. From each zoospore suspension, 50 μL was taken and examined for germination. The germination tube length was measured after 1 and 5 h of incubation at room temperature using a fluorescence microscope (DMRA2, Leica Mycrosystems, Wetzlar, Germany) and a monochrome camera (Evolution QEI, MediaCybernetics, Rockville, MD, USA), with the use of Image Pro software (V2. 2., MediaCybernetics, Rockville, MD, USA). Measurements were repeated ten times for each concentration [36].

2.7. Effect of SeNPs on Biomass Content of P. capsici

The biomass content was determined based on the weight of mycelium obtained from cultures with SeNPs. The culture of P. capsici was similar to the steps described in Section 2.3. Samples per SeNP concentration were added in 50 mL tubes and then placed in a centrifuge (Multifuge X3R, Thermo Scientific, Austin, TX, USA) at 10,000 rpm for 20 min. The supernatant was removed from the samples and placed in a vacuum centrifuge concentrator (Vacufuge Plus Concentrator, Eppendorf North America, Inc. Framingham, MA, USA) for 3 h at 60 °C in AQ-mode. The weight of each pellet was recorded in triplicate for each treatment using an analytical balance (BP121S, Sartorius, Sigma Aldrich, St. Louis, MO, USA).

The treatment with 400 µg/L of SeNPs was discarded due to the lack of P. capsici growth in the biomass content, glycerol, exopolysaccharide, and membrane permeability.

2.8. Effect of SeNPs on Mycelial Glycerol Content

The determination of glycerol content was measured according to the cupric glycerinated colorimetry method described by Duan et al. [29]. To determine the glycerol content of the oomycete, the mycelium grown in the different concentrations of SeNPs was dried under vacuum for 3 h at 30 °C in AQ-mode until dryness. An amount of 0.05 g of the dehydrated mycelial tissue was taken and pulverized in an MM400 mixer mill (Retsch GmbH, Haan, Germany) at 25 Hz for 1 min. To each ground sample, 2 mL of sterile distilled water was added, heated at 80 °C for 15 min, and centrifuged at 9000 rpm for 10 min. The glycerol content was determined in the supernatants. Then, in a 2 mL tube, 500 µL of each sample was mixed with 50 µL of CuSO₄ (0.05 g/L) and 175 µL of NaOH (0.05 g/mL). The tubes were then shaken at 100 rpm for 12 min, and the samples were filtered through filter paper. The absorbance was measured with a microplate spectrophotometer (xMark™, Bio-Rad, Hercules, CA, USA) at 630 nm, the maximum absorbance frequency (Amax) of cupric glycerinate. According to the glycerol standard curve, the higher the absorbance, the higher the concentration.

2.9. Effect of SeNPs on Exopolysaccharide Content (EPS)

To ascertain the EPS content of P. capsici, liquid cultures with the concentrations of SeNPs described in Section 2.3 were centrifuged at 10,000 rpm for 20 min, and the supernatant was taken. The EPSs were precipitated from 1 mL of each supernatant using three volumes of absolute ethanol and were concentrated under a vacuum centrifuge concentrator (Vacufuge Plus Concentrator, Eppendorf North America, Inc. Framingham, MA, USA) for 6 h at 45 °C in AL-mode and 3 h in AQ-mode to dryness. The EPSs were dissolved in 8 mL of distilled water and quantified with the standard curve. The assay had three biological replicates and two technical replicates of each treatment.

To determine the EPS content, the modified phenol–sulfuric acid method was used [29]. An amount of 100 µL of each sample and 100 µL of phenol (5%) were added to a 2 mL tube and homogenized in a mixer (Vortex-Genie 2 Mixer, Scientific Industries, Bohemia, NY, USA). Subsequently, 500 µL of concentrated sulfuric acid (H₂SO₄) was added directly to the center of the liquid surface within 10 s, mixed gently, and left at room temperature until the reaction mixture cooled. The samples were then sealed with laboratory film and mixed using a vortex for 10 s. The absorbance of the reaction was measured on a microplate spectrophotometer (xMark™, Bio-Rad, Hercules, CA, USA) at 490 nm. Glucose was used to prepare the standard curve.

