Green Synthesized Silver Nanoparticles: A Novel Approach for the Enhanced Growth and Yield of Tomato against Early Blight Disease

Tomato plants are among the most widely cultivated and economically important crops worldwide. Farmers’ major challenge when growing tomatoes is early blight disease caused by Alternaria solani, which results in significant yield losses. Silver nanoparticles (AgNPs) have gained popularity recently due to their potential antifungal activity. The present study investigated the potential of green synthesized silver nanoparticles (AgNPs) for enhancing the growth and yield of tomato plants and their resistance against early blight disease. AgNPs were synthesized using leaf extract of the neem tree. Tomato plants treated with AgNPs showed a significant increase in plant height (30%), number of leaves, fresh weight (45%), and dry weight (40%) compared to the control plants. Moreover, the AgNP–treated plants exhibited a significant reduction in disease severity index (DSI) (73%) and disease incidence (DI) (69%) compared to the control plants. Tomato plants treated with 5 and 10 ppm AgNPs reached their maximum levels of photosynthetic pigments and increased the accumulation of certain secondary metabolites compared to the control group. AgNP treatment improved stress tolerance in tomato plants as indicated by higher activities of antioxidant enzymes such as PO (60%), PPO (65%), PAL (65.5%), SOD (65.3%), CAT (53.8%), and APX (73%). These results suggest that using green synthesized AgNPs is a promising approach for enhancing the growth and yield of tomato plants and protecting them against early blight disease. Overall, the findings demonstrate the potential of nanotechnology-based solutions for sustainable agriculture and food security.


Introduction
Tomatoes (Solanum lycopersicum L.), belonging to the family Solanaceae, are among the most important and commonly used crops at the domestic and industrial levels [1][2][3]. The productivity of tomatoes is around 200 million tons globally, economically benefitting the farmers [4]. After potatoes and sweet potatoes, it is the largest vegetable crop in the world [5]. In the food sector, tomatoes are used in a variety of ways. They are used to make soups, salads, pickles, sausages, ketchup, puree, chutney, jam, and many more goods, both nanoparticle synthesis due to the simple and one-step procedure that avoids the timeconsuming process of maintaining cell culture and a non-aseptic environment [39]. Plants are widely available and can be sourced locally, reducing the need for imported materials. They are easy to handle, are a rich source of many metabolites, and include pharmacological ingredients that work as reducing and capping agents in the creation of nanoparticles, preventing the development of undesirable by-products and enhancing nanoparticle stability [40]. Using this procedure, one may synthesize nanoparticles with unique properties such as improved stability, biocompatibility, and antibacterial activity that are both economical and environmentally benign [41,42]. Nanoparticles synthesized using plants have many applications, including in biomedical, environmental, and energy-related fields [43]. Traditional chemical synthesis methods often require hazardous chemicals, but plant-mediated synthesis eliminates this need, making the process safer and more sustainable [44].
Common plants used in the synthesis of nanoparticles include Azadirachta indica [45], Ocimum basilicum [46], Moringa oleifera [47], and Citrus limon [48]. This study aimed to develop a green method for synthesizing silver nanoparticles and investigated their efficacy as a nano-fungicide against early blight in tomato plants. The impacts of silver nanoparticles on the growth rates, biomass output, yield, and certain biochemical traits of the plants were assessed during the interaction between the plants and nanoparticles.

Materials
Leaves of the neem plant were collected to prepare the plant extract for the synthesis of silver nanoparticles (AgNPs). Seeds of two tomato varieties named nadar and naqeeb were brought from the vegetable research institute of the Ayyub Agricultural Research Institute (AARI) in Faisalabad. The required chemicals were purchased through Science Traders from Sigma-Aldrich, St. Louis, MO, USA.

Synthesis of Silver Nanoparticles
The Ahmed et al. [49] modified procedure was used to prepare the plant extract. The leaves of the neem plant were washed, dried at room temperature under shade, and crushed with an electric grinder to make a powder material. The powder material in an amount of 10 g was added to 100 mL of distilled water, brought to a boil for 30 min, allowed to cool, and then filtered through Whatman's No. 1. Then, 20 mL of the leaf extract was mixed with 10 mL of 1 mM silver nitrate in a vial and incubated for 3 h at 70 • C. The color of the reaction mixture changed, and the resultant solution was examined using UV-vis spectroscopy with a wavelength range of 300-800 nm (model BMS: 2800). The reaction mixture was taken in Eppendorf's tube and centrifuged at 10,000 rpm for 10 min. Prior to obtaining pure AgNPs, the centrifugation cycle was repeated several times. Silver nanoparticles were kept in storage at 4 • C for further use.

