Antiviral Activity of TiO₂ NPs against Tobacco Mosaic Virus in Chili Pepper (Capsicum annuum L.)

Abstract

Tobacco mosaic virus is the etiological agent of one of the most critical diseases limiting chili pepper production. Various practices have been used to manage the disease, e.g., the use of resistant varieties and interference with the vector through chemical control. However, these practices are not helpful once the virus has been established in the plant. There is still no effective method for the sustainable management of the disease; therefore, exploring new options is required. Currently, some studies have reported the activity of TiO₂ NPs against viruses in plants, although not against TMV in chili pepper. The present work aims to determine a possible direct action of TiO₂ NPs against TMV and if there is a relationship between the amount of virus and symptoms. The application of TiO₂ NPs at 150 μg/mL in infected pepper plants reduced symptoms and viral load and improved the morphological characteristics compared to the control. Incubation of 150 µg/mL TiO₂ NPs with the virus for 6 and 8 h before infection decreased viral concentration significantly after infection compared to the control. In this work, it is reported, for the first time, that the use of TiO₂ NPs is a novel practice for the control of TMV in chili pepper.

1. Introduction

Nanotechnology has received significant attention in developing particles with dimensions less than <100 nm [1]. Their physical and chemical properties depend on the high area/volume relationship. The smaller they are, the greater the surface area, presenting more significant activity compared to their bulk materials [2]. In agriculture, at specific doses, they have been used in a promising way in disease management and the improvement of plant health. Therefore, the application of nanotechnology can contribute to guaranteeing global food security [3,4]. Studies have highlighted the potential applications of metal oxide NPs as elicitors or biostimulants, improving the nutraceutical and nutritional quality of plants [5]. On the other hand, they also have been shown to be a promising practice for integrated strategy disease management due to their antimicrobial, antifungal, and antiviral activity against different types of these histological agents. Tobacco mosaic virus (TMV) belongs to the Tobamovirus genus [6,7]. TMV disease is one of the most critical limitations in the production of chili crops. TMV are viruses with a size of 300 x 15 nm, which can encode for four proteins: the 126 and 183 kDa replication-associated proteins, the structural capsid or coat protein, and movement protein [8]. This virus in plants causes mosaic, mottling, necrosis, chlorosis, and leaf distortion in plants such as Capsicum annuum, Solanum lycopersicum, and Nicotiana tabacum virus [9]. Currently, various chemical compounds (ex. ribavirin and oseltamivir) [10,11] and biological compounds (ex. seco-pregnane steroids, ningnanmycin, tagitinin C (Ses-2), and 1β-methoxydiversifolin-3-0-methyl ether (Ses-5)) limit the infection and proliferation of the TMV [12]. However, most of these strategies are established in plants cultivated under in vitro conditions since they are not very effective once the virus has been established in plants cultivated in the open field.

Considering the above, it is appropriate to develop new practices focused on reinforcing the integrated management of TMV diseases. The scientific references for incorporating NPs into TMV management practices have reported evidence that these compounds possess antiviral activity against TMV. For example, chitosan Schiff–base nano-silver (S-cos-Ag) has been reported to prevent and treat the TMV virus and reduce the number of lesions in TMV-infected tobacco leaves, showing a degree of control of up to 78% [7]. On the other hand, pretreatment of ZnO NPs and SiO₂ NPs exhibited substantial aggregation of TMV particles and lysis in vitro, and inoculated tobacco plants showed significantly lower virus colonization [6]. TiO₂ NPs are very popular since they are produced on a large scale due to their properties, including low toxicity and a wide range of applications in the industry. Although there are no reports of the antiviral activity of this type of nanoparticles against TMV, they have been found to have antiviral activity against the virus as the turnip mosaic virus (TuMV) in Nicotiana bethamiana plants [13] and broad bean stain virus in Vicia faba L. [14]. The antiviral activity of TiO₂ NPs against TMV in chili pepper is currently unknown. Considering that ZnO NPs and SiO₂ NPs may be feasible practices for managing the disease caused by TMV in a solanaceous such as tobacco, the purpose of this work is to expand the options for the integrated management of this pathogen in another solanaceous, namely, chili. It is also desirable to determine if there is a direct action of TiO₂ NPs on the virus and if there is a relationship between the amount of virus and symptoms.