2.10. Effect of SeNPs on Cell Membrane Permeability

The culture of P. capsici was performed using the steps described in Section 2.3. Subsequently, the cultures with the oomycete grown under different concentrations of SeNPs were centrifuged, and the supernatant was removed. Per sample, 0.5 g of mycelium was taken, and 10 mL of distilled water was added. To evaluate the degree of leakage of cell contents through the cell membranes, after 180 min of incubation, electrical conductivity was measured with a conductivity meter (CONDUCTRONIC pH140, Puebla, Mexico). The samples were then autoclaved for 5 min, allowed to cool to room temperature, and the final conductivity was measured. Relative conductivity was calculated according to the equation from Wang et al. [30].

(3)$\mathrm{Relative}\mathrm{conductivity}(\%)={\displaystyle \frac{\mathrm{Initial}\mathrm{conductivity}}{\mathrm{Final}\mathrm{conductivity}}}\times 100$

2.11. Statistical Analysis

All experiments were performed with two independent assays in three replicates. To find out if there were significant differences in effects between SeNP concentrations on the oomycete and across incubation time, each experiment underwent a two-way analysis of variance (ANOVA) between treatments and days, or one-way to compare the different concentrations of SeNPs, as well as Fisher’s least significant difference (LSD) test in Minitab software (v.20, Minitab, LLC, State College, PA, USA).

3. Results

3.1. UV–Vis and Dynamic Light Scattering (DLS) of SeNPs

As part of the characterization of SeNPs, a peak absorbance maximum was found at a 289 nm UV–vis wavelength, a characteristic optical property of these NPs. The SeNPs showed an average hydrodynamic diameter of 87.70 nm, a polydispersity (PdI) of 0.168, and a zeta potential of −52.72 mV (Figure 1).

3.2. Phylogenetic Analysis

The strain used in this study was assigned to the code JAL-M60 and is part of the CIATEJ internal collection. Molecular identification was performed at the species level based on the amplification of the regions between primers ITS 1 and ITS 4. Strain JAL-M60 is grouped in the genus Phytophthora, Kingdom Stramenopila, Phylum Oomycota, Class Oomycetes, Order Peronosporales, and Family Pythiaceae. This strain is phylogenetically related to the species Phytophthora capsici with 100% similarity (Figure 2). The sequence obtained was deposited in GenBank with the accession number PQ796471.1.

3.3. SeNPs Inhibited Mycelial Growth in P. capsici

From the first day of the treatments with SeNPs, inhibition of the growth of the mycelium in P. capsici was observed, with a reduction of 7 to 73% of the diameter of the colony with SeNPs. In this work, the inhibitory effect of Na₂SeO₃, being the ionic precursor of SeNPs, was also compared, finding inhibition percentages of 18–85%, with concentrations of 12.5, 50, and 100 μg/mL, and 100% inhibition with 300 and 400 μg/mL. Cycloheximide (CHX) was included as a positive control. On the third day, changes in growth type and pathogen morphology were observed, with inhibition percentages of 1–64% with SeNPs and 15–82% with Na₂SeO₃, with a concentration range of 12.5 to 200 μg/mL. At the same time, 100% inhibition of mycelial growth was maintained with 300 and 400 μg/mL concentrations of both SeNPs and Na₂SeO₃ (Figure 3).

Mycelial growth was arrested on the sixth day with 6–62% inhibition with SeNPs and 28–85% with Na₂SeO₃, except the highest SeNP concentrations (300 and 400 μg/mL) that obtained 100% inhibition values from the beginning of the assay until the seventh day. However, with the 300 μg/mL concentration of Na₂SeO₃, slow and premature but continuous growth was observed from the fifth to the last day (Figure 3). The CHX control maintained a percentage of 100% until the fifth day, then decreased to 95% until the seventh day. SeNPs showed a higher inhibition rate at the highest concentrations tested, concerning Na₂SeO₃ (Figure 4).

For Fisher’s least significant difference (LSD) analysis and analysis of variance (ANOVA), differences were observed between treatments (p < 0.001) and between days (p < 0.001).

3.4. SeNPs on Sporangia Production and Zoospore Release

The total number of sporangia decreased, and the percentage of inhibition increased as the concentration of SeNPs rose from 0 to 400 µg/mL throughout the assay. The inhibition in sporangium production was variable, with percentages from 7 to 30% at 24 h of incubation with the different concentrations of the SeNPs and increased to the range of 25 to 50% at 120 h of treatments. However, Fisher’s LSD test indicated that there was no significant difference in the number of sporangia nor the percentage of inhibition at any concentration of SeNPs concerning the control (p ≥ 0.05) (Table 1).