Source of Alternaria solani
The isolate of Alternaria solani L. was isolated from the infected leaf of a tomato plant with the accession number OP984330.

In Vitro Antifungal Studies of Silver Nanoparticles
Commercially available potato dextrose agar (PDA) media of Sigma-Aldrich was used to prepare Petri plates for the antifungal activity of AgNPs. To inhibit the bacterial growth, a 2% solution of streptomycin was applied to the medium. A standard solution of pure AgNPs was used to make concentrations of 5 ppm, 10 ppm, 15 ppm, 20 ppm, 25 ppm, and 50 ppm. The PDA media and 1 mL solution of different concentrations of AgNPs were poured into respective Petri plates and incubated at 25 ± 1 • C, 75 ± 5% R. H for 48 h. The plate without AgNP solution was taken as a control. An agar plug of equal size was placed in the center of each Petri plate, using a sterile cork borer from the pure culture of A. solani incubated for 14 days at ±27 • C in an incubator (model WIG-32, WiseCube Co. Ltd., Seoul, Korea). The experiment was performed in triplicate, and the plates were labeled. All the work was performed in a laminar airflow cabinet under a sterilized environment to avoid contamination. The radial growth of the colony was measured, and the rate of inhibition was calculated using the following formula: where "R" denotes the expansion of the fungus' mycelium on control plates, and "r" denotes the expansion of the fungus' mycelium on AgNP-treated plates [50].
2.5. In Vivo Antifungal Studies of Silver Nanoparticles 2.5.1. Experimental Design A pot experiment was performed to determine the potential of AgNPs against the early blight disease of tomato caused by A. solani. Using a randomized complete block design (RCBD) with three replicates, the experiment was conducted on the highly susceptible varieties nadar and naqeeb. Loamy soil and farmyard manure were mixed to prepare the pots. To prevent soil water loss and excessive drainage, pebbles were used to partially plug the pores at the bottom of the pots. The pots were filled with 7 kg/pot soil and were arranged according to varieties, respective treatments, and replicate numbers, and they were provided similar environmental conditions (25 ± 1 • C, 75 ± 5% R. H and 12 h photoperiod). Already prepared seedlings of tomato varieties were transplanted in prepared pots, and one plant per pot was kept. Standard agricultural practices were used to irrigate and maintain the plants. Weeds were removed manually during the experimentation. The pots were regularly examined throughout the season.

Inoculation Preparation and Application of A. solani
The conidial suspension was prepared from the 10-day-old culture of A. solani. To collect the conidia, the plates were flooded with ddH 2 O (Fisher Scientific, 0.01% Tween 20 surfactant, Waltham, MA, USA). The prepared conidial suspension at a rate of 10 3 mL −1 in water was sprayed on leaves until runoff [51].

Application of Silver Nanoparticles
Six different concentrations of AgNPs, i.e., 5,10,15,20,25, and 50 ppm, were sprayed until runoff on leaves of tomato plants at intervals of 15 days till harvest. The first treatment was given 35 (vegetative stage 1 according to BBCH scale) days after transplantation (DAT). Positive controls were provided with the standard dose (6 ppm) of commercially available fungicide (cabrio top). The active ingredients in the cabrio top are pyraclostrobin and metriram, which work together and provide both preventative and curative protection against a range of fungal pathogens. Tap water was given to plants taken as the negative control.

Measurement of Disease Parameters of Tomato Plant
The disease parameters, i.e., lesion size, number of lesions/plant, and disease incidence, were recorded at 55 DAT (vegetative stage 19, according to BBCH scale) and 85 DAT (inflorescence emergence stage 5, according to BBCH scale).

Growth and Yield Analysis of Tomato Plant
In order to evaluate the effects of NPs on plant development and biomass yield at the vegetative, reproductive, and mature phases, plants protected against disease attack by AgNPs were also examined. The plants at vegetative stage 19 and inflorescence emergence stage 5 were taken out of the pots, intact with their roots, and were brought in bags to the lab to measure the morphological characteristics (shoot length, root length, number of leaves, and leaf area) and fresh biomass. Yield characteristics, including fruit fresh weight and size, number of fruits/plant, and fruit yield, were measured at fruit ripening stage 88 according to the BBCH scale. To assess how AgNPs affected the growth of dry biomass, the plants were dried. The plants were placed in paper bags and dried for 3 days in a Wiseven oven (Model WOF-105, Korea) at a temperature of 70 • C, and the weights were measured with a digital weighing balance (Cubis_II_analytical balance, Sartorius, Göttingen, Germany).

