Evaluation of In Vitro and In Vivo Antifungal Activity of Green Synthesized Silver Nanoparticles against Early Blight in Tomato

Abstract

Silver nanoparticles have gained considerable interest in recent decades due to their antimicrobial activity and are used in water disinfection, wound healing, food packaging, and plant protection. This study tested the potential of silver nanoparticles synthesized using the neem (Azadirachta indica) leaf extract against Alternaria solani causes early blight disease in tomato plants. The pathogen was isolated from infected tomato plants and identified using morphological and molecular features. The results showed significant variation among isolates. Isolates, Shk-1 and Ksr-1 were highly pathogenic, causing up to 80% disease incidence. The potential of silver nanoparticles against each isolate was determined using different concentrations of silver nanoparticles. During in vitro and in vivo experiments, the growth inhibition rate of the pathogen was 70–100% at 50 ppm. Lower concentrations of silver nanoparticles (5 and 10 ppm) increased phenolics, PO, PPO, and PAL production by more than 50% as compared to the untreated control. These defensive mechanisms clearly demonstrate the fungicidal potential of AgNPs and recommend their utilization in different crop protection programs.

1. Introduction

Nanotechnology has received much attention due to its applications in many industries, such as medical imaging, therapy, drug delivery, and energy generation [1]. Nanotechnology is a field of science and technology that focuses on the study, design, creation, manipulation, and use of materials and devices on the nanoscale level [2]. A nanometer is one billionth of a meter, which means that nanotechnology deals with materials and devices that are typically between 1 and 100 nanometers in size [3]. Due to their smaller size, nanoparticles have a higher surface-to-volume ratio, making them more effective with different properties than their bulk material [4]. Nanotechnology has broad applications in many fields, including electronics, energy, medicine, and agriculture [5].

Applications of nanomaterials, primarily biological applications, depend on their synthesis methods. Nanoparticles are generally synthesized using different physical or chemical approaches [6,7]. However, physical processes usually involve the maintenance of high temperatures and pressures and require more energy, due to which the quality of the product is also compromised. Moreover, chemical methods utilize hazardous synthetic chemicals that persist in the environment and cause pollution [8,9]. Therefore, green synthesis is more of a focus now because using plants to synthesize nanoparticles is a simple, single-step, fast, economic, and eco-friendly approach [10,11]. Green nanoparticle synthesis refers to synthesizing nanoparticles using natural materials such as plants and avoiding harmful chemicals and high temperatures [12]. Green synthesis methods synthesize biocompatible and biodegradable nanoparticles, making them suitable for agricultural applications [13,14]. Plants produce biomolecules like carbohydrates, proteins, polyphenols, and co-enzymes that can reduce silver to silver nanoparticles [15,16].

Silver nanoparticles are the most promising among all the metallic nanoparticles due to their unique properties, physiochemical activities, and potential biological applications [13], including antimicrobial [17], anti-inflammatory, anti-infectious, and antiseptic properties, even at lower concentrations [18]. AgNPs are used as drug delivery agents, cancer therapeutics, and wound healing agents. AgNPs have been investigated for their potential use in environmental remediation, such as water purification and air filtration [19]. AgNPs are being studied for their potential applications in electronics, particularly in developing electronic devices, due to their high electrical conductivity and thermal stability [20]. Moreover, AgNPs have also shown promising results in agriculture for their potential use as an alternative to chemical fertilizers and pesticides [21]. AgNPs improve crop yield and enhance plant growth [22]. AgNPs have also been found to possess antifungal and antibacterial properties, making them suitable for controlling different plant diseases [23].

Silver nanoparticles release silver ions, disrupting the cell membrane and inhibiting the growth of bacteria, fungi, and viruses. The small size of silver nanoparticles allows for high surface area-to-volume ratios and releases more silver ions to counter microbial activity [24]. This property makes silver nanoparticles a promising alternative to traditional antibiotics for treating infectious diseases and a potential ingredient in antimicrobial coatings, textiles, and medical devices [1,18]. It is observed that silver nanoparticles do not affect living cells at lower concentrations, so they cannot provoke microbial resistance [25]. Silver nanoparticles have a high surface area to volume ratio, which allows them to interact with a large number of microbial cells at once. Silver nanoparticles penetrate the cell wall of the microbe and interact with the cell membrane, leading to disruption of the membrane [14]. Silver nanoparticles generate reactive oxygen species (ROS) that damage cellular components, such as DNA and proteins. The mechanism of action is particularly effective against bacteria and fungi, which have a simpler defense system against ROS compared to higher organisms [26]. Silver nanoparticles also interfere with cell signaling pathways, preventing them from communicating with each other and coordinating their response to stressors, which ultimately results in their inability to adapt to changing environments [24]. Silver nanoparticles have a broad spectrum of activity against many different types of microorganisms, which makes it less likely for any one microbe to develop resistance to it [27]. Even if a few microbes do develop resistance, the diverse mechanisms of action of silver nanoparticles make it difficult for them to spread their resistance to other microbes [28].