In the case of zoospore production, the total number decreased, and the percentage of inhibition increased in a SeNP concentration-dependent manner. The inhibition in zoospore release was 11–21% at 24 h from the start of SeNP treatments and 12–43% with 120 h of SeNP exposure. Fisher’s LSD test showed significant differences in the number and percentage of spore inhibition in treatments with concentrations of 200, 300, and 400 µg/mL (p ≤ 0.05) (Table 1).

3.5. SeNPs on Germ Tube Growth

During the first hour of exposure to SeNPs, the zoospore germination inhibition percentages were 100% at 200, 300, and 400 µg/mL concentrations. While at the highest doses (300 and 400), the inhibition effect lasted until 5 h of treatment, where only spores with damage and 0% germination were observed, in comparison with the other SeNPs treatments that obtained between 89–93% inhibition, as compared to the control (Table 2, Figure 5). Likewise, Fisher’s LSD test showed that there were significant differences in all the concentrations used during the hours analyzed (p = 0.001).

3.6. Biomass Content

The biomass content in all treatments with SeNPs was lower than that of the control. The dry weight of the control biomass was 0.0742 g, whereas that of the CHX was 0.0519 g. The biomass remained from 0.045 to 0.048 g with the SeNPs, except for the treatment with 12.5 µg/mL, which had 0.0514 g (Figure 6). According to Fisher’s LSD test, all treatments were significantly different (p = 0.001).

3.7. Mycelial Glycerol Content

The glycerol content was 0.16 g/L in the control treatment without added SeNPs. At the same time, the glycerol content increased significantly in all treatments with SeNPs, except for the lowest dose (12.5 µg/mL), which was statistically similar to the control and CHX. However, the content decreased by 245 and 152% with the 200 and 300 µg/mL doses, respectively, concerning the highest glycerol content (100 µg/mL). Fisher’s LSD test showed that there were significant differences between treatments concerning the control (p ≤ 0.005) (Figure 7).

3.8. Exopolysaccharide Content (EPS)

On the fifth day, the EPS content for the control was 0.80 g/L, and for the antifungal cycloheximide 100 and 400 µg/mL, it was 0.78 and 0.71 g/L, respectively. In contrast, with the SeNPs treatments, values between 0.58 and 0.78 g/L were obtained. During the five days of recording, the EPS content with SeNPs was variable; however, a trend of decreasing EPSs was observed as the concentration of SeNPs increased over the days (Figure 8). Compared to the control, on day five, EPSs were reduced by 6, 21, 18, 24, and 27% in the treatments with 12.5, 50, 100, 200, and 300 µg/mL, respectively. Fisher’s LSD test showed that there were significant differences between treatments concerning the control (p = 0.001).

3.9. Cell Membrane Permeability

On day seven, after the treatments with SeNPs were started, there was an increase in the relative conductivity of 26–40%, compared to the control (17%), as the concentration increased in the treatments (12.5, 50, and 100 µg/mL). However, the relative conductivity decreased with the 200 and 300 µg/mL SeNPs treatments, whereas the antifungal had a 27% increase compared to the control (Figure 9). Fisher’s mean test analysis revealed significant differences between the treatments (p ≤ 0.001).

4. Discussion

SeNPs were previously characterized by Hernández-Díaz et al. [12], reporting a maximum UV–vis absorbance peak at 265 nm. In this work, the maximum absorbance was found at 289 nm, supporting the NPs formation. According to Abdelsalam et al. [31], SeNPs present characteristic peaks in the UV–vis wavelength region from 200 to 300 nm. Also, a size range of 40 to 60 nm and a spherical shape identified by transmission electron microscopy (TEM) were reported [12]. DLS analysis obtained a larger average size (87.70 nm), which could be since this size includes the water layer of the NPs, as has been reported in other SeNPs [27]. However, zeta potential values higher than ±30 mV indicate that nanomaterials are more stable in colloidal solutions, as in the case of these SeNPs (Figure 1). This could indicate that they exhibit lower aggregation due to higher electrostatic repulsion [27,31].

The different concentrations of SeNPs showed inhibitory activity on the filamentous vegetative growth stage (mycelial growth), spore germination, and reproductive cycle (the formation of sporangia and zoospores), which are fundamental in the pathogenicity and virulence of P. capsici. In all the assays, the percentage of mycelial growth inhibition increased as the concentration of SeNPs increased. These results agree with the findings reported by Nguyen et al. [36], who reported a similar effect in this oomycete with OCAgNPs (oligochitosan-coated silver nanoparticles) at 3 and 9 ppm (µg/mL). With SeNPs, the inhibition percentages ranged from 6 to 81% at all concentrations, except for the highest concentrations (300 and 400 µg/mL), which had 100% inhibition in the seven days of incubation (Figure 3). Similar behavior was reported with silver nanoparticles (AgNPs) synthesized with extracts of Artemisia absinthium, which had a 100% inhibitory effect on all life stages of P. capsici [28]. In another study with copper nanoparticles (CuNPs), a complete inhibition in the growth of this oomycete was reported on the second day after the beginning of treatments [23], or percentages of up to 93% with 125 ppm (µg/mL) on the third day [24].

