In Vitro Antibacterial Activity of Green Synthesized Silver Nanoparticles Using Mangifera indica Aqueous Leaf Extract against Multidrug-Resistant Pathogens

An estimated 35% of the world’s population depends on wheat as their primary crop. One fifth of the world’s wheat is utilized as animal feed, while more than two thirds are used for human consumption. Each year, 17–18% of the world’s wheat is consumed by China and India. In wheat, spot blotch caused by Bipolaris sorokiniana is one of the major diseases which affects the wheat crop growth and yield in warmer and humid regions of the world. The present work was conducted to evaluate the effect of green synthesized silver nanoparticles on the biochemical constituents of wheat crops infected with spot blotch disease. Silver nanoparticles (AgNPs) were synthesized using Mangifera indica leaf extract and their characterization was performed using UV-visible spectroscopy, SEM, XRD, and PSA. Characterization techniques confirm the presence of crystalline, spherical silver nanoparticles with an average size of 52 nm. The effect of green synthesized nanoparticles on antioxidative enzymes, e.g., Superoxide dismutase (SOD), Catalase (CAT), Glutathione Reductase (GR), Peroxidase (POX), and phytochemical precursor enzyme Phenylalanine Ammonia-Lyase (PAL), and on primary and secondary metabolites, e.g., reducing sugar and total phenol, in Bipolaris sorokiniana infected wheat crop were studied. Inoculation of fungal spores was conducted after 40 days of sowing. Subsequently, diseased plants were treated with silver nanoparticles at different concentrations. Elevation in all biochemical constituents was recorded under silver nanoparticle application. The treatment with a concentration of nanoparticles at 50 pp min diseased plants showed the highest resistance towards the pathogen. The efficacy of the green synthesized AgNPs as antibacterial agents was evaluated against multi drug resistant (MDR) bacteria comprising Gram-negative bacteria Escherichia coli (n = 6) and Klebsiella pneumoniae (n = 7) and Gram-positive bacteria Methicillin resistant Staphylococcus aureus (n = 2). The results show promising antibacterial activity with significant inhibition zones observed with the disc diffusion method, thus indicating green synthesized M. indica AgNPs as an active antibacterial agent against MDR pathogens.


Introduction
Wheat (Triticum spp.), the so-called "King of cereals" is considered one of the oldest staple food crops grown worldwide, with an annual production of 734 million tonnes [1].

Preparation of the M. indica Aqueous Leaf Extract
The aqueous M. indica leaf extract was prepared following the protocol described by Narayana et al. with minor modifications. Thus, 10 g of M. indica leaves powder was boiled in 100 mL of distilled water for 30 min (10% aqueous plant extract) and filtered through Whatman No. 1 filter paper. The filtrate was collected and stored in the refrigerator at 4 • C until further use.

Green Synthesis of Silver Nanoparticles (AgNPs) Using M. indica Aqueous Leaf Extract
The AgNO 3 solution was used as a precursor for the synthesis of silver nanoparticles (AgNPs). Hence, 0.02 M solutions were prepared by the addition of 1.7 g of AgNO 3 to 500 mL of distilled water. For the green synthesis of AgNPs we used 0.02 M silver nitrate (AgNO 3 ) as a precursor and 10% M. indica aqueous leaf extract as both reducing and capping agent [14]. Leaf extract and AgNO 3 (0.02 M) solutions were mixed in the ratio of 9:1 and kept for reaction in the dark for approximately 24 h. The plant extract reduced the silver nitrate and led to the formation of AgNPs. The change in solution color from brown to blackish indicated the formation of AgNPs. The reduction process was monitored by using UV-Visible spectroscopy.

Characterization of Green Synthesized Silver Nanoparticles (AgNPs)
The synthesized nanoparticles were characterized in terms of shape, size, and morphology using UV-Visible spectrophotometry (UV-Vis), particles size analyzer (PSA), scanning electron microscope (SEM), and X-ray diffraction (XRD). Spectral analysis of AgNPs was performed using an ELICO UV1900, Hyderabad, India, Double Beam Spectrophotometer with spectrum scanning in the wavelength range of 190-1100 nm with a band width of 2 nm. Green nanoparticles were analyzed at a wavelength ranging from 200 to 700 nm. Characterization of AgNPs in terms of mean diameter and distribution was performed using PSA (Nicomp, NANOZ Z3000 PSS, Billerica, MA, USA). To assess the topology of green synthesized nanoparticles, SEM (Carl Zeiss-EVO-18-Cambridge, UK) was used. An X-ray Diffractometer (Powder) (Model: SmartLab SE, Tokyo, Japan) with fully automated alignment under computer control SAXS capabilities, with Optional D/teX Ultra-high-Antibiotics 2022, 11, 1503 4 of 20 speed, position-sensitive detector system with 2D: 2-150 • , was used to understand the structural information of AgNPs.