Many researchers have reported the antimicrobial potential of silver nanoparticles against various pathogens [29,30,31], including Alternaria, Corynespora, and Fusarium spp. [32]. Genus Alternaria Nees is a broad fungal group. Its species, especially solani Sorauer, is the most destructive one that causes an Early Blight disease in tomato and potato plants worldwide [33,34]. Alternaria solani colonizes the leaves, stems, and fruits [35,36] of plants belonging to the family Solanaceae, causing up to 78% of crop losses [37]. The genetic variation in A. solani results in morphological, physiological, and pathogenesis differences among isolates [38]. Many practices have been introduced, such as crop rotation, resistant cultivars, sanitation, and synthetic fungicides to manage Early Blight disease, but have failed [39,40]. Silver nanoparticles can be used due to their antimicrobial potential; however, there are concerns about their potential toxicity and environmental impacts. Nanoparticles usually cause irreversible damage to living cells by oxidative stress, which solely depends on the size, composition, and concentration of the NPs. Hence, research regarding the identification of optimal doses of NPs against plant diseases is needed [4].

The present study aimed to evaluate the in vitro and in vivo antifungal activity of green synthesized silver nanoparticles (AgNPs) against Alternaria solani. The identification and occurrence of A. solani were also investigated to understand the distribution and prevalence of the pathogen in tomato crops, which is critical for disease management strategies. In addition, the characteristics of the AgNPs using various analytical techniques were also studied.

2. Materials and Methods

2.1. Materials

Leaves of the Azadirachta indica A. Juss. (neem) plant were collected from the ground (31.497185°-N, 74.298172°-E) of University of the Punjab (Lahore, Pakistan), washed, and dried for further use. Seeds of tomato (Solanum lycopersicum L.) varieties were obtained from the Vegetable Research Institute of Ayyub Agricultural Research Institute (AARI), Faisalabad, Pakistan. The required chemicals, i.e., silver nitrate (AgNO₃), potato dextrose agar (PDA), methanol, Folin–Ciocalteu reagent, sodium carbonate, Catechol, phosphate buffer, Guaiacol, hydrogen peroxide, sodium phosphate, trichloroacetic acid, and L-phenylalanine were bought from Sigma-Aldrich, United States through Science Traders.

2.2. Green Synthesis of Silver Nanoparticles

The silver nanoparticles were synthesized using leaves extract of neem plant prepared in distilled water. The dried leaves of the neem plant were ground using an electric grinder to make fine powder material. Ten (10) grams of the powder material was added to 100 mL of distilled water, boiled for 30 min, cooled at room temperature, and filtered using Whatman’s No. 1 filter paper. The plant extract of 20 mL was then reacted with 10 mL of 1 mM silver nitrate solution and incubated for 3 h at 70 °C. The production of AgNPs synthesis was confirmed by observing the solution’s color change. Then, the resulting solutions were subjected to UV–vis spectroscopy within 300–800 nm wavelength. Each solution was taken in Eppendorf’s tube and centrifuged. The pellet was again centrifuged after dissolving in distilled water. The cycle of centrifugation was repeated several times until purified AgNPs were acquired. The synthesized silver nanoparticles were stored at 4 °C to study their characteristic features.

2.3. Characterization of Silver Nanoparticles

Green synthesized silver nanoparticles using leaf extract of A. indica (neem) plant were subjected to various techniques, i.e., UV–spectrophotometry, Fourier-transform infrared spectroscopy (FTIR) analysis, zeta sizer, X-ray diffraction (XRD), and scanning electron microscopy (SEM) to determine their size, surface structure, morphology, and other characteristics.

2.3.1. UV–Spectrophotometry of AgNPs

This technique was used to confirm the synthesis of silver nanoparticles. The 1 mL of the sample was taken in Eppendorf’s tube and centrifuged at 14,000 rpm for 10 min. After discarding the supernatant, the pellet was dissolved in distilled water again. The procedure was repeated three times to get rid of debris. The purified silver nanoparticles were dissolved in 1 mL of distilled water using a vortex meter and subjected to UV–vis analysis. The absorption spectrum within the 300–800 nm wavelength range on a UV–visible spectrophotometer was taken.

2.3.2. Fourier-Transform Infrared Spectroscopy (FTIR) Analysis of AgNPs

Fourier transform infrared spectroscopy (FTIR) was calibrated to study the organic functional groups attached to the surface of AgNPs. The selected nanoparticles, synthesized at optimized conditions, were purified using repeated centrifugation at 14,000 rpm for 10 min and dried at 60 °C. The dried samples were mixed with a fine powder of potassium bromide (Kbr) and analyzed by FTIR.

2.3.3. X-ray Diffraction (XRD) Analysis of AgNPs

X-ray diffraction (XRD) was used to examine the overall oxidation state and crystal structure of AgNPs. Synthesized nanoparticles were purified by centrifugation, and the resulting pellets were dispersed into 10 mL of deionized water. After freeze-drying the purified AgNPs, the structure was analyzed with XRD.

2.3.4. Scanning Electron Microscopy (SEM) of AgNPs

Scanning electron microscopy (SEM) was performed to envision the morphology of synthesized particles at the submicron scale and elemental information at the micron scale. It provided the exact magnitude and figure of AgNPs. Thin films of the sample were prepared on a carbon-coated copper grid. A drop of AgNPs was placed on carbon-coated copper grids and allowed to stand for two min, and the excess solution was removed using a blotting paper. Then, the film on the grid was allowed to dry at room temperature and exposed to an electron beam.