2. Materials and Methods

2.1. Plant Establishment

The plant material used in the experiment was a variety of Jalapeño Don Pancho pepper obtained from Mexico’s National Institute for Forestry, Agriculture, and Livestock Research (INIFAP). The plants were germinated and transplanted when they had 6 to 8 true leaves in 9 L bags with tezontle substrate, humus, and soil where chili is usually grown, in a 2:2:5 ratio. Plant nutrition consisted of a 90% Stainer solution. The experiment was carried out in a greenhouse where the average temperature was 21.3 °C and with an RH% of 59%.

2.2. Experimental Design

The experimental design arrangement was in randomized blocks with seven plants per experimental unit, five treatments, and seven replicates. The concentrations of TiO₂ NPs evaluated were 50, 100, 150, and 200 µg/mL. The application of the TiO₂ NPs was carried out at 72, 144, and 216 h after the plants had been inoculated. The negative control was water, and the positive control was the plants infected with the TMV virus.

2.3. Synthesis and Characterization of TiO₂ Nanoparticles

Titanium dioxide nanoparticles were synthesized using the sol–gel method, in which 5 mL of titanium isopropoxide (97%) and 100 mL of 2-propanol (99.5%) were placed in a beaker with slight agitation. Subsequently, 30 mL of deionized water was added to the solution, and the mixture was kept in reaction with slow stirring for 24 h. After this, the solid was recovered by filtration and washed several times with deionized water. The solid was dry at room temperature and was finally subjected to muffle drying at 110 °C for 18 h and calcined at 500 °C for 4 h [15]. The characterization of the nanoparticles was carried out with a HITACHI model SU8230 High-Resolution Scanning Electron Microscope at the Center for Applied Physics and Advanced Technology (CFATA) of the UNAM-Juriquilla to determine the morphology of the material obtained and the size of the nanoparticles. In addition, the crystalline structure of the material from 20 to 80° 2θ was analyzed through X-ray diffraction using Cu Kα radiation at a wavelength of 1.54 Ǻ with a Bruker model Advance D8 instrument at the facilities of the Autonomous University of Queretaro, Campus Aeropuerto. Additionally, the diffraction pattern was refined to determine the average crystal size and the weight concentration of the phases found.

2.4. TMV Inoculation in Capsicum annuum L. Plants

The TMV virus was provided by the Autonomous University of Mexico (UNAM) Campus Iztacala. A 1:10 solution of infected material and extraction buffer (GEB) from the Agdia DAS-ELISA kit was prepared. When the plants presented six true leaves, the virus was inoculated mechanically with a swab impregnated with the viral solution and Carburundum 100.

2.5. Measurement of Morphological Variables and Evaluation of Symptoms

The following morphological variables were measured: plant height, stem diameter, number of leaves, flowers, flower buds, and fruits. For the evaluation of the symptoms, the severity scale reported by Torres-Pacheco [16] was used. This scale was adapted to TMV symptoms and includes values between 1 and 5 (

Table 1. Symptoms scale in infected plants proposed by Torres-Pacheco [16].

Symptom Description Level	Category	Percentage in Leaf (%)
A few apical wrinkles Small yellow spots were approximately 1 mm in diameter, only visible with light exposure	1	0–10
Light yellow spots on apical leaves were clearly visible	2	10–20
These spots appeared to form networks primarily at the base of the apical leaves	3	20–30
Evident network and little protuberances	4	30–40
The protuberance rolled up the leaves and necrosis	5	40–50

).

2.6. Immunoassay DAS-ELISA

Leaves were collected 20 days after virus inoculation and frozen with liquid nitrogen. The leaves were taken at the base of the plant and the apical part to determine the virus’s movement. Viral load was performed with the Agdia DAS-ELISA PathoScreen Kit according to the manufacturer’s instructions [17]. Color measurement was performed spectrophotometrically using a Thermo Scientific MULTISKAN GO spectrophotometer model SN 1510-02385C at 405 nm.