Biochemical Analysis
The seeds were procured from the Durum Wheat Main Agricultural Research Station of the AICRP (All India Coordinated Research Projects), at the University of Agricultural Sciences in Dharwad. The tests were performed in a factorial complete-replicate design with three replicates. The variety of durum wheat employed in this study was Bijaga Yellow which is a tall durum wheat variety cultivated in rain fed regions.
Wheat plant was evaluated by pot culture experiments in a polyhouse. Blast disease was inoculated artificially after 40 days of sowing. The AgNPs treatment was given after the development of disease as described in the study design (Table 1). Leaves samples were collected after 2-3 days of treatment for determining the reducing sugar and total phenol and antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), peroxidase (POX), and phenylalanine ammonia lyase (PAL).

Extraction of Enzyme and Determination of Protein Content
In the present work, enzymes were extracted from freshly collected leaf tissues at 0-4 • C. The antioxidant enzyme activities of SOD, CAT, POX, GR, and PAL were determined by extracting 0.5 g of leaf tissues with 2 mL of respective buffers (0.05 M sodium phosphate buffer of pH 7.8 for SOD, pH 7.0 for CAT and POX, 0.1 M Tris-HCl buffer of pH 7.8 containing 2 mM dithiothreitol for GR and 0.1 M Tris buffer of pH 8.5 for PAL). The homogenates were centrifuged at 14,000 rpm for 20-min at 4 • C. The resulting supernatant was used as an enzyme source for determining antioxidant activities and the protein content was measured using Lowry's method.
2.7. Antioxidant Enzyme Assays 2.7.1. Catalase CAT (EC 1.11.1.6) activity of Wheat leaves was analyzed using the Beers and Sizers spectrophotometric technique [15]. To commence the reaction, 20 µL of enzyme extract was added to a reaction mixture containing 2.98 mL of 16.65 mM hydrogen peroxide in 50 mM phosphate buffer, pH 7.0. The decrease in absorbance was monitored at 240 nm against substrate blank for a period of 3 min. Specific activity was expressed as µM min −1 mg −1 protein and one unit of CAT was defined as 1 µM of H 2 O 2 degraded per minute at pH 7.0 and 25 • C.

Superoxide Dismutase
Using the Beauchamp and Fridovich method [16], the SOD activity in wheat leaves was measured photochemically at 560 nm. The composition of 3 mL of reaction mixture included 50 mM phosphate buffer (pH 7.8), 20 µL enzyme extract, 10 mM L-methionine, 33 µM p-nitroblue tetrazolium chloride (NBT), 0.66 µM ethylene diamine tetra acetic acid (EDTA), and 3.3 µM riboflavin. The addition of riboflavin was immediately followed by illuminating the glass tube with the light of a 15 W fluorescent lamp at 25 • C for 20 min. Absorbance at 560 nm was read to determine the concentration of blue formazan formed by NBT photoreduction against a blank which comprised of same reaction mixture excluding the exposure to light. The specific activity of SOD is measured in international units (IU) per milligram of protein, and one unit of SOD is defined as the quantity of enzyme needed to inhibit 50% of the NBT photo-reduction per minute.

Peroxidase Activity
Peroxidase activity (POX, EC 1.11.1.7) in wheat leaves was measured kinetically by the method of Chance and Maehly [17]. The addition of 20 µL of the enzyme extract to reaction mixture, containing 2.88 mL of 0.1 M potassium phosphate buffer (pH 7.0), 50 µL of 0.02 M guaiacol, and 50 µL of 0.042% hydrogen peroxide, initiated the reaction and the increase in optical density at 436 nm was recorded for 5 min. The specific activity of POX was quantified as µM of oxidized guaiacol produced per minute per milligram of protein.