2.4. Isolation of Pathogen

Pathogen (Alternaria solani) was isolated from infected leaves of the diseased tomato plants grown in tunnels and fields of Lahore, Kasur, Faisalabad, Bahawalpur, and Multan. The leaves showing distinctive symptoms of early blight disease were collected in sterilized polythene bags, labeled, and brought to the laboratory for further procedure. The infected parts of leaves were cut into 1–2 cm pieces and placed in one of the surface disinfectant solutions (1% sodium hypochlorite) for 30 s, washed with distilled water, and blotted. These surface-sterilized pieces were placed on PDA media, three to five per Petri plate, with sterilized forceps. The Petri plates were incubated at 27 ± 2 °C for 7 days, and fungal isolates were sub-cultured for purification and identification [41]. Freshly prepared Petri plates were used to purify the pathogen culture from the original culture plate using single spore culture method. A single fungal colony that appeared on the original plate free from contamination was selected to transfer the fungal spore with a sterile inoculation loop. The fresh cultural plates were incubated at 27 ± 2 °C for future use.

2.5. Identification of Pathogen

The isolated fungal pathogen was identified using morphological characteristics, i.e., color, zonation, margin, and diameter of the colony. The size and shape of conidia were observed microscopically at 10×, 40×, and 100× [42], and photographs were taken to compare with the literature [43,44,45]. Molecular identification was performed by extracting DNA using the 2% CTAB method [46]. The primer pair ITS1 (5-TCCGTAGGTGAACCTGCGG-3), ITS4 (5-TCCTCCGCTTATTGATATGC-3), and Taq DNA polymerase (Tiangen Biochemical Technology Co., Beijing, China) were used for the amplification of the Internal Transcribed Spacer (ITS) region using the PCR technique. The thermocyclic conditions were kept as initial denaturation at 94 °C for 5 min followed by 37 cycles of 94 °C for 1 min, 52 °C for 40 s, 72 °C for 1 min, and a final extension at 72 °C for 10 min [47]. Amplified DNA products were then sequenced from Beijing Genome Institute, China. The obtained sequences with accession numbers (OP984328, OP984329, OP984330, OP984331, OP984332, OP984333, and OP984334) were then BLAST searched at National Center for Biotechnology Information (NCBI) and closely related Alternaria species were retrieved from GenBank to carry out the phylogenetic analysis.

2.6. Pathogenicity Test of Alternaria solani

The inoculum for the pathogenicity test of each isolate was prepared from 10 days old culture grown on PDA media. Conidia suspension was harvested by flooding the plates with ddH₂O containing 0.01% of surfactant Tween 20 and brushing the agar surface with a paintbrush. Leaves of two months old plants of moderately susceptible tomato variety Reograndi were sprayed with the prepared conidial suspension at 10³ mL⁻¹ rate until run-off. The data, including disease incidence (%), number of lesions/leaf, and lesion size (mm²), were recorded at regular intervals after 5, 10, and 15 days of inoculation to select the highly virulent pathogen for further use and fulfilling Koch’s postulate test.

2.7. In vitro Antifungal Studies of Silver Nanoparticles

Different concentrations of AgNPs, i.e., 5, 10, 15, 20, 25, and 50 ppm, were prepared to determine their effects on the growth of A. solani. The PDA media and 1 mL solution of different concentrations of AgNPs were poured down into respective Petri plates and incubated for 48 h. Petri plates were inoculated with an agar plug (placed in the center of each Petri plate) from pure culture and incubated at 27 ± 2 °C for 14 days. Three replicates were used to experiment, and no treatment was given to the control. The fungus growth was monitored regularly, and radial growth of the colony was noted after 14 days. The inhibition rate was calculated using the following equation.

$$\mathrm{Rate} \mathrm{of} \mathrm{Inhibition} \left(\%\right)=\frac{\mathrm{R}-\mathrm{r}}{\mathrm{R}}\times 100$$

where ‘R’ represents the radial growth of fungal mycelium in control plates, and ‘r’ is the radial growth of fungal mycelium in AgNPs treated plate [48].

2.8. Screening of Tomato Varieties against Alternaria solani

Earthen pots of 35.56 cm were washed, cleaned, and filled with a mixture of sandy and loamy soil in 1:3 ratio. Farmyard manure and DAP were added to the soil mixture to enrich it with nutrients. Pots were placed in a wired house covered with a plastic sheet to protect tomato plants from frosting. Already prepared seedlings of different varieties of tomato plants were transplanted in prepared pots, and leaves were sprayed with a conidial suspension of A. salani at a rate of 10³ mL⁻¹ in water until run-off [49]. Plants were irrigated regularly and monitored for the development of symptoms and severity of infection. The data were recorded after 15 days of inoculation.