When comparing the growth inhibition of P. capsici between SeNPs and Na₂SeO₃, it was found that concentrations of 25 µg/mL of Na₂SeO₃ inhibited the growth of the oomycete by over 50% from the third day of exposure. In contrast, SeNPs inhibited the growth of the oomycete by around 10% (Figure 3). This is because inorganic Se, particularly Na₂SeO₃ and selenate (Na₂SeO₄), have a higher bioavailability and solubility, causing a more significant toxic effect due to their oxidation state (+4 and +6, respectively) [26]. Unlike SeNPs, which result from a reduction process, elemental Se (Se⁰) predominates. Se⁰ is less toxic and has higher biological activity than the other forms of Se. The antimicrobial properties of Se⁰ in the form of SeNPs are due to its size and shape, and upon release, it interacts with cellular structures, triggering ROS formation and causing cell damage or death [39].

A study conducted with SeNPs against Fusarium mangifera also revealed a higher percentage of growth inhibition (48.31%) at a concentration of 300 μg/mL [40]. SeNPs have been reported to be antimicrobial agents against several pathogens of agricultural importance [11,20,40,41] because the ions released react upon contact with the thiol protein group (SH) or sulfhydryl group of the amino acids of the pathogen, which can affect cell permeability. This can damage the function of essential membrane proteins and generate oxidative stress as a consequence of the formation of superoxide radicals, followed by a malfunction in the mitochondrial membrane by increasing the levels of ROS, causing the alteration of cell shape, the absorption of nutrients from the environment, and severe damage to the DNA until cell death [20,39,41,42]. The results obtained in this work demonstrated the in vitro antifungal effect of SeNPs, providing an environmentally friendly and reliable alternative to chemical fungicides.

Specifically, studies with SeNPs have been effective at different applied concentrations and suppressing different diseases caused by pathogens in solanaceous crops. For example, early blight disease caused by Alternaria solani has been 100% inhibited with doses of 800 ppm (µg/mL) [43]. Likewise, an inhibitory effect was reported with doses of 50 and 100 ppm against the pathogens Colletotrichum capsici and A. solani on chili and tomato leaves, respectively [34]. In another investigation, a concentration of 100 ppm against Phytophthora infestans at the in vitro level affected its growth and spore production [44]. Also, the mechanism of SeNPs, derived from Trichoderma harzianum, where the effect of the various organic acids and their derivatives that coat them on their surface significantly reduced the production of fumonisin B1 (63%) and toxins of A. alternata, as well as the expression of key biosynthetic genes that are involved in the damage and oxidative stress of their structural components, was reported [45].

In this study, there was a reduction effect on the biomass content of P. capsici compared to the control, with a decrease of 30% on the seventh day of exposure with SeNPs (Figure 6). A similar reaction was observed with AgNPs in contact in a liquid medium with Colletotrichum gloeosporioides, where the dry weight of the mycelium was significantly different from the control. At the same time, as concentration increased, the inhibition of the growth and development of fungal hyphae became more significant [46]. On the other hand, a change in the coloration of the oomycete to intense red was clearly observed in the sedimented biomass of all treatments with SeNPs, compared to the control, which retained its white color. In the case of the treatments, this is likely to be due to the accumulation of selenium in the mycelial hyphae [47]. Accumulation may involve two modes: surface binding of nanoparticles to the cell wall of living and dead cells and uptake into the cell through the cell membrane only in living cells since this mode depends on energetic metabolism [48].

The intracellular glycerol content increased significantly, from 51 to 387%, compared to the control (Figure 7). This is consistent with its function in pathogen osmoregulation, which involves controlling the amount of water, ion concentration, and solute synthesis in response to osmotic stress. Glycerol accumulation favors oomycete resistance to SeNPs [49]. An interesting result to highlight is that 100 µg/mL of SeNPs significantly enhanced this effect compared to the commercial antifungal (cycloheximide). The results suggested that SeNPs play an important role in osmotic regulation and can induce damage to the cell membrane of P. capsici when the glycerol content accumulates on the cell membrane, providing resistance to osmotic stress caused by SeNPs.