Glutathione Reductase
The spectrophotometric quantification of GR (EC 1.8.1.7) activity in wheat leaves was performed following the method of Mavis and Stellwagen [18]. Composition of reaction mixture included 0.1 mL of 30 mM oxidized glutathione, 1.5 mL of 100 mM potassium phosphate buffer with 3.4 mM EDTA, pH 7.6, 0.35 mL of 0.8 mM reduced ß-nicotinamide adenine dinucleotide phosphate (NADPH), and 0.95 mL of distilled water. The addition of 100 µL of enzyme extract commenced the reaction and the reduction in absorbance at 340 nm was monitored for 5 min. GR specific activity is represented as µM min −1 mg −1 protein, and one GR unit is equal to the quantity of enzyme required to oxidize 1 µM of NADPH per minute at pH 7.6 and 25 • C.

Phenylalanine Ammonia Lyase
Using the spectrophotometric approach developed by Paltonen and Karjalainen [12], the PAL (EC.4.3.1.5.) activity in wheat leaves was measured. The assay mixture containing 0.5 mL of enzyme extract and 2.5 mL of 0.2% L-phenylalanine in 0.1 mL of Tris buffer, pH 8.5 was incubated for 1-h at 40 • C and the reaction was terminated with 0.2 M HCl. The absorbance was measured at 290 nm against a substrate blank. The specific activity of PAL was reported in units of µM of trans-cinnamate released per minute per milligram of protein. One unit of PAL is defined as the quantity of enzyme necessary to liberate 1 µM of trans-cinnamate from L-phenylalanine per minute.

Estimation of Reducing the Sugar
For the quantitative analysis of reducing sugars in wheat leaves, 1 g of dried leaf material was introduced in hot ethanol (80%) for 15 min and then homogenized with pestle and mortar. The homogenate thus obtained was filtered using muslin cloth, the residue was re-extracted two or three times, and the filtrate were made up to final volume of 10 mL with 80% ethanol. Reducing sugars were quantified by employing Nelson Somogi's method (Norton Nelson, 1944) in which the reducing sugars in alkaline medium reduce cupric ions to cuprous ions which form a chromophore with arsenomolybdate reagent and have a λmax at 510 nm. The reducing sugar was expressed as mg per gram dry weight.

Estimation of Total Phenols
The Folin-Ciocalteau reagent (FCR) method was used to estimate the total phenols present in wheat leaf samples. The reaction between phenols and the oxidizing agent phosphomolybdo-phosphotungstate in FC reagent results in the formation of blue-colored chromophore with a maximum absorption at 660 nm. The amount of phenol has a direct correlation with the development of color with the reagent. Total phenols were extracted using 10.0 mL of hot 80% alcohol with 1 g of dried leaf as the starting material (Sadasivam and Manikam, 1992). The colorimetric method of (Bray and Thorpe, 1954.) was used for the determination of total phenols using the Folin-Ciocalteau reagent. The phenol content was expressed as mg per gram dry weight.

Bacterial Strains Used in This Study
We used 15 multidrug-resistant (MDR) bacteria comprising gram negative bacteria-Escherichia coli (n = 6), Klebsiella pneumoniae (n = 7) and gram-positive bacteria-Methicillin Resistant Staphylococcus aureus (n = 2). These clinical strains were phenotypically and genotypically characterized in our previous study and found to possess wide spectrum of beta lactamase genes responsible for antibiotic resistance [17]. ATCC 25922 E. coli was used as a control strain. The antibacterial activity of MNPs against the selected MDR bacterial (n = 15) strains was carried out using the Kirby-Bauer disc diffusion method [18]. The bacterial strains were grown until attaining McFarland standard O.D of 0.5. The inoculum was spread in Muller Hinton Agar (MHA) (Himedia, India) using sterile cotton swabs and sterile antimicrobial susceptibility disks were loaded on the plates with 10 µL of MNP. The plates were incubated at 37 • C for 48 h and zone of inhibition was recorded.

Statistical Analysis
The data of the experiment were analyzed statistically by the procedure described by Gomez and Gomez [19]. The addition of M. indica leaf extract to the silver nitrate solution led to the change in color from brown to dark blackish-brown, indicating the formation of AgNPs due to the reduction of silver ions by the reducing agents present in the M. indica leaf extract. Further confirmation of AgNPs formation was performed using the UV-visible spectrophotometer. Surface plasmonic resonance absorbance range between 410 and 480 nm was, in particular, used as an indicator to confirm the reduction of Ag+ to metallic Ag o [20]. The strong and broad surface plasmonic resonance (SPR) peak centered at 475 nm and 410 nm confirmed the formation of AgNPs ( Figure 1).