The formula for the percentage of the Early Blight Index is given below.

$$\mathrm{PEBI} \left(\%\right)=\frac{\mathrm{Sum} \mathrm{of} \mathrm{all} \mathrm{readings}}{\mathrm{No}. \mathrm{of} \mathrm{leaves} \mathrm{samped}\times \mathrm{Maxi} \mathrm{disease} \mathrm{scale}}\times 100$$

EB severity on each leaf of the plants was recorded on a scale of 0 to 5, where 0 (immune) = no visible lesions on the leaf; 1 (highly resistant) = up to 10% leaf area affected; 2 (resistant) = 11–25%; 3 (tolerant) = 26–50%; 4 (susceptible) = 51–75%; and 5 (highly susceptibility) = more than 75% leaf area affected, or leaf abscised [50].

2.9. Greenhouse Experiment for In Vivo Efficacy of Silver Nanoparticles

A pot experiment was performed to determine the potential of AgNPs against the Early Blight disease of the tomato plant. Healthy seedlings of two susceptible varieties were transplanted into prepared pots and kept one plant per pot. The pots were arranged according to a randomized complete block design (RCBD). Leaves of plants were inoculated with A. solani and regularly monitored for the development of symptoms. Six different concentrations of AgNPs, 5, 10, 15, 20, 25, and 50 ppm, were prepared and applied as foliar spray twice at 15 days intervals on inoculated tomato plants. Tap water was given to plants taken as a negative control. Disease incidence, amount of phenols, and a few stress enzymes were recorded after 55 days of seedlings transplantation.

2.9.1. Quantification of Total Phenolics

A test tube was taken and filled with 5 mL dH₂O, 1 mL methanolic leaf extract, and 250 mL Folin–Ciocalteu reagent (50% solution) and placed in the dark. After half an hour, 1 mL of 50% sodium carbonate (Na₂CO₃) solution was added and incubated for another 10 min in the dark. The absorption rate was determined at ∆725 nm using a double-beam spectrophotometer (BMS: 2800). Catechol was used to draw the standard curve. By comparing the standard curve, total phenolics were given as catechol µg mg⁻¹.

2.9.2. Quantification of Peroxidases, Polyphenol Oxidases, and Phenylalanine Ammonia–Lyase

Leaves of treated plants (1 g) were crushed in a mortar containing ice-cold 100 mM phosphate buffer. The homogenized material with pH 7 was centrifuged at 4 °C for 15 min at 5000 rpm. The clear supernatant was collected and used to quantify enzymes.

Peroxidase activity was calculated using the Fu and Huang [51] method. Guaiacol reagent serving as the substrate was mixed with 10 mL sodium phosphate (10 mM) buffer. Finally, 3 mL of the enzyme mixture was added and incubated for 5 min at room temperature. At 470 nm, the absorbance was measured, and calculated PO activity was using ∆470 nm gfw⁻¹ min⁻¹ [50].

Polyphenol oxidase (PPO) was determined using the Mayer et al. [52] method. A total of 1.5 mL of sodium phosphate (10 mM) buffer (pH 6.0) was added to 150 mL of 0.1 M catechol solution (used as a substrate to measure enzyme activity). In this reaction mixture, 200 mL enzyme mixture was added and incubated for 1 h at room temperature. At ∆495 nm, the absorbance was measured, and PPO activity was estimated to be ∆495 nm min⁻¹ mg⁻¹ protein [51].

The Burrell and Rees [53] protocol was used to assess phenylalanine ammonia–lyase activity (PAL). L-phenylalanine (250 mL and 0.03 M) was added in 2.5 mL of sodium borate (Na₂H₂₀B₄O₁₇) buffer and 200 mL of the reaction mixture and maintained at pH 8.8. For 1 h, this reaction mixture was placed in a 37 °C water bath. After incubation, 1 M of trichloroacetic acid (C₂HCl₃O₂) solution was added, and the absorption rate was determined at ∆290 nm and measured in µg of trans-cinnamic acid h⁻¹ mg⁻¹ protein [52].

2.10. Data Analysis

The data collected during lab and field work were evaluated by one-way analysis of variance (ANOVA). Duncan’s multiple range test (DMRT) at 0.05% level of significance was used to separate the treatment means. PCA analysis was performed using Origin 2018 to determine the effect of AgNPs on growth attributes of tomato plant and correlation between disease incidence and other factors of plants studied.

3. Results

3.1. Characterization of Silver Nanoparticles

The synthesis of silver nanoparticles was initially determined by color change. The yellow-colored reaction mixture changed to dark brown, indicating AgNP synthesis. Silver nanoparticles were then purified and subjected to UV–spectrophotometry. A clear and sharp characteristic peak at 424 nm giving an absorption rate of 0.368, can be seen in Figure 1a due to the surface plasmon resonance phenomenon verifying the synthesis of AgNPs. The particle size was recorded within 22–30 nm when subjected to Zeta sizer, as shown in Figure 1b.

FTIR analysis was performed to examine the attached functional groups of biomolecules on the surface of AgNPs acting as capping/stabilizing agents. The FTIR spectrum showed the absorption bands at 678.9 cm⁻¹, 1639.5 cm⁻¹, 2144.8 cm⁻¹, 3298.2 cm^−1, and 3518 cm⁻¹ (Figure 1c). The peaks near 678.9 cm⁻¹ assigned to CH out of plane bending vibrations are substituted ethylene systems –CH=CH (cis). The band at 1639.5 cm⁻¹ corresponds to amide-I arising from carbonyl stretch –C=O) in proteins. The peaks at 2144.8 cm⁻¹ are assigned to the stretching vibration of –C–N of amide I, while the band at 3298.2 cm⁻¹ is assigned to C–H (methoxy compounds). The stretching vibration of aromatic compounds and 3518 cm⁻¹ corresponds to –OH stretching vibration, indicating the presence of alcohol and phenol in capping and stabilizing AgNPs.