2.7. Reactivity Determination

The reactivity test was carried out to determine the direct effect of the TiO₂ NPs on the virus. To carry out this test, the following treatments were applied: (1) water control (without NPs, without viruses); (2) control NPs (TiO₂ NPs 150 μg/mL); (3) virus control (only viruses without TiO₂ NPs); (4) treatment (TiO₂ NPs 150 μg/mL with the virus). The viral extract was mixed with the solution with TiO₂ NPs 150 µg/mL TMV extract and allowed to incubate for 6 h; then, the pepper plants were inoculated. The next stage involved incubating the mixture of TiO₂ NPs at 150 µg/mL and TMV extract for 12 h and inoculating the pepper plants. The treatments were then inoculated in plants with 6–8 leaves. Viral quantification was performed at 48, 96, 144, and 192 h inoculation with the Agdia DAS-ELISA Reagent Set Kit [17]. Five viral load measurements were taken every 48 h to see how the viral load evolved.

2.8. Hydrogen Peroxide Determination

The content of hydrogen peroxide in the leaves of the infected plants treated with the NPs was determined using the technique of Junglee et al. [18]. The method was based on the colorimetric determination of the hydrogen peroxide content in plants using potassium iodide. A total of 100 mg of sample was homogenized with 0.25 mL of 10 mM K/Na-phosphate buffer (pH 5.8), 0.25 mL of 0.1% trichloroacetic acid, and 0.5 mL of 1 M KI. The supernatant obtained was centrifuged at 4 °C, for 15 min, at 16,000× g and kept for 20 min in the dark. The samples were measured at 350 nm. Reported units were nanomoles of peroxide per milligram of protein.

2.9. Enzymatic Activity

For the extracts, 300 mg of the samples were weighed, and 2 mL of 0.05 M potassium phosphate extraction buffer, pH 7.8, at 4 °C was added. It was made up to 250 mL with distilled water to be later centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was removed and stored at 4 °C for later use. The amount of protein was determined according to the methodology reported by Bradford [19], using bovine serum albumin as a standard for protein concentration (Sigma-Aldrich, St. Louis, MO, USA).

2.9.1. Phenylalanine Ammonia-Lyase (PAL) Activity

PAL activity was performed according to the methodology reported by Toscano et al. [20] with modifications. Briefly, 1.15 mL of borate buffer (pH 8.8 containing 10 mM L-phenylalanine) was placed, 100 µL of enzyme extract was added, and the mixture was calibrated to 2 mL. The mixture was incubated for 60 min in a water bath at 40 °C. After 60 min, 0.5 mL of 1N HCl was added (to stop the reaction), and the mixture was allowed to stand for 10 min. The calibration curve consisted of a cinnamic acid solution of 2–12 µg cinnamic acid/ml. Absorbance at 290 nm was measured on a MULTISKAN GO, Thermo Scientific spectrophotometer, SN 1510-02385C. The blank was made with 1.5 mL of the reaction buffer solution of borate of 0.1 M and L-phenylalanine of 10 mM, pH 8.8. PAL activity was expressed as µg cinnamic acid/mL produced under the specific conditions and expressed as U/mg protein content.

2.9.2. Catalase (CAT) Activity Assay

Spectrophotometric determination of CAT activity was performed according to the method reported by Afiyanti and Chen [21] with modifications. Briefly, for the assay, 2 mL of reaction buffer (50 mM potassium phosphate buffer, pH 8.0), 0.2 mL of H₂O₂, and 0.1 mL of enzyme extract were placed directly in the quartz cell. The mixture was stirred, and the change in absorbance at λ 240 nm was measured instantly for 6 min (one reading every minute) in a MULTISKAN GO Thermo Scientific spectrophotometer, SN 1510-02385C. The change in absorbance at 240 nm was measured for 1 min and used to determine the rate of decomposition of H₂O₂ by CAT; 1 U CAT decomposes 1 mmol H₂O₂/min at pH 8.0 and 25 °C. Data are expressed as U/mg protein.