Determination of Photosynthetic Pigments of Tomato Plant
Healthy leaves were taken from potted plants at 55 and 85 DAT. The measurements of the photosynthetic pigments, including chlorophyll a and b, were measured in accordance with the instructions provided by Davies [52] and Arnon [53]. The Kirk and Allen [54] approach was used to determine the sample's carotene content (1965). The alkaloids were quantified using the method of Ulubelen et al. [55] by thin layer chromatography (TLC). Alkaloids were extracted from 2 g samples of the dried plant organs with n-hexane (3 × 20 mL) followed by MeOH (3 × 20 mL). The extraction was conducted at room temperature, and each cycle with hexane or MeOH lasted 24 h. The extract was evaporated in a rotary film evaporator (RFE). As the last step, each tube was rinsed with 15 mL of distilled water, which was added to the flask. The non-alkaloids in the mixture were removed with CH 2 Cl 2 (3 × 1/3 of the total volume). The acidic aqueous solution was made alkaline (pH 8 to 10) by adding 10% NaOH, and the contents were extracted with CH 2 C1 2 to obtain the alkaloids [55]. At this stage, the aqueous phase was discarded, and the organic phase was dried in RFE. The residue was dissolved in 1 mL CH 2 C1 2 and stored until further use.
With a few adjustments, the approach of Krizek et al. [56] was used to determine the total flavonoid content in the leaves. Briefly, 1 g of leaves was ground with 3 mL of 1% acetic acid-ethanol (1:99, v:v), followed by centrifugation at 12,000× g for 15 min. The supernatant was transferred into a polypropylene tube and incubated in a water bath at 80 • C for 10 min. The spectrophotometer was used to measure the absorbance at 270, 300, and 330 nm. Total flavonoid content was calculated using the extinction coefficient of 0.033 mM/cm. Using the Nelson [57] procedure, the total amount of soluble sugar was calculated (1944). Fresh leaf in the amount of 0.5 g was taken in 10 mL of alcohol for 60 min at 60 • C and filtered to estimate the total soluble sugar. The remaining plant residues were again mixed in alcohol, and the volume was made to 25 mL. An amount of 1 mL of extract was mixed with 1 mL of 5% phenol and 5 mL sulfuric acid. Using 80% ethanol as a blank, the absorbance at 485 nm was measured using a spectrophotometer.
The Lowry [58] technique was used to determine the total soluble protein content. By mixing phosphate buffer (0.1 M, pH-7, 1:4 (w/v)) in a pre-chilled pestle and mortar, frozen leaves were crushed. For 1 g of plant leaves, 4 ml of phosphate buffer was added. Then, at 10,000 rpm at 4 • C, the sample was centrifuged for 10 min. In 0.4 mL of supernatant, 0.2 mL of Folin's mixture was poured, and at room temperature, the test tube was placed for 15 min. Then, for the development of color, 0.2 mL of Folin's Phenol reagent was added, mixed, and placed at room temperature for 45 min. A double-beam spectrophotometer was used to measure the absorbance at 750 nm.

Assessment of Proline and Phenolics Activity of Tomato Plant
The proline concentration was assessed spectrophotometrically at 520 nm after the combination was extracted with toluene, in accordance with the Bates [59] ninhydrin technique. In 3 mL of 3% sulfosalicylic acid, fresh leaf samples (300 mg) were homogenized. Filtrate was poured in test tubes and placed in a water bath at 100 • C, and then, it was reacted with 1 mL each of acid ninhydrin and glacial acetic acid for 1 h. By using L-pro as a standard, the absorbance was measured spectrophotometrically at 520 nm after the extraction of the mixture with toluene.
Using the method described by Lim et al. [60], the Folin-Ciocalteu reagent was used to assess the total phenols in plant extracts after treatment. By using methanol in the amounts of 0.01, 0.02, 0.03, 0.04, and 0.05 mg/g, the concentration of gallic acid was prepared. In methanol in the amount of 0.1 ml, the plant extract was also prepared. In all test tubes of 0.5 mL samples, 2 mL of 7.5% sodium carbonate and 2.5 mL of Folin reagent were added. By using parafilm, test tubes were sealed and placed at room temperature for 30 min. The absorbance was measured at 760 nm using a spectrophotometer. The Folin-Ciocalteu reagent created a blue color after the reaction due to its sensitivity to the reducing compounds, including polyphenols.