The XRD analysis indicates the synthesis of the crystalline nature of silver nanoparticles (Figure 1d). The size of samples was 24.98, determined using the Debye–Scherrer formula, with values close to the determined by the DLS technique by zeta sizer. A single sharp peak recorded at 2θ degrees of 30.06 is assigned to plane 111, suggesting a face-centered crystalline structure. A scanning electron microscope was used to study the surface morphology of green synthesized silver nanoparticles. Smooth and spherical AgNP nanoparticles can be seen in Figure 1e.

3.2. Morphological and Molecular Identification of Pathogen (Alternaria solani)

The mycelial growth of all seven isolates was significantly diverse when grown on PDA media except for Shk-1 and Fsd-1. The growth range was between 48–70 mm in 7 days old culture, and maximum mycelial growth was shown by Bhl-1 (69.21 ± 0.090 mm) while minimum by M-1. The data of mycelial growth for all isolates, their color, and the nature of growth are given in

Table 1. Morphological characteristics of different A. solani isolates obtained from different areas of Punjab.

Isolates	Colony Size (mm)	Colony Color	Mycelial Growth	Conidia Length (µm)	Conidia Width (µm)	Beak Length (µm)
Lhr-1	64.26 e ±0.188	Whitish grey	Cottony Circular	16.14 c ±0.866	9.29 bc ±1.641	19.56 a ±1.867
Ksr-1	65.05 d ±0.159	Whitish brown	Cottony Circular	13.85 c ±2.066	7.66 bc ±0.613	23.15 a ±2.292
Shk-1	68.59 b ±0.111	Whitish brown	Cottony Circular	21.20 ab ±1.170	10.61 ab ±0.924	17.63 ab ±0.918
Fsd-1	68.56 b ±0.436	Whitish brown	Cottony Circular	23.33 a ±2.212	9.56 bc ±0.299	22.89 a ±0.966
Fsd-2	66.01 c ±0.076	Whitish brown	Cottony Circular	18.21 bc ±1.141	8.51 bc ±1.164	11.77 bc ±0.681
Bhl-1	69.21 a ±0.090	Whitish grey	Cottony Circular	23.68 a ±0.321	12.81 a ±0.574	6.53 c ±1.443
M-1	48.21 f ±0.015	Olive green	Not Cottony Circular	16.27 c ±0.841	7.19 c ±0.565	13.18 b ±1.098

Treatment mean for each treatment is average of the three replicates; ± represents standard error; letters represent variation by isolates using Duncan’s multiple range test.

. The conidia of isolates were straight, mostly oblong with a beak attached, and length without a beak varied between 13–24 µm. The conidia width was within the range of 7–12 µm, the minimum by M-1 (7.19 ± 0.565 µm), while the maximum was by Bhl-1 (12.81 ± 0.574 µm). The beak was straight to flexuous, and its length was more remarkable in Ksr-1 (23.15 ± 2.292 µm). The conidia of isolates had longitudinal (3–5 in Lhr-1, Ksr-1, Fsd-1, and M-1) and transverse septa (1–3 in Shk-1, Fsd-2, and Bhl-1). The color of conidia also varied from light to dark brown.

Seven ITS sequences were obtained from seven isolates using ITS1 and ITS4 as forward and reverse primers. The homology analysis of the sequences retrieved from GenBank using the basic local alignment search tool (BLAST) and obtained sequences was performed. All the obtained sequences showed 99.5–99.7% identity with Alternaria solani. Stemphylium sarciniforme was chosen as the outgroup. The acquired and retrieved sequences were aligned using MAFFT software with default settings. The final data set contained 495 positions, of which 469 were conserved, 75 variable sites, 70 parsimony uninformative, and 31 informative. The maximum likelihood method, implemented in MEGA11 based on the Jukes–Cantor model, inferred the phylogenetic tree. In the phylogram, the obtained sequences clustered with different sequences of Alternaria solani have an identity of 99.1–99.5% in the same clade, supporting our morphological results (Figure 2). The results are based on ITS regions; further analyses could discriminate more clearly the isolates from other species, such as A. tomatophila.

3.3. Pathogenicity Variability among Alternaria solani Isolates

The pathogenicity test for all seven isolates was conducted using a highly susceptible Rio Grande variety. The symptoms started appearing after three days of inoculation, but the data was recorded at regular intervals after 5, 10, and 15 days. The results revealed that isolates Shk-1, Fsd-1, and Fsd-2 were highly virulent and severely attacked the plants. Other isolates, i.e., Lhr-1, Ksr-1, Bhl-1, and M-1, were also virulent, but their disease incidence was lesser than the isolates mentioned above. The percentage of disease incidence increased with time, and the maximum was by Shk-1 (88.22% ± 1.103) and the minimum by M-1 (69.69% ± 3.059) after 15 days of inoculation. There was no significant difference in the size and number of lesions that appeared on leaves, as given in

Table 2. Pathogenicity study of each A. solani isolate against Rio Grande variety of tomato plant.