2.9.3. Superoxide Dismutase (SOD) Activity Assay

SOD activity was performed by nitro blue tetrazolium (NBT) photochemical reduction inhibition according to the method reported by Hayat et al. [22]. Briefly, 1.5 mL of reaction buffer (50 mM potassium phosphate buffer, pH 7.8), 0.3 mL of 0.1 mM EDTA-Na₂, 0.3 mL of 0.13 M methionine, 0.3 mL of 0.75 mM NBT, 0.3 mL of 0.03 mM riboflavin, 0.05 mL of enzyme extract, and 0.25 mL of distilled water was mixed by inverting clear glass tubes. The mixture was exposed to sunlight for 30 min, waiting for the development of a blue color. The blank was made with the previous solutions replacing the enzyme extract with distilled water. Absorbance at λ560 nm was measured on a MULTISKAN GO Thermo Scientific spectrophotometer. A total of 1 U SOD inhibited the reduction of NBT by 50% at pH 7.8 and 25 °C. Data are expressed as U/mg protein.

2.9.4. Peroxidase Activity (POD)

The spectrophotometric determination of POD activity was performed with the Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit from Invitrogen. The methodology suggested by the brand was followed. A total of 1.1 μL of the enzymatic extract and 48.9 μL of the reaction buffer were added directly to the microplate, 50 μL of the 100 μM Amplex^® Red–2 mM H₂O₂ solution was added, and the mixture was incubated in the dark for 30 min. The absorbance was measured at 560 nm. The negative control consisted of 50 µL of 1X reaction buffer, and the positive control consisted of 50 µL of 2 mU/mL HRP.

2.10. Antioxidant Activity

The determination of antioxidant activity in each treatment was evaluated through 1,1-diphenyl-2-picrylhydrazyl (DPPH•−) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+). The methanolic extracts were prepared by adding 9 mg of sample to 15 mL of methanol, and the mixture was kept under stirring for 24 h.

2.10.1. DPPH• Radical Scavenging Activity (DRSA)

DRSA was determined according to the method of Chen et al. [23] with modifications. A total of 0.5 mL of the methanolic extract and 0.5 mL of the 0.1 mM DPPH• radical were mixed (3.94 mg were weighed and made up to 100 mL with methanol). The mixture was shaken vigorously and allowed to incubate in the dark for 30 min at room temperature. Finally, the absorbance was taken at a wavelength of 515 nm; the reaction blank was methanol, and the control was distilled water. %DRSA was calculated by the following equation:

%DRSA = [1 − (A_sample − A_blank)/A_control)] × 100

(1)

2.10.2. ABTS •+ Radical Scavenging Activity (ARSA)

The determination of ARSA was carried out according to the method of Re et al. [24] with modifications. The ABTS•+ radical was generated by mixing the 7 mmol/L ABTS stock solution with 2.45 mmol/L potassium persulfate under darkness (ABTS•+ radical generation) 12 h prior to analysis. The ABTS•+ solution was then diluted in phosphate-buffered saline (PBS) at 0.15 M, pH 7.4, until a reading of 0.7 Abs was obtained at a wavelength of 734 nm. Subsequently, 3 mL of this final solution was mixed with 150 µL of the methanolic extract in a glass cell; finally, the change in absorbance at a wavelength of 734 nm was measured every minute for 6 min. The reaction blank used was PBS, and the control was distilled water. %ARSA was calculated by the following equation:

%ARSA = [1 − (A_sample − A_blank)/A_control)] × 100

(2)

2.11. Statistical Analysis

Data were subjected to analysis of variance (ANOVA) and Tukey test (α = 0.05) using the Statgraphics Centurion XVI statistical software (StatPoint Technologies, Bedford, MA, USA).