Estimation of Antioxidative Activities of Tomato Plant
The superoxide dismutase (SOD) activity was evaluated using the method developed by Sairam et al. [61]. Using the unit/mg protein/30 min method, the SOD activity was determined. For that purpose, 2 g of leaf sample was mixed with a buffer solution (0.05 M phosphate buffer), ascorbic acid, and polyvinylpyrrolidone with a pH of 7.0. The mixture was then centrifuged at 12,000 rpm for 1 2 hour at 4 • C. The reaction mixture was composed of 100 µL of Na 2 CO 3 , 200 µL if methionine, 100 µL of NBT, 100 µL of EDTA, 1.5 mL of potassium buffer (pH 7.5), 1.0 mL of d/w, and 150 µL of enzyme.
To assess the peroxidase (PO) activity of treated plants, the Fu and Huang [62] methodology was employed. A total of 470 nm/g fresh weight/minute was used to express the activity of PO. Leaf samples (0.2 g), completely washed, were kept frozen using liquid nitrogen and were crushed with mortar and pestle in 5 mL of 100 mM phosphate buffer solution until homogenized. The solution was then centrifuged for 25 min at 12,000 rpm. The obtained supernatant was taken in a beaker and allowed to react with 20 µL of 40 mM hydrogen per oxide. The oxidation of guaiacol was noted at 470 nm min −1 with the help of a spectrophotometer.
The Mayer et al. [63] approach was used to gauge the activity of polyphenol oxidase (PPO). The enzyme activity was measured using the substrate catechol. To make the reaction mixture, 1.5 mL of 10 mM sodium phosphate buffer (pH 6.0) was added to 150 mL of 0.1 M catechol solution. Following that, 200 mL of the enzyme mixture was added to the tube and incubated for 1 h at room temperature. PPO activity was calculated to be 495 nm/min/mg protein based on the observed absorbance at 495 nm.
The phenylalanine ammonia-lyase (PAL) activity was evaluated using the procedure developed by Burrell and Rees [64]. In micrograms of trans-cinnamic acid per hour per milligram of protein, the enzyme activity was calculated. L phenylalanine (250 mL and 0.03 M) was added in 2.5 mL of sodium borate (Na 2 H 20 B 4 O 17 ) buffer and 200 mL of the reaction mixture and maintained at a pH of 8.8. For 1 h, this reaction mixture was placed in a 37 • C water bath. After incubation, 1 M of trichloroacetic acid (C 2 HCl 3 O 2 ) solution was added, and the absorption rate was determined at D290.
Using a modified version of the Nakano and Asada [65] approach, the ascorbate peroxidase (APX) activity of the relevant material was measured. First of all, 2 g of leaf sample was mixed with a buffer solution (0.05 M phosphate buffer), ascorbic acid, and polyvinylpyrrolidone with a pH of 7.0. The mixture was then centrifuged at 12,000 rpm for 30 min at 4 • C. For the assay of enzyme activity, the rate of the hydrogen peroxidedependent oxidation of ascorbic acid was determined in a reaction mixture that contained 0.25 mL of 0.05 M phosphate buffer with a pH of 7.0, 0.25 mL of 0.0001 M ethylenediamine tetra-acetic acid (EDTA), 0.25 mL of 0.05 M ascorbate, and 150 µL of supernatant. The reaction was initiated by the addition of 0.25 mL of 0.0001 M H 2 O 2 , and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm for 1 min. Using an attenuation value of 2.8 mM −1 cm −1 , the APX activity was measured as mol ascorbate oxi./mg protein/2 min.

Data Analysis
The data collected during lab and field work were evaluated by one-way analysis of variance (ANOVA) (ANOVA). Duncan's multiple range test (DMRT) at the 0.05 percent level of significance was used to separate the treatment means. All statistical analyses were carried out using Statistix 8.1 package (Analytical Software, Tallahassee, FL, USA).

In Vitro Antifungal Studies of Silver Nanoparticles
The radial growth of the tested pathogen was used to assess the antifungal activity of green synthesized silver nanoparticles under in vitro conditions. The results determined the potential of green synthesized silver nanoparticles prepared from the neem plant leaf extract as a nano-fungicide against potential fungal pathogens. It was observed that various concentrations of silver nanoparticles, i.e., 5, 10, 15, 20, 25, and 50 ppm, significantly inhibited the fungal culture growth compared to the control. The inhibition rate increased from 20% to 81% with the increase of the concentration of NPs, as given in Figure 1. The maximum inhibition rate was observed at 50 ppm of AgNPs with a 1.22 ± 0.033 cm zone.