Isolate Name	Disease Incidence (%)			No. of Lesions/Leaf	Size of Lesion (mm2)	Virulency
	5 Days	10 Days	15 Days
Lhr-1	46.39 cd ±2.217	57.58 cd ±3.785	63.03 d ±2.389	5.00 a ±0.578	16.41 a ±2.132	Virulent
Ksr-1	52.93 b ±1.070	63.54 bc ±1.470	76.17 bc ±1.875	5.67 a ±0.334	19.67 a ±2.502	Virulent
Shk-1	61.69 a ±2.174	73.21 a ±2.566	88.22 a ±1.103	6.33 a ±0.883	20.77 a ±2.299	Highly Virulent
Fsd-1	51.00 bc ±0.958	69.54 ab ±2.058	85.58 ab ±4.616	6.00 a ±0.578	21.16 a ±1.860	Highly Virulent
Fsd-2	48.43 bcd ±1.681	68.37 ab ±3.278	85.60 ab ±3.640	6.67 a ±0.667	20.48 a ±0.168	Highly Virulent
Bhl-1	52.52 b ±0.667	69.62 ab ±2.935	75.32 c ±3.771	5.33 a ±0.667	19.24 a ±0.688	Virulent
M-1	44.48 d ±2.496	54.70 d ±0.818	69.69 cd ±3.059	5.67 a ±0.334	18.86 a ±1.199	Virulent

Treatment mean for each treatment is average of the three replicates; ± represents standard error; letters represent variation by isolates using Duncan’s multiple range test.

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3.4. In Vitro Antifungal Analysis of Silver Nanoparticles against Alternaria solani

Silver nanoparticles have shown antifungal activity and proved to be a potential fungicide against each isolate, inhibiting 70–80% growth, as shown in Figure 3 and Figure 4. The inhibition rate increased gradually as the concentration of silver nanoparticles increased from 5 to 50 ppm. The silver nanoparticles showed the maximum rate of inhibition against Shk-1, i.e., 81.10% compared to control, followed by Fsd-2 (79.39%), Bhl-1 (78.51%), Ksr-1 (76.97%), Lhr-1 (76.33%), Fsd-1 (75.34%), and M-1 (65.44%).

3.5. Screening of Susceptible Tomato Varieties against Alternaria solani

Six local and six hybrid varieties obtained from Ayyub Agriculture Research Institute, Faisalabad, Roshan seeds, Lahore, and Sheikhupura were tested against a highly virulent isolate, Shk-1 (Figure 5). The plants showed symptoms during the early stage of infection, but the data were recorded after 15 days of inoculation. Nagina and Red diamond varieties were tolerant against the Shk-1 isolate, showing 38.67% and 39.33% disease index, respectively. The other six varieties, Roma, Sultan, Sahel, Amber, Red stone, and Clara, were ranked as susceptible with 48%, 56%, 54.67%, 50.67%, 45%, and 40.67% disease index, respectively. Naqeeb was the highly susceptible variety, with an 80% disease index, followed by Nadar (74.67%), Rio Grande (73.33%), and Cristal (66.67%). The plants of the Naqeeb variety were highly injured, with 56.62% tissue damage. Infection also highly occurred among the Rio Grande, Sultan, and Cristal variety plants. There was a significant difference between lesions size appearing on different tomato varieties given in

Table 3. Response of different tomato varieties against A. solani.

	Parameters	No. of Lesions per Leaf	Size of Lesion (mm2)	Infected Area of Leaf (%)	Disease Incidence (%)	Disease Index (%)	Disease Reaction
Varieties
Local Varieties	Nadar	5.07 b ±0.372	23.20 def ±1.656	38.24 bc ±0.614	47.12 c ±2.935	74.67 bc ±1.766	Highly Susceptible
	Naqeeb	6.00 a ±0.116	30.14 bc ±1.063	54.98 a ±0.694	56.62 a ±3.636	80.00 a ±2.312	Highly Susceptible
	Nagina	3.80 cd ±0.347	25.40 cde ±1.731	29.16 efg ±1.223	28.06 fg ±0.502	38.67 g ±3.532	Tolerant
	Roma	3.27 d ±0.176	41.61 efg ±0.301	35.28 bcd ±2.849	25.10 fgh ±3.443	48.00 ef ±2.312	Susceptible
	Rio Grande	6.00 a ±0.2	41.53 a ±3.729	33.77 cde ±0.799	52.31 ab ±3.339	73.33 b ±1.335	Highly Susceptible
	Sultan	4.20 c ±0.116	34.13 b ±2.835	26.58 fg ±1.018	40.44 cd ±2.233	56.00 d ±2.312	Susceptible
Hybrid Varieties	Sahel	3.67 cd ±0.133	26.87 cd ±3.325	36.81 bcd ±0.727	31.58 ef ±1.878	54.67 de ±2.669	Susceptible
	Cristal	4.07 c ±0.176	29.36 bcd ±1.474	39.96 b ±4.139	45.71 bc ±0.371	66.67 c ±1.337	Highly Susceptible
	Amber	4.13 c ±0.291	17.82 fg ±0.367	31.59 def ±1.763	30.01 fg ±1.525	50.67 def ±1.335	Susceptible
	Red Stone	4.33 c ±0.241	16.76 g ±1.135	24.27 g ±2.505	23.74 gh ±2.305	45.33 fg ±2.669	Susceptible
	Red Diamond	3.37 d ±0.145	17.03 fg ±1.422	17.74 h ±0.894	18.98 h ±0.809	39.33 g ±2.909	Tolerant
	Clara	3.30 d ±0.067	18.27 fg ±0.754	31.54 def ±0.683	25.52 fgh ±0.656	40.67 g ±1.335	Susceptible

Treatment mean for each treatment is average of the three replicates; ± represents standard error; letters represent variation by verities using Duncan’s multiple range test.