AgNPs Particle Size Distribution Study by Particle Size Analyzer (PSA)
Nanoparticles size distribution and mean diameter of green synthesized AgNPs using M. indica aqueous leaf extract were characterized using PSA (Nicomp NANOZ Z3000 PSS). PSA showed a mean diameter at 52.8 nm and nanoparticle size distribution from 5 nm to 220 nm ( Figure 2). ter. Surface plasmonic resonance absorbance range between 410 and 480 nm was, in particular, used as an indicator to confirm the reduction of Ag+ to metallic Ag o [20]. The strong and broad surface plasmonic resonance (SPR) peak centered at 475 nm and 410 nm confirmed the formation of AgNPs ( Figure 1).

SEM Micrographs of Green Synthesized AgNPs
Scanning electron microscopy imaging was carried out to view the morphology and size of the silver nanoparticles. SEM micrograph obtained for the AgNPs synthesized using M. indica leaf extract showed high-density nanoscale particles (52 nm) with spherical shape (Figure 3). SEM is a surface imaging technique that can resolve micro-and nanoscale differences in particle size, particle size distribution, nanomaterial type, and particle surface morphology. Through scanning electron microscopy (SEM), we can analyze particle morphology and create a histogram from images, either manually by measuring and

SEM Micrographs of Green Synthesized AgNPs
Scanning electron microscopy imaging was carried out to view the morphology and size of the silver nanoparticles. SEM micrograph obtained for the AgNPs synthesized using M. indica leaf extract showed high-density nanoscale particles (52 nm) with spherical shape (Figure 3). SEM is a surface imaging technique that can resolve micro-and nanoscale differences in particle size, particle size distribution, nanomaterial type, and particle surface morphology. Through scanning electron microscopy (SEM), we can analyze particle morphology and create a histogram from images, either manually by measuring and counting the particles or automatically using specialized software. Our findings are in line with previous studies involving nanoparticles synthesized using Raphanus sativus and Vitex leucoxylon extract [21,22].

Reducing Sugar
Levels of reducing sugar in the AgNPs -treated plants showed a slight increase when compared to the diseased wheat plant (2.73 g % dry weight) ( Table 2). Among the treatments, plants treated with a higher concentration of silver nanomaterials (30 ppm: 6.94 g % and 50 ppm: 7.82 g % dry weight) showed a phenomenal increase in reducing sugar content compared to plants treated with lower concentration (5 ppm: 4.44 g %, 10 ppm: 5.00 g %, 15 ppm: 5.45 g %, and 20 ppm: 5.87 g % dry weight) and diseased plant (2.34 g % dry weight). In comparison to control, the disease inoculated wheat plant leaves recorded a reduction in reducing sugar content (2.73 g % dry weight) as compared to control leaves (10.61 g % dry weight).   Table 2). Among the treatments, plants treated with a higher concentration of silver nanomaterials (30 ppm: 6.94 g % and 50 ppm: 7.82 g % dry weight) showed a phenomenal increase in reducing sugar content compared to plants treated with lower concentration (5 ppm: 4.44 g %, 10 ppm: 5.00 g %, 15 ppm: 5.45 g %, and 20 ppm: 5.87 g % dry weight) and diseased plant (2.34 g % dry weight). In comparison to control, the disease inoculated wheat plant leaves recorded a reduction in reducing sugar content (2.73 g % dry weight) as compared to control leaves (10.61 g % dry weight).

Total Phenol
Total phenol content in all nano-treated plants showed a slight increase when compared to the diseased wheat plants (1.65 g % dry weight). Among the treatments, plants treated with a higher concentration of AgNPs (30 ppm: 1.98 g %, 50 ppm: 2.09 g % dry weight) showed a phenomenal increase in total phenol content compared to low concentration (5 ppm: 1.72 g %, 10 ppm: 1.76 g %, 15 ppm: 1.81 g %, and 20 ppm: 1.85 g % dry weight) and diseased plant (1.65 g % dry weight). In comparison to control, the disease inoculated wheat plant leaves recorded a higher total phenol content (1.65 g % dry weight) as compared to control leaves (1.2 g % dry weight) ( Table 3).