In Vivo Antifungal Studies of Silver Nanoparticles
The in vivo antifungal activity of green synthesized silver nanoparticles against A. solani was determined in pots under greenhouse conditions. Results demonstrated that green synthesized silver nanoparticles controlled the growth of A. solani and played an active role in disease reduction. A 62.92% disease incidence was noted in control plants of the nadar variety after 55 DAT, while 60.46% was in plants of the naqeeb variety. It increased with time and rose to 88% and 83.68% at 85 DAT in plants of both varieties, respectively. The plants sprayed with the standard dose of fungicide reduced the disease incidence by 9% compared to the control and showed up to a 75% disease incidence. The lower concentration of silver nanoparticles, i.e., 5 ppm, reduced the pathogen growth, and the plants showed a 45-49% disease incidence in both varieties at 55 DAT. Further treatments of 5 ppm AgNPs reduced the disease incidence by more than 50%. An increase in the concentration of AgNPs (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)

Effect of Silver Nanoparticles on Growth Traits of Tomato Plant
The present study also looked at how silver nanoparticles affected tomato plant development characteristics such shoot length, root length, leaf area, and number of leaves measured at 55 and 85 DAT. When compared to the control, the results revealed that tomato plants of both types grew much better when exposed to 10-20 ppm AgNPs (Table 1).
In 30 days under greenhouse circumstances, the shoot and root length of the control plants of the nadar variety increased from 31.58 cm and 18.67 cm to 38.37 cm and 26.57 cm, whereas they increased from 30.96 cm and 16.83 cm to 44.80 cm and 27.77 cm in the case of the naqeeb variety. Plants of both varieties treated with fungicide had shoot and root lengths that were not significantly different from the control. In comparison to the control, the lower concentrations of silver nanoparticles 5 to 20 ppm increased the shoot and root length by 20-80% in both varieties at both stages. Plants of the nadar variety treated with 10 ppm silver nanoparticles at 55 DAT had maximum shoot and root lengths of 52.81 cm and 29.30 cm, respectively, whereas plants of the naqeeb variety had maximum shoot and root lengths of 53.85 cm and 29.30 cm, respectively. Nevertheless, plants treated with 5 ppm silver nanoparticles at 85 DAT showed the longest shoot and root lengths. When AgNP concentrations were increased further to 25 and 50 ppm, the shoot and root lengths were 20% shorter than they were in the control. As silver nanoparticles were sprayed on the leaves, the quantity of leaves on each plant, along with their size, was counted. With a leaf area of 27.87 cm 2 for the nadar variety and 29.67 cm 2 for the naqeeb variety, respectively, the number of leaves in control plants rose by 40% from 55 DAT to 85 DAT. The number of leaves increased by 70% in plants treated with 10 ppm at 55 DAT and 5 ppm at 85 DAT compared to the control in both varieties and reduced up to 15% at higher concentrations, suggesting the lower concentrations and less exposure of silver nanoparticles were more effective in plant growth improvement. The maximum leaf area of 46.10 cm 2 was recorded in plants of the naqeeb variety with 5 ppm AgNPs at 85 DAT.

Effect of Silver Nanoparticles on Biomass Production of Tomato Plant
The application of green synthesized silver nanoparticles also affected the fresh and dry biomass of the tomato plant. The shoot fresh and dry biomass was 28.71 g and 8.36 g in control plants of the nadar variety at 55 DAT, which increased to 39.63 and 8.24 g at 85 DAT. The silver nanoparticles increased the shoot fresh and dry biomass by 75% with 5 and 10 ppm concentrations at 55 and 85 DAT in both varieties of tomato plants. The maximum fresh and dry biomass of roots at 55 DAT was recorded to be 14.26 g and 5.24 g in plants of the nadar variety treated with 10 ppm silver nanoparticles, which increased up to 21.77 g and 5.47 g, respectively. A similar pattern was followed by plants of the naqeeb variety, as shown in Table 2. The higher concentration of silver nanoparticles (25 and 50 ppm) reduced the fresh and dry biomass in plants of both varieties compared to the control.

Effect of Silver Nanoparticles on Photosynthetic Pigments of Tomato Plant
Green synthesized silver nanoparticles also affected the photosynthetic pigments, including total chlorophyll content, chlorophyll a and b, and carotenoids ( Table 3). The results demonstrated that lower concentrations of silver nanoparticles (5-10 ppm) improved the photosynthetic pigments of both varieties by more than 50%. The higher concentrations of AgNPs, such as 25 and 50 ppm, reduced chlorophyll and carotenoids, resulting in less food production and plant growth.