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3.6. In Vivo Efficacy of Silver Nanoparticles against A. solani

Different concentrations of silver nanoparticles, 5, 10, 15, 20, 25, and 50 ppm, demonstrated an effective reduction in disease incidence compared to the control. The disease was reduced from 50% to 5% in both varieties by increasing the concentration of silver nanoparticles from 5 ppm to 50 ppm. The plants exposed to the pathogen showed more than 80% disease incidence compared to plants treated with silver nanoparticles. The amount of phenolics compounds, PAL, PO, and PPO, was also quantified. The amount of phenolic compound was significantly increased in silver nanoparticles treated plants at lower concentrations than in control. The production decreased at a higher concentration of silver nanoparticles but was more than the control (Figure 6a). The PO activity increased in treated tomato plants in the same way that phenolic activity increased. Lower concentrations of silver nanoparticles resulted in the highest PO activity in Nadar and Naqeeb (Figure 6b). The most increased activity was observed at 5 and 10 ppm compared to the control. Other concentrations ranging from 15 to 20 ppm exhibited 25% and 10% more activity than control in both varieties. However, activity was comparatively reduced at 25 and 50 ppm.

In the case of PPO activity, it was found in excess in tomato plants with lower concentrations of AgNPs (Figure 6c). Various concentration ranges from 5 to 15 ppm exhibited 20–60% activity in Nadar and 10–60% activity in Naqeeb, with maximum activity observed at 10 ppm (0.449±0.013 ∆495 nm/min/mg protein) in Nadar and 5 ppm (0.437±0.014 ∆495 nm/min/mg protein) in Naqeeb. PPO activity was lower at higher concentrations, i.e., 20, 25, and 50 ppm, than the control. These findings suggested that AgNPs could increase PPO activity, allowing plants to fight pathogenic stress and grow healthy. In plants treated with AgNPs, the same pattern was observed for PAL activity (Figure 6d). Plants treated with lower concentrations of silver nanoparticles showed increased PAL activity compared to the control. In comparison, plants treated with higher concentrations of silver nanoparticles showed the least PAL activity.

3.7. Principal Component Analysis (PCA)

Data variability of 95.71% (PC1 = 70.72%, PC2 = 24.99%) was revealed by PCA, as shown in Figure 7. Disease indices and growth rate of the pathogen on leaves were negatively correlated with phenolic compounds, PO, PPO, and PAL. The foliar application of 5 and 10 ppm of silver nanoparticles was most effective in crop protection, enhancing the defective mechanism and reducing the pathogen growth.