Estimation of CAT Activity
CAT activity increased by nearly 22.22% in the diseased plants compared to the control plants. All AgNPs treated plants showed elevation in CAT activity in all different treatments. At 30 ppm: 492 U/mg protein and 50 ppm: 583 U/mg protein, the AgNPs treated plants showed significant elevation in enzyme activity and a reduction in disease severity. In T2, T3, T4, and T5, crops showed elevation in enzyme activity but no significant reduction in disease severity. At 5 ppm: 24.49%, 10 ppm: 22.76%, 15 ppm: 30.29%, and 20 ppm: 28.05% elevation in enzyme activity was recorded (Table 5).

Estimation of POX Activity
POX activity increased by nearly 40.61% in the diseased plants compared to the control plants. All AgNPs treated plants showed elevation in POX activity in all different treatments. At 30 ppm: 3.00 U/mg protein (61.00%) and 50 ppm: 3.24 U/mg protein (63.89%), the nanoparticle treated plants showed significant elevation in enzyme activity and a reduction in disease severity. In T2, T3, T4, and T5, plants showed elevation in enzyme activity but no significant reduction in disease severity. At 5 ppm: 52.24%, 10 ppm: 56.5%, 15 ppm: 57.30%, and 20 ppm: 59.52% elevation in enzyme activity was recorded (Table 6).

Estimation of GR Activity
GR activity increased by nearly 39.37% in the diseased plant compared to the control plant. All AgNPs treated plants showed elevation in GR activity in all different treatments. At 30 ppm: 3.79 U/mg protein and 50 ppm: 4.00 U/mg protein, the nanoparticle treated plant showed significant elevation in enzyme activity and a reduction in disease severity. In T2, T3, T4, and T5, wheat plants showed elevation in enzyme activity but no significant reduction in disease severity. At 5 ppm: 49.00%, 10%: 49.50%, 15 ppm: 53.0%, and 20 ppm: 54.70% elevation in enzyme activity was recorded (Table 7).

Estimation of PAL Activity
PAL activity increased by nearly 32.98% in the diseased plant than in the control plant. All AgNPs treated plants showed elevation in PAL activity in all different treatments. At 30 ppm: 6.36 U/mg protein and 50 ppm: 6.78 U/mg protein, the nanoparticle treated plants showed significant elevation in enzyme activity and a reduction in disease severity. In T2, T3, T4, and T5, crops showed elevation in enzyme activity but no significant reduction in disease severity. At 5 ppm: 34.92%, 10 ppm: 34.92%, 15 ppm: 33.45%, and 20 ppm: 39.37% elevation in enzyme activity was recorded (Table 8).

Antibacterial Activity against MDRs
The AgNPs showed antibacterial activity against all the MDR pathogens included in this study. The results of disc diffusion assay are summarized in Table 9. The clearly visible zone of inhibition exhibited by MNP against the Gram-negative and -positive MDR pathogens is depicted in Figure 5. The scientific name of the bacterial strain used in the study is presented in Table 10. K. pneumoniae KP-2 11 10.
MRSA-2 16 Table 10. Scientific name/ strain for the organisms used in the study.

Green Synthesis of AgNPs by Using M. indica Leaf Extract and Their Characterization
The present study reports the successful synthesis and characterization of green synthesized AgNPs using M. indica aqueous leaf extract. Green synthesis of AgNPs using different plant sources has been well documented in the literature [14,25]. In the present study, we chose M. indica leaf extract for the green synthesis of AgNPs because it has a significant amount of reducing activity and capping properties due to the presence of phytochemicals, e.g., alkaloids, phenols, flavonoids, alkaloids, fats, amino acids, saponins, and oils. These bioactive compounds efficiently reduce silver salts (Ag + ) to Ag 0 [26]. Another reason for M. indica leaf selection is that some of the phytochemicals present in the plant extracts exhibit antimicrobial properties. In addition, M. indica leaf extracts not only efficiently synthesize silver nanoparticles, but also do so through capping.
Green synthesized AgNPs were characterized for their mean size, distribution, shape, morphology, and topography of nanoparticles. In this study, phytosynthesized AgNPs were initially identified by the change in color from light brown to blackish, indicating the formation of AgNPs. The present results were reported from colorless to brown grey in previous studies [27,28].
Further confirmation of AgNPs formation was ascertained by a surface plasmon resonance peak centered at 475 nm with a median nanoparticle size of 52 nm. A similar AgNPs size was observed in other studies. For instance, M. indica leaf extract based AgNPs size was reported to be 20-55 nm with plasmon resonance peak at 452 nm, 5-40 nm with plasmon resonance peak at 470 nm [29], 10-26 nm with surface plasmon resonance peak at 450 nm [30], and 32 nm with a surface plasmon peak at 393 nm [31]. Catharanthus roseus based silver nanoparticles were evaluated as 20-50 nm with a peak absorbance at 500 nm [27], 49 nm with surface plasmon resonance absorption peak at 425 nm [32], and 40-60 nm [33]. AgNPs other than the spherical shape can also contribute to the shift in surface plasmon resonance peak towards 500 nm and above.
SEM and XRD results confirmed the presence of AgNPs. SEM micrographs showed spherical shape AgNPs and the presence of crystalline AgNPs through the XRD spectrum analysis [34,35]. The SEM micrograph revealed phytosynthesized AgNPs using Catharanthus roseus leaf extract present as a bunch form in shape [33].