Effect of Silver Nanoparticles on Alkaloids, Flavonoids, Total Soluble Sugar, and Total Soluble Protein of Tomato Plant
Silver nanoparticles improved the growth of plants and affected the production of various essential biochemicals such as alkaloids, total flavonoids, total soluble sugar, and total soluble protein ( Figure 3). The alkaloid production increased by over 50% with 5 and 10 ppm of silver nanoparticles in both varieties at 55 DAT. The 5 ppm silver nanoparticles increased the amount of alkaloids from 0.33 mg CE/g to 0.583 mg CE/g in plants of the nadar variety and 0.34 mg CE/g to 0.625 mg CE/g within 30 days with 5 ppm silver nanoparticles. The plants of the naqeeb variety produced more alkaloids and were healthier than those of the nadar variety. The minimum production of alkaloids was observed with a 50 ppm concentration of silver nanoparticles in both varieties.
The total flavonoid content also increased significantly when plants of both varieties were exposed to lower concentrations of silver nanoparticles than control plants. The total flavonoid content of the nadar and naqeeb plant control group at 55 DAT was 2.62 mg QE/g and 2.54 mg QE/g, which increased to 3.721 mg QE/g and 3.636 mg QE/g at 85 DAT, respectively. The maximum amount of alkaloids of 4.23 mg QE/g and 4.06 mg QE/g was produced at 55 DAT in nadar and naqeeb variety leaves using 10 ppm concentrations. With further treatments of AgNPs, the maximum amount of alkaloids of 5.833 mg QE/g and 5.778 mg QE/g was produced using 5 ppm in leaves of nadar and naqeeb varieties. Figure 3c shows the significant change in total soluble sugar production in both varieties of tomato plants. The maximum production of total soluble sugar was 0.724 mg g −1 and 0.805 mg g −1 in plants of the nadar and naqeeb variety, respectively, using 10 ppm Ag-NPs, which was more than 60% compared to the control at 55 DAT. The production of total soluble sugar was improved with further treatments of AgNPs in both varieties at 85 DAT. The higher concentrations of AgNPs reduced its production compared to the control.
A similar trend was observed in the accumulation of protein content in both varieties of tomato plants. The lower concentrations of AgNPs (5-15 ppm) produced more protein content in both varieties than in the control plants. On the other hand, higher concentrations reduced the protein content at 55 and 85 DAT, as shown in Figure 3d.

Effect of Silver Nanoparticles on Total Phenolic Content and Stress Enzymes of Tomato Plant
Silver nanoparticles produced more phenolic content, PO, PPO, PAL, CAT, SOD, and APX to reduce the pathogenic stress in plants. The higher concentrations of AgNPs caused more stress in plants than in the control, producing a higher amount of proline. A lower amount of proline was detected in plants treated with lower concentrations (5-10 ppm), indicating stress reduction, as shown in Figure 4b. The maximum phenolic contents of 1.483 µg mg −1 , 1.479 µg mg −1 , 1.691 µg mg −1, and 1.750 µg mg −1 were quantified in plants of both varieties treated with 5 ppm AgNPs at 55 and 85 DAT, respectively. The PO activity was also improved in both varieties of tomato with the exposure to lower concentrations of AgNPs compared to the control. More PO activity was observed in plants of the nadar and naqeeb varieties at 55 DAT than 85 DAT, suggesting that the exposure to AgNPs inhibits the pathogen attack, resulting in the production of less reactive oxygen species (ROS), lowering the PO activity. A similar trend was observed in PPO activity, as shown in Figure 4d Figure 5a suggests that further treatments of AgNPs improved the PAL activity after 30 days. The production of superoxide dismutase (SOD) was induced by applying silver nanoparticles in a lower concentration. The activity of SOD was increased up to 65.31% and 71% with 10 ppm AgNPs at the first stage in plants of nadar and naqeeb varieties, respectively, compared to the control. A further increase in concentrations started in the reduction of SOD activity, indicating the severe stress conditions in plants. Catalase (CAT) is another important antioxidant enzyme that reduces plant stressinduced toxicity. In contrast to the control and fungicide, the activity of CAT was markedly increased in plants treated with AgNPs. In comparison to the control, plants exposed to lower concentrations of silver nanoparticles (5-15) had more activity. Nevertheless, at both stages and in both kinds, activity began to decline with an increase in NP concentration. The maximum activity of 3.903 and 4.313 was recorded with 10 ppm AgNPs at 55 DAT and 3.615 and 3.485 at 85 DAT in nadar and naqeeb plants, respectively. The control plants showed activity within the range of 2.266-2.521, and the lowest activity was demonstrated with 50 ppm AgNPs. A similar trend was observed with APX activity in plants of both varieties. The activity was increased by more than 50% from 0.027 to 0.045 and 0.040 with 10 ppm AgNPs in nadar and naqeeb variety plants at the first stage, respectively. The higher concentrations of silver nanoparticles reduced the activity, creating more stress in plants at both stages. The above-mentioned results indicate that AgNPs induce more resistance in plants against A. solani than the standard dose of used fungicide.