3. Results

3.1. Characterization of TiO₂ Nanoparticles

The characterization of the morphology of the synthesized TiO₂ nanoparticles was carried out by scanning electron microscopy (SEM). The formed nanoparticles appeared hemispherical with an approximate size of 20 nm, according to Figure 1. The nanoparticles aggregated in irregular clusters of approximately 0.6 microns, forming interstitial voids (Figure 1a,b). The obtained images made it possible to identify a uniform size of the nanoparticles formed, which can benefit the spraying process of the material and enhance its interaction with plant cells. On the other hand, the X-ray diffraction study was used to determine the crystalline phase corresponding to the prepared TiO₂ nanoparticles. A combination of polymorphs of the anatase phase was identified, with a minor brookite phase, as observed in the diffraction pattern obtained in Figure 2. In addition, a quantitative analysis was performed using the Rietveld method with the GSAS-II software, and it was found that the weight percentage of the polymorphs corresponded to 91% for anatase and 9% for brookite, with average crystal sizes of 11.6 nm and 13.5 nm, respectively. Figure 1c,d shows the accumulation and distribution of TiO₂ nanoparticles as black particles within plant cells, including chloroplasts.

3.2. Evaluation of the Morphological Characteristics and Symptomatology Scale of the Plants Infected and Treated with TiO₂ NPs

Concentrations of TiO₂ NPs from 50 to 250 μg/mL were evaluated to determine the impact of TiO₂ NPs on some morphological characteristics of pepper plants infected with TMV, and height, diameter, and symptom scale were measured (

Table 2. Morphological characteristics and symptom level in plants infected with TMV and treated with TiO₂ NPs.

Treatment	High (mm)	Diameter (mm)	Disease Symptoms
Water	21.0 b	4.3 a	0.88 d
TiO2 150 μg/mL	20.2 b	3.7 b	1.00 d
TMV	13.7 c	3.02 c	3.65 a
TMV + TiO2 50 μg/mL	22.2 b	4.1 ab	3.50 ab
TMV + TiO2 100 μg/mL	22.6 b	4.0 ab	3.58 a
TMV + TiO2 150 μg/mL	30.4 a	4.5 a	2.10 c
TMV + TiO2 200 μg/mL	21.4 b	3.7 b	2.1 b

The letters indicate the significant difference between treatments (α = 0.05).

). The application of TiO₂ NPs seemed to increase the height in infected plants by 122% compared to plants infected with TMV without treatment. Likewise, compared to the control (water), there was no significant difference. Once again, the application of TiO₂ increased the diameter in infected plants, with the treatment of TiO₂ 150 μg/mL becoming significantly equal to the control. Plants infected with TMV showed symptoms from day 9 after inoculation. After 20 days, the disease’s symptomatology level was measured according to the health scale proposed by Torres-Pacheco [16] (Table 1). The treatments that decreased the symptoms caused by the virus were the highest concentrations of TiO₂ NPs, 150 μg/mL and 250 μg/mL, since they did not present severe symptoms such as the mosaic pattern, deformed leaves, and curling. Although the best treatment was 150 μg/mL, the application of the NPs cannot completely suppress the symptoms at the same level shown by the control plant sprayed with water (Figure 1). In general, for this study, the results show that the treatment of 150 μg/mL improved the antiviral activity, reducing the appearance of the symptoms caused by TMV in pepper plants. In addition, as shown in Figure 3, this antiviral effect of TiO₂ in TMV-infected plants was maintained in the most advanced stages of vegetative development. Figure 4 shows the severe symptoms of the virus in plants infected with TMV and plants with TMV treated with TiO₂ NPs 150 μg/mL.

3.3. TMV Detection and Reactivity Test

According to Figure 5a, TiO₂ NPs showed a decrease in the virus concentration in the leaves as the nanoparticle concentration increased. The concentrations that showed the least amount of the virus were 150 and 200 μg/mL, reducing it by approximately 90%. From the results obtained above, the concentration of TiO₂ at 150 μg/mL was chosen to carry out the reactivity test to observe the direct antiviral activity with the virus. For this, the nanoparticles were incubated with the TMV virus for 6 and 12 h before infecting the plant. Once the virus was incubated with the NPs, it was observed that viral replication and proliferation were significantly inhibited at 2, 4, 6, and 8 days later in the plant compared to plants infected without treatment.

The invasion of pathogens causes defense responses in plants associated with the generation of ion fluxes; the induction of kinase cascades and nitric oxide (NO); and the accumulation of ROS, and among the most crucial molecules, H₂O₂. The accumulation of H₂O₂ was detected to understand the role of ROS in resistance against TMV. As shown in Figure 6, TMV induced hydrogen peroxide production while the application of TiO₂ NPs in infected and uninfected plants showed a lower concentration, indicating that the nanoparticle can reduce reactive oxygen species (ROS) accumulation in leaves caused by TMV, thus reducing oxidative damage in the plant.