Exopolysaccharides are produced as a protective mechanism when microbial cells are under environmental stress conditions and are essential in the life of the microorganism to carry out different chemical reactions and nutrient capture [50]. These macromolecules are also an indicator of the pathogenicity and virulence factors of fungi, since, when EPSs decrease, the ability of the oomycete to adhere to the host and to form biofilms is affected. In addition, the pathogen’s resistance to the host immune system decreases, which reduces its ability to cause disease and its degree of virulence [50]. In this work, the assays for determining the EPSs had a positive effect since the EPS content decreased to 6–27% concerning the control, as the concentration of SeNPs increased (Figure 8). These results were similar to those reported by Liu et al. [51], in which there was a reduction in the EPS content compared to the control when increasing the concentration of a flavonoid with an antifungal effect on P. capsici. Of particular interest, concentrations higher than 50 µg/mL were more efficient in reducing the EPS content than a two-fold concentration of cycloheximide (100 µg/mL). This proves that EPS production in this pathogen depends on gradual stress conditions in the culture medium [52].

The electrical conductivity values also increased in the range of 26 to 40% with increasing SeNP concentration (Figure 9), indicating ion discharge and increased membrane permeability in P. capsici, with these events being responsible for cell death at the highest doses tested (300 and 400 µg/mL). A similar effect was observed with turmeric oil, where glycerol and electrical conductivity increased significantly as the oil concentration increased, causing damage to the cell membrane of P. capsici [30]. It is possible that an elevated level of these factors causes a large influx of external water into the environment and leads the oomycete to cell expansion, rupture, and death [53]. On the other hand, the electrostatic interaction of the cationically charged molecules of SeNPs on the negatively charged components of the cell membrane triggers an osmotic imbalance, causing hydrolysis of cell wall peptidoglycans, releasing electrolytes and intracellular nutrients [54].

It should be noted that changes were observed in the morphology of P. capsici in the V8 medium poisoned with SeNPs, which could be because its cell wall is more complex, with respect to other phytopathogens, to the sensitivity of the oomycete on its the growth conditions and its resistance to the components of the growth medium, since, when in contact with SeNPs, it can produce extracellular enzymes that degrade and metabolize the substrate, which it converts into its food instead of inhibiting its growth. This phenomenon may occur when the SeNPs become non-toxic when in contact with the media [55].

In this study, the effect of SeNPs was observed by invading and altering the integrity of the cell membrane of P. capsici by endocytosis, causing a decrease in biomass and EPS content and an increase in glycerol content and electrical conductivity, leading to cell death and denaturation during its life cycle at the in vitro level. This could indicate that SeNPs have fungicidal potential against oomycetes.

5. Conclusions

SeNPs demonstrated strong antifungal activity against P. capsici, inhibiting its growth and development at various stages of its life cycle. Inhibition increased with SeNP concentration, reaching 100% at the highest doses tested (300 and 400 µg/mL). These results prove that efficient doses and incubation time influence the outcome of the maximal inhibition of P. capsici growth in vitro. This suggests that SeNPs have an antifungal potential for the control of diseases caused by this phytopathogen, causing a decrease in biomass and EPS content, as well as an increase in glycerol content and conductivity as part of its mechanism of damage to the cell membrane of P. capsici, causing its death. Therefore, their use can be recommended and sustainable, being a biologically synthesized product. However, additional studies under field conditions and at the level of plant–pathogen interaction are required to evaluate its effectiveness fully.

Author Contributions

Conceptualization, S.G.-M. and A.d.J.L.-G.; methodology, A.d.J.L.-G. and I.B.-M.; software, A.d.J.L.-G.; formal analysis, A.d.J.L.-G.; investigation, A.d.J.L.-G., J.A.Q.-Z., I.B.-M., M.I.M.-C. and S.G.-M.; resources, J.A.Q.-Z., I.B.-M., M.I.M.-C. and S.G.-M.; data curation, A.d.J.L.-G.; writing—original draft preparation, A.d.J.L.-G. and S.G.-M.; writing—review and editing, S.G.-M.; visualization, I.B.-M. and S.G.-M.; supervision, I.B.-M., J.A.Q.-Z., M.I.M.-C. and S.G.-M.; funding acquisition, S.G.-M. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data is contained within the article.