Reducing Sugars
Sugars constitute the primary biomolecule providing energy and structural material for defense responses in plants, and also act as signal molecules interacting with the plant immune system. Sugars cause oxidative burst at the early stages of infection, increasing the lignification of cell walls, stimulating the synthesis of flavonoids, and inducing certain PR proteins. Some sugars act as priming agents, inducing higher plant resistance to pathogens reported [36].
In the present study, the response of AgNPs applied to wheat crops infected with spot blotch was studied to correlate the role of reducing sugar content upon spot blotch infection and nanoparticle application. As per the results obtained (Table 1), the control plant recorded the highest reducing sugar content, and the lowest reducing sugar content was observed in the spot blotch infected plant(T 1 ). Spot blotch induced a decrease in sugar content. All other treatments showed elevation in reducing sugar content after the application of silver nanoparticles.
Carbohydrates and proteins are the primary metabolites that are exploited by biotic stress inducers for their growth and development. These primary metabolites also function as a precursor for secondary substances, which play a major role in plant defense [37]. Alberto (2014) [38] observed a decrease in reducing sugar content in the disease-infected plant compared to the healthy plants in their study on biochemical changes in Theiumcepa L. infected with anthracnose. Kumar et al. (2011) [39] reported that reducing sugar was decreased after infection of spot blotch infection in barley. Yanik F and Vardar F (2019) [40] observed the elevation in reducing sugar content in wheat plants applied with silver nanoparticles at different concentrations. Krishnaraj et al. (2012) in their study on biologically synthesized AgNPs found that these particles exerted a slight stress condition on the growth and metabolism of B. monnieri [41] accompanied by an elevation in total sugar content based on concentration applied.

Total Phenol
Phenols play an important role in the plant defense system against plant pathogens, insects, and the cyclic reduction of reactive oxygen species (ROS), such as superoxide anion and hydroxide radicals, H 2 O 2 , and singlet oxygen [42].
In the present study, the response of AgNPs applied to wheat plants infected with spot blotch was studied to correlate the role of total phenol content upon spot blotch infection and nanoparticle application. As per the results obtained (Table 2) among control and disease infected wheat crops, the healthy plant showed a low amount of total phenol. AgNPs treated spot blotch infected wheat plants showed elevation in total phenol content correlating the concentration of nanoparticles applied, and reduction in severity was also observed at higher concentration nanoparticle application.
Similar results were reported earlier whereby rust-resistant sorghum genotypes recorded higher phenols as the age of the crop increased [43]. A similar study on spot blotch (Bipolaris sorokiniana) in healthy and infected leaves of barley (Hordeum vulgare) indicated that resistant genotypes had more amount of total phenols content than susceptible genotypes [44]. Phenols inhibit disease development through different mechanisms like inhibition of extracellular fungal enzymes (cellulase, pectinase, laccase and xylanase), fungal oxidative phosphorylation, nutrition deprivation and antioxidant activity in plant tissue [45]. A similar study on the effect of nanoparticle application on spot blotch infected barley biochemical systems also showed an elevation in total phenol content associated to nanoparticle application concentration [39].