3.4. Enzymatic Activity

CAT, SOD, and POD are related enzymes in cellular ROS homeostasis. Antioxidant activity was evaluated in leaves treated with TiO₂ NPs in healthy and TMV-infected plants. As shown in Figure 6, TMV infection induced SOD synthesis in plants with and without NP treatment, being higher in plants that did not have the nanoparticles. For those plants not infected, both the control and those treated with TiO₂ NPs did not show the synthesis of this enzyme. As far as POD is concerned, nanoparticle treatments significantly decreased the effect of the enzyme compared to control and infected plants.

In contrast, the activity of the CAT enzyme increased in the plants sprayed with metal oxide nanoparticles, both in the infected and non-infected plants. The foregoing shows that TiO₂ NPs enhanced disease tobacco resistance by setting their defensive enzyme activity differently. On the other hand, the total protein content was decreased in plants infected with TMV. NPs also can increase PAL activity, an enzyme responsible for promoting phenylalanine metabolism substances. PAL is related to plant resistance to pathogens, including viruses.

3.5. Antioxidant Activity

Finally, even though PAL is strongly related to antioxidant activity since it induces the synthesis of bioactives such as phenols and flavonoids, no differences in antioxidant activity were observed by the DPPH• and ABTS•+ methods of the different treatments, even though TiO₂ 150 μg/mL induced the expression of this enzyme (data not shown).

4. Discussion

Viral infections in plants induce a hypersensitive response associated with the accumulation of ROS and changes in different oxidoreductase enzymes [25]. In this work, TMV infection in chili plants not treated with TiO₂ NPs accumulated H₂O₂, which is the molecule responsible for initiating the defense process and central cellular signaling molecules [26]. Moreover, the H₂O₂ generation was followed by increases in the activities of antioxidant enzymes such as SOD (Figure 6). However, it is notable that the defense response of the pepper plants did not appear or was insufficient to contend against the effects of the virus; that is, it did not reduce the symptoms in plants (Figure 3). In other words, applying TiO₂ NPs was needed to observe a significant decrease in the syndrome. These results agree with those reported by Bhatia et al. [27], wherein the authors indicated that in tobacco plants infected with TMV, the superoxide dismutase and peroxidase increased while the activity of catalase decreased. Other reports indicated that CAT does not increase in the presence of TMV and may even decrease its activity [28], which correlates with our results. This is attributed to the fact that the production of salicylic acid (SA) as a defense response inhibits CAT, leading to the accumulation of H₂O₂, and this then switches on the expression of defense genes and activation of systemic acquired resistance (SAR) [29]. Moreover, other workers reported that TiO₂ NP 150 μg/mL has the ability to induce the CAT enzyme in plants with or without virus infection; this enzyme plays an essential role in the rapid removal of ROS [26]. Therefore, in this work, this enzyme was probably responsible for maintaining H₂O₂ levels at a level similar to the control treated with water.

In general, for this research, the result suggests that the treatment of 150 μg/mL improved the antiviral activity, reducing the appearance of the symptoms caused by TMV in pepper plants. In addition, as shown in Figure 3, this antiviral effect of TiO₂ in TMV-infected plants was maintained in the most advanced stages of vegetative development. This work also suggests that the TiO₂ NPs inhibited viral symptoms, which may have been due to a direct action on viral proliferation and was reflected in a decrease in the concentration of viral load (Figure 5a) and correlated with the low level of severe symptoms in plants infected with TMV, such as yellow spots, as well as rolling and protuberance characteristics of the presence of the virus (Figure 3).

According to the reactivity test (Figure 5b), the best treatment was the incubation of TMV with TiO₂ 150 μg/mL for 8 h, since it showed a lower concentration of the virus. However, foliar application of the NPs post-infection at 72, 144, and 216 h had a greater decrease in the amount of virus than the treatments in the reactivity test. This may have been because, as observed in Figure 1c,d, the metal nanoparticles can accumulate and distribute themselves in the cells of the chili pepper leaves, which can facilitate their interaction with viruses.