4. Discussion

Green nanoparticle synthesis is a more advanced, widespread, and remarkable area of nanotechnology [54]. Using plants to synthesize nanoparticles is easy, single-step, nonpathogenic, cost-effective, non-toxic, and sustainable as it uses renewable recourses and is environment friendly [10,55]. Various plants like neem [56,57], moringa [58], java plum [59], fig, rosemary [60], and many others have been reported to be used in the synthesis of silver nanoparticles. Silver nanoparticles possess unique optical properties due to surface plasmon resonance (SPR) generated from free electron movement. Therefore, interact with a specific wavelength of visible light reported between 400–500 nm and develops brown color in the solution depicting the synthesis of silver nanoparticles [61,62,63]. In the current study, AgNPs of 22–30 nm size and spherical shape were synthesized using leaves extract of the Neem plant and evaluated for their antifungal activity against Alternaria solani. The absorption peak of the AgNPs synthesized was also shown at 424 nm. FTIR analysis makes us understand the role of biomolecules in plant leaf extract for reducing silver to silver nanoparticles and acting as capping agents [64]. The FTIR analysis reveals the involvement of alkanes, alkynes, amines, carboxylic groups [65], methylene [66], and various other biomolecules in reducing and stabilizing green synthesized silver nanoparticles [67]. XRD pattern and SEM analysis of AgNPs synthesized by neem and other plants also suggest the synthesis of crystalline structure spherical silver nanoparticles [68,69,70]. Mohamed and Elshahawy [71] synthesized silver nanoparticles of 32–47 size using peel extract of orange (Citrus sinensis L.). UV–Vis spectrum showed an absorption peak at 435 nm corresponding to silver nanoparticles [66]. Chakravarty et al. [59] synthesized spherical-shaped silver nanoparticles using fruit extract of Syzygium cumini L., which showed a characteristic peak at 443 nm. The presence of OH and C=O confirmed the presence of secondary plant metabolites by FTIR spectroscopy. Green synthesized nanoparticles have tremendous biological applications in medicine and agriculture as antibacterial, antifungal, and anti-cancerous agents [72,73]. The current findings highlight the importance of green synthesized silver nanoparticles in disease reduction. Early Blight disease of tomatoes is widespread worldwide and devastatingly causes massive crop yield loss yearly. In the present study, we successfully isolated and identified A. solani, responsible for Early Blight disease in tomato plants. The identification of the pathogen is important for developing effective disease management strategies. The colony color, pattern, and muriform conidia with beak confirmed the presence of A. solani, and the variation among different isolates was seen clearly. The molecular characterization using PCR amplification and sequencing of the ITS region of the genome confirmed the identification of the isolated fungus as A. solani, with 99% sequence similarity to the A. solani reference sequences. The results of this study indicated that A. solani was highly pathogenic to tomato plants, causing significant damage to both the leaves and the fruits. The symptoms of Early Blight disease caused by A. solani included the formation of brown, circular lesions with concentric rings on the plant’s leaves. The virulence factors produced by A. solani allow the fungus to invade and colonize the plant tissues, leading to disease symptoms. The cultural, morphological, and genetic variability of A. solani, their virulency among different isolates, and resistance in some tomato varieties have been reported in recent decades. Riaz et al. [74] revealed the morphological and genetic variation among different isolates of A. solani in various areas of Punjab, Pakistan. All isolates showed radial growth, colony character, and conidial morphology variation. A field survey revealed the variation ranging from 9–74% incidence with 6.11–24% severity among different areas of Punjab, Pakistan [74]. Disease severity and incidence are so high in various regions because of continuous farming without rotation of crops, lack of proper disease management, and uninterrupted and rigorous farming. Lack of awareness among farmers has introduced genetic variation and resistant pathogens because of the unlimited use of fungicides due to lack of awareness among farmers [75,76,77]. Isolation and identifying actual disease pathogens could be fundamental tools for understanding disease progression and exploring the curative agents. As genus A. solani poses a significant threat to vegetables nowadays, thus it is of utmost importance to identify its different species to explore potential control measures [78]. Riaz et al. [74] also collected different isolates of A. solani, which showed smooth, circular, and greenish-black colony growth with variations in size. The conidia were single, usually flexuous or straight, with beak attached varied in size within the range of 68 to 88 μm [74].

Disease severity also depends upon the level of pathogenicity and its variation among different isolates of A. solani [79,80], as shown in the present study and a few previous reports [49,81,82]. It has become essential to control these resistant pathogens effectively using various methods [83]. Scientists have reported using green synthesized silver nanoparticles as a promising tool against different phytopathogenic fungi as a more effective substitute for synthetic fungicides commonly used by farmers [84,85,86]. Silver nanoparticles prepared using orange peel and pomegranate extract inhibited the mycelial growth of A. solani by almost 60–80% when treated with different concentrations [87]. Tyagi et al. [32] also reported in 2020 that biosynthesized nanoparticles can inhibit the absolute growth (100%) of A. solani at 100 ppm concentration. However, the inhibition rate varies with the change in the concentration of silver nanoparticles. Silver nanoparticles probably disrupt DNA replication and inactivate the functions of cellular proteins and enzymes, preventing the normal functioning of the pathogen [31,88]. Silver nanoparticles inhibit pathogen growth, prevent plants from establishing infection, and combat collateral damage [89]. The main components of several anti-pathogenic compounds secreted by plants, which serve as the first line of defense, are phenolic compounds. Silver nanoparticles stimulate the production of phenols in plants, which aids in the reduction of disease severity [90]. Foliar application of biosynthesized silver nanoparticles improved tomato plants’ first line of defense, as evidenced by increased phenolics and antioxidative enzyme production (PO, PPO, and PAL). Plants with increased resistance are better prepared to reduce ROS production after a pathogen attack, resulting in lower stress enzyme activity in plants treated with silver nanoparticles [91].

5. Conclusions

Early Blight disease of the tomato plant is a serious challenge for farmers, which affects crop yield due to pathogen variability. The identification and occurrence of A. solani in tomato crops were also investigated in the current study. The results showed that the pathogen is prevalent in tomato crops, highlighting the importance of developing effective disease management strategies. Green synthesized nanomaterials are a safe and effective alternative to chemical fungicides in controlling Early Blight disease and are regarded as the best option in this scenario. Green synthesis is a simple, environment-friendly, cost-effective, and easily scaled procedure for the large-scale synthesis of nanoparticles. The application of silver nanoparticles reduced the pathogenic growth of A. solani depending on the concentration. The different treatments of silver nanoparticles on tomato leaves increased the resistance in plants by producing phenolics and other antioxidants. Silver nanoparticles can be used for managing diseases, rapid disease detection, and enhancing the ability of plants to absorb nutrients. The findings of this study have significant implications for the development of safe and sustainable plant disease management strategies that can minimize the use of chemical fungicides and promote eco-friendly agricultural practices. Using green synthesized silver nanoparticles as an antifungal agent can reduce the risk of environmental pollution and toxicity associated with chemical fungicides. Overall, this study adds to the research on the behavior of nanoparticles in the ecosystem by providing important information about the effects of silver nanoparticles on plant oxidative responses.