Effect of Silver Nanoparticles on Yield Attributes of Tomato Plant
The fruit was examined for fresh weight, size, quantity of fruits per plant, and fruit yield when collected 115 days after transplantation (Figures 6 and 7). With more silver nanoparticle treatment than the control, the quantity of fruits produced per plant rose. The number of fruits per plant increased with the increase of the treatment of silver nanoparticles compared to the control. The production was increased from 20 to 36 and 21 to 37 in the case of the nadar and naqeeb plants, respectively. The plants treated with 10 ppm silver nanoparticles produced the highest number of fruit in the case of both varieties. The higher concentrations of silver nanoparticles caused the reduction of the fruit number compared to the control. More than 85% of the obtained fruit was marketable, with an average weight of 38 g. The weight of the fruit was significantly affected by applying silver nanoparticles.
A maximum weight of 61.33 g with 10 ppm AgNPs in the fruit of the nadar variety and 57.67 g with 5 ppm AgNPs in the naqeeb variety was observed. The fruit harvested from plants exposed to doses of 25 and 50 ppm were not considered marketable since their average weight (18 to 20 g) was less than 30 g, and their size was 35% less than that of the control, as shown in Figure 7a. The maximum fruit yield (kg/plant) was obtained from plants treated with 10 ppm silver nanoparticles by 35-43% more than control plants. The lower concentrations of silver nanoparticles (5 and 10 ppm) significantly affected the overall growth and fruit yield of both varieties of tomato plants. In comparison, the higher concentrations caused stress and resulted in the production of unmarketable fruit.