The reactivity test also shows that TMV-infected plants increased virus concentration over time (2–8 days). However, plants infected with viruses in which the viruses were incubated with 150 μg/mL TiO₂ nanoparticles for 6 and 8 h showed no increase. However, in pepper plants infected with TMV, the induction of H₂O₂, SOD, and POD failed to suppress the virus infection; the application of TiO₂ in infected plants induced the activity of these in comparison with the control (Figure 6). This suggests that the inhibitory effect of the symptoms was mainly due to the direct action of the TiO₂ nanoparticles on the virus and, to a lesser extent, to the induction of defense responses in the plant. That is, in the reactivity test, the incubation of the viruses with TiO₂ nanoparticles repressed the multiplication of the viruses but it remained at low levels over time, probably due to the cooperation that occurred due to the change in the antioxidant response caused by the variability of H₂O₂, SOD, and CAT induced by NPs.

Although there were results on the possible action effect of TiO₂ NPs on the TMV virus, more research is required to determine if the compound block viral replication, protein synthesis, or the assembly process. It may be that the effect of TiO₂ nanoparticles has a more general character of action on viruses, which implies an interesting challenge to determine the generic element of the mechanism of action. For example, it has been reported that in the influenza virus (H3N2), TiO₂ NPs destroy the virion envelope and cause aggregation of viral particles [30]. In plant studies, only data have been reported suggesting that the effect of TiO₂ NPs against turnip mosaic virus infection in tobacco (Nicotiana benthamiana) is very effective in limiting viral infection and inhibiting viral replication in plant cells [13]. On the other hand, TiO₂ NPs also reduce the disease severity of the broad bean stain virus (BBSV) in Vicia faba L. plants [14]. Regarding the antiviral action of other metallic nanoparticles on the TMV virus, studies have only been reported on Nicotiana benthamiana with the use of Schiff base nanosilver NPs [7] and zinc oxide NPs [6], which reduced the invasion and damage by TMV. The above information suggests that the foliar application of TiO₂ NPs may be incorporated as a practice on TMV-integrated disease management because it has a protective effect once the virus has established itself.

In addition, our dates also suggest a possible biostimulant activity that can influence in a certain way to inhibit the symptoms and viral load and increase the defense of chili pepper against TMV. The treatment with TiO₂ showed behavior in the morphological variables similar to that of the uninfected plants. The above because infected and non-infected plants, to which the treatment with nanoparticles was applied at a concentration of 150 μg/mL, did not show damage, and the magnitudes of their morphological variables such as height and diameter increased compared to the control (Table 2). This last piece of information is consistent with that provided in some reports that state that foliar application of TiO₂ nanoparticles improves some growth characters in Hordeum vulgare L., Coriandrum sativum L., Vitis vinifera L., Triticum aestivum L., Helianthus annuus L., Dracocephalum moldavica, and Lycopersicum sculentum L. [31,32,33]. Some reports indicate that the above may be due to the photocatalytic capacity of TiO₂ NPs that favors photosynthesis and plant growth under specific wavelengths [33].

The results generated in this study suggest that a TiO₂ concentration of 150 μg/mL has a protective effect. Therefore, a new approach is opened to managing concentration and/or exposure variables that allows for the obtaining of favorable results without negatively affecting the plant and even promoting the biostimulant or elicitor effect. On the other hand, on the basis of the results of reactivity where the interaction of the nanoparticle and the virus decreases its infectivity, the spraying of nanoparticles in the pre-infection period can also be considered since contact with the virus and the nanoparticle would be more direct and faster.

5. Conclusions

In this study, the results suggest that the foliar application of TiO₂ NPs at 150 μg/mL is a promising new practice to control the TMV virus in the chili pepper Capsicum annuum L. In addition, the study showed a biostimulant effect in infected and non-infected pepper plants from a concentration of 150 μg/mL. The foregoing is a reference for future research in controlling another type of geminivirus in other crops of economic importance against TMV.