Acknowledgments

We thank CONAHCYT-Mexico for the PhD scholarship awarded to AJ López-Gervacio (grant 925124). Joaquín Alejandro Qui- Zapata and S García-Morales are members of the PLANTECC National Laboratory. M.C. Consuelo López Vielma, a member of Innovaciones Biotecnológicas de la Sierra de Occidente S.A. de C.V., is acknowledged for providing the purified P. capsici strain.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, the collection, analysis, or interpretation of data, the writing of the manuscript, or the decision to publish the results.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. Characteristics of SeNPs. (a) UV–vis spectra of SeNPs; (b) size distribution of SeNP; and (c) zeta potential.

Figure 2. Phylogenetic neighbor-joining tree of the species of the genus Phytophthora reported in the GenBank, with the strain JAL-M60. The sequence of Pythium ultimum was used as an outgroup.

Figure 3. Percent inhibition of mycelial growth in P. capsici in media amended with SeNPs (a) or Na2SeO3 (b) for 7 days. CHX = cycloheximide: 400 µg/mL. Means that do not share a letter indicate significant differences, based on Fisher’s LSD test at a 95% confidence level.

Figure 4. Effect of different concentrations of SeNPs (a) and Na2SeO3 (b) on the growth of the Phytophthora capsici’s colonial mycelium at day 7 after the initiation of treatments. CHX = cycloheximide (400 µg/mL).

Figure 5. Effect of SeNPs at different concentrations on the germ tube length of the zoospores in P. capsici at 5 h. (A) = Control 0 µg/mL, (B) = 100 µg/mL, (C) = 200 µg/mL, (D) = 300 µg/mL, and (E) = 400 µg/mL. Scale = 10 µm. The yellow letters indicate the size of the germ tube length (µm).

Figure 6. Effect of different concentrations of SeNPs on biomass production in P. capsici, seven days after exposure with SeNPs. CHX = Cycloheximide, 400 µg/mL. Letters indicate differences between treatment means, based on Fisher’s LSD test (95% confidence level).

Figure 7. Effect of different concentrations of SeNPs on the mycelial glycerol content in P. capsici, with seven days of exposure. CHX = cycloheximide, 400 µg/mL. The different letters indicate the differences between the treatment means, based on Fisher’s LSD test (95% confidence level).

Figure 8. Effect of different concentrations of SeNPs on exopolysaccharide (EPS) content during 5 days with treatments. CHX = Cycloheximide, 400 µg/mL. Letters indicate differences between treatment means per day, based on Fisher’s LSD test (95% confidence level).

Figure 9. Effect of different concentrations of SeNPs on ion release in the cell membrane of P. capsici for 7 days. CHX = Cycloheximide, 400 µg/mL. The letters indicate differences between treatment means, based on Fisher’s LSD test (95% confidence level).

Table 1

Effect of SeNPs on the total number and percentage of inhibition of the sporangia and zoospores at 120 h.

SeNPs (µg/mL)	TNS (mL)	IS (%)	p-Value (TNS)	TNE (mL)	EI (%)	p-Value (TNE)
0	2.5 × 103 a ± 2315	-	-	1.2 × 104 a ± 2086	-	-
100	1.8 × 103 a ± 1768	25	0.522	1.1 × 104 ab ± 1336	12	0.161
200	1.8 × 103 a ± 2216	25	0.522	1.0 × 104 bc ± 2086	19	0.028
300	1.5 × 103 a ± 1860	37.5	0.338	8.7 × 103 cd ± 2673	31	0.001
400	1.2 × 103 a ± 1336	50	0.204	7.1 × 103 d ± 2478	43	0.001

TNS = total number of sporangia, TNE = total number of spores, IS = inhibition of sporangia, and EI = spore inhibition. Means that do not share a letter indicate significant differences, based on Fisher’s LSD test at the 95% confidence level. ± means standard deviation.

Table 2

Germ tube length for 1 and 5 h under different concentrations of SeNPs.

SeNPs (µg/mL)	GTL (µm) 1 h	IP (%)	GTL (µm) 5 h	IP (%)
0	31.54 a ± 15.44	-	189.4 a ± 41.3	-
100	6.344 b ± 0.645	79.8	19.39 b ± 8.12	89.7
200	0 c ± 0	100	13b c ± 5.28	93.1
300	0 c ± 0	100	0 c ± 0	100
400	0 c ± 0	100	0 c ± 0	100

GTL = germ tube length, IP = inhibition percentage, ± = standard error. Treatment means were compared using Fisher’s LSD test at 95% confidence level (p ≤ 0.05).

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