Discussion
Silver nanoparticles (AgNPs) are often characterized by their unique physical, chemical, and biological properties [66]. These characteristics have to do with particle size, shape, and surface chemistry, all of which may be altered using synthesis techniques [67]. Using plant extracts as reducing and stabilizing agents during the nanoparticle production process is known as "green synthesis". This method is eco-friendly, is cost-effective, and can produce nanoparticles with unique properties [68]. Many studies have been conducted on silver nanoparticles (AgNPs), which are known to exhibit a broad spectrum of antifungal actions [69]. AgNPs' antifungal efficacy is influenced by several variables, including particle size, shape, surface charge, and concentration [41]. Due to their increased surface area-to-volume ratio, smaller AgNPs tend to have better antifungal activity than bigger particles, which improves their capacity to enter fungal cells and interact with cellular components [70]. According to Hernandez-Daz et al. [71] and Mansoor et al. [72], AgNPs interact with Alternaria solani's cell membrane and induce structural damage that results in membrane permeabilization, oxidative stress, and cell death.
In the current study, the green synthesized silver nanoparticles of 22-30 nm size were proven to be an effective antifungal agent in the laboratory and under greenhouse conditions against Alternaria solani causing early blight disease. Even at lower concentrations, the AgNPs inhibited the development of the fungus by more than 50% compared to the control and reduced the symptoms. The higher concentrations of silver nanoparticles completely prevented the fungus growth. AgNPs applied as a foliar spray reduce the fungal growth, mycelial development, disease severity, and oxidative damage caused by A. solani, leading to improved plant growth and development [73]. Silver nanoparticles have been found to increase the accumulation of certain phytochemicals, such as phenols and flavonoids, which have been shown to have antifungal properties [74]. By controlling the synthesis of stress-related hormones, enhancing the plant's antioxidant defense system, and influencing the expression of stress-responsive genes against pathogenic stress, AgNPs increase plants' ability to withstand stress [75,76]. AgNPs lessen oxidative stress-related harm by having a high surface area to volume ratio, being highly reactive, and being able to interact with cellular components [77].
Silver nanoparticles enhance the activity of stress-tolerant enzymes, such as peroxidases (PO) and polyphenol peroxidase (PPO), and phenylalanine ammonia-lyase (PAL), which can help reduce oxidative damage and improve the plant's stress tolerance [78][79][80]. PO is an enzyme that catalyzes the oxidation of various substrates using hydrogen peroxide as an oxidizing agent. In plants, it plays a role in the defense against biotic stress, such as pathogen attack, by generating ROS to help inactivate the pathogens [81]. Another enzyme, PPO, is involved in lignifying cell walls to reinforce plant tissues against stress [82]. Secondary metabolites including flavonoids and phenylpropanoids are biosynthesized by PAL [83]. The formation of secondary metabolites, which can function as antioxidants and can aid in the reduction of ROS, was increased as a result of AgNPs activating the PAL activity [84]. Another study found that applying AgNPs to tomato plants increased the activity of the enzymes PO, PPO, and PAL, which are crucial for the manufacturing of phytochemicals and the body's response to pathogenic assaults [68].
The use of silver nanoparticles enhances the activity of the enzymes superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in plants, boosting the plant's capacity to resist oxidative stress and maintain cellular homeostasis under stressful circumstances [85]. SOD protects the plant from oxidative stress by converting superoxide radicals into hydrogen peroxide and molecular oxygen [86]. CAT acts to decompose hydrogen peroxide into water and oxygen [87]. In comparison, APX is involved in the ascorbate-glutathione cycle and helps neutralize the plant's hydrogen peroxide and other reactive oxygen species [88]. These enzymes are crucial in the plant's defense mechanism against pathogen infections. By maintaining the balance of reactive oxygen species and reducing oxidative damage, SOD, CAT, and APX help the plant to overcome stress conditions and improve its overall health and productivity. It has been found that applying AgNPs to tomato plants resulted in upregulating SOD, CAT, and APX activities, reducing oxidative damage levels, and improving plant growth under early blight stress conditions [89]. AgNPs can improve the tomato crop's growth, yield, and quality by enhancing the plant's overall health and protecting it from disease.
The green synthesis of silver nanoparticles has positively affected tomato plant growth, development, productivity, and increased resistance to pathogens when applied as a foliar spray. In several plant species, including tomato, the use of green produced AgNPs has been demonstrated to improve the shoot length, root length, number of leaves, and leaf area [90,91]. Shoot length, root length, and plant height all increase as a result of the AgNP stimulation of cell division and elongation [92]. By promoting root growth and boosting nutrient uptake from the soil, AgNPs increase plant growth [93]. Improvements in soil nutrient uptake, greater chlorophyll content in the leaves, and better plant resiliency to external stressors are assumed to be the causes of a rise in growth characteristics [94].
Applying AgNPs increases leaves' chlorophyll content, leading to improved photosynthetic efficiency [95,96]. AgNPs enhance photosynthetic activity by lowering oxidative stress [97] and enhancing the uptake of vital nutrients such as nitrogen, phosphorus, and potassium. They also assist in maintaining the structural integrity of chloroplasts and other cellular components involved in photosynthesis [98]. The synthesis of several secondary metabolites, including sugar, protein, alkaloids, flavonoids, and phenolic compounds, is improved when resources are more readily available [99]. It is indicated that AgNPs influence the levels of plant hormones, such as auxins and gibberellins, which regulate alkaloid biosynthesis [100]. AgNPs have been shown to modulate the activity of certain enzymes involved in the biosynthesis of alkaloids, leading to changes in alkaloid production [101]. It has been suggested that silver nanoparticles can trigger the plant's stress response, leading to increased flavonoid synthesis [102]. These compounds have antiinflammatory, antioxidant, and antimicrobial properties and play important roles in plant defense against pathogens and environmental stress, and they also have potential health benefits for humans [5,100].
The use of AgNPs has been reported to increase tomato output [73,103,104]. The growth and fruit output of tomato plants were shown to be enhanced by AgNPs at a dosage of 5 ppm. The study concluded that greater plant vigor and stronger antioxidant defense systems were responsible for the increased output [105]. Fruit output in tomato plants has been demonstrated to be impacted by the application of silver nanoparticles (AgNPs). Depending on the dose and length of exposure, AgNPs can have either favorable or detrimental impacts on the fruit output of tomato plants. According to certain research, utilizing AgNPs can boost plant growth and physiological functions including photosynthesis, nutrient absorption, and stress tolerance, which in turn can enhance tomato plants' fruit output [106]. By causing oxidative stress, lowering photosynthesis, and disrupting the plant's hormonal balance, larger concentrations of AgNPs, such as 100 or more, might, on the other hand, negatively affect tomato plant development and fruit output.

Conclusions
The present study provides compelling evidence for the potential of green synthesized AgNPs as a novel approach for enhancing the growth and yield of tomato plants and protecting them against early blight disease. The AgNPs synthesized using leaf extract of the neem tree were found to be effective in promoting plant growth, as evidenced by the significant increase in plant height, fresh and dry weight, chlorophyll, alkaloids, total soluble sugar, flavonoids, and total soluble protein of the tomato plants. Moreover, the plants treated with AgNPs exhibited a significant reduction in disease severity and disease incidence compared to the control plants, increasing the enzymatic activities of antioxidants PO, PPO, PAL, SOD, CAT, and APX, indicating their potential as an eco-friendly and sustainable solution for plant protection. These findings highlight the potential of using green synthesized AgNPs as a safe, effective, and environment friendly alternative to conventional fungicides for crop protection and yield enhancement.  Data Availability Statement: The paper contains the original contributions that were presented in the study. The appropriate author can be contacted for more information.