Superoxide Dismutase
SOD catalyzes the dismutation of superoxide radicals to hydrogen peroxide and oxygen, and constitutes the most important enzyme in cellular defense, because its activation directly modulates the amount of superoxide anionand H 2 O 2 [46]. SOD activity may reduce the risk of hydroxyl radical formation. SODs are classified into three major groups based on their metal cofactor, and they are iron SOD (Fe-SOD) and copper-zinc SOD (Cu/Zn-SOD) are localized in the chloroplast, cytosol; manganese SOD (Mn-SOD) is found mainly in mitochondria and peroxisomes [47].
In the present study, biochemical changes in AgNPs treated wheat plants infected with spot blotch disease were analyzed. The wheat plants showed elevation in the SOD activity in all silver nanoparticles treated conditions when compared with the control. As per the results obtained (Table 4), all the AgNPs treated wheat plants showed an elevation in SOD activity, whereas only the 30 ppm and 50 ppm silver AgNPs treatment plants showed a reduction in spot blotch severity and elevation in SOD activity.
Similar results were observed [41] in studies on the effect of AgNPs application on B. monnieri biochemical activity and the treated plants showed an elevation in SOD activity at 100 ppm AgNPs treatment. Similar results were observed by Du et al. (2015), who studied the effect of AgNPs application on wheat plants and recorded an elevation in SOD activity at 100 mg/kg nanoparticle application [48].

Catalase
Plants possess several antioxidant enzymes that eliminate reactive oxygen species (ROS). CAT enzymes have a defensive role against ROS. They are responsible for the removal of toxic H 2 O 2 in the cells, thereby protecting the cells from getting damaged. Up to a certain level, ROS production under stress may work as a signal for triggering defense responses via transduction pathways [49,50]. A high amount of ROS production in the cell causes cell death.
In the present study, the wheat plants showed elevation in the catalase activity in all AgNPs treated conditions when compared with a control. As per the results obtained (Table 4), all the silver nanoparticle treated wheat plants showed elevation in CAT activity but there was no reduction in disease severity in other treatments, except at 30 ppm and 50 ppm AgNPs treatment, where the infected plants showed a reduction in spot blotch severity and elevation in catalase activity.
Similar results were obtained in the study on AgNPs induced morphological, physiological, and biochemical changes in the wheat plant. Elevation in all of the antioxidative enzymes and phenol content was recorded [51]. A similar study conducted by Mohamed et al., in 2017, which also showed corresponding biochemical changes in a crop treated with nanoparticles [51,52].

Peroxidase (POX)
A heme-containing protein called peroxidase (POX) preferentially oxidizes aromatic electron donors at the expense of H 2 O 2 . By using oxidative polymerization, POX facilitates the conversion of cinnamyl alcohol to lignin. Vascular plants have many POX genes, and each class of these genes may have a unique physiological function in preventing oxidative damage to the cell membrane [53].
In the present study, the wheat plants showed elevation in the peroxidase activity in all of the AgNPs treated conditions when compared with a control. As per the results obtained (Table 5), all the AgNPs treated wheat plants showed elevation in POX activity, but there was no reduction in disease severity in other treatments except at 30 ppm and 50 ppm AgNPs treatment. With 30 ppm and 50 ppm silver nanoparticle treatment, crops showed a reduction in spot blotch severity and elevation in peroxidase activity was recorded. The present study indicates the crop showed more resistance towards disease at 30 and 50 ppm silver nanoparticles, but all treatments showed elevation in all plant biochemical constituents.
Numerous fungal species, including Trichosporium vesiculosum, Macrophomin phaseolina, Coprinus comatus, Mycophaerella arachidicola, Fusarium exosporium, and Botrytis cenere, have been shown to be resistant to POX in some studies [53,54]. Following inoculation with stem and leaf rust pathogens, low infection type isogenic lines of wheat showed a quicker increase in POX activity than sensitive isogenic lines of wheat [55].

Glutathione Reductase
Another important pathway involved in the control of ROS levels in the plant tissue is the ascorbate/glutathione cycle in which Glutathione reductase plays a major role [56]. GR is a flavoprotein oxidoreductase that catalyzes the reduction of glutathione disulphide to the sulfhydryl form GSH. This enzyme employs NADPH as a reductant.
As per the results obtained (Table 6), all treatments showed elevation in GR activity, but a reduction in disease severity was observed in only 30 ppm and 50 ppm AgNPs treatment conditions. In the present study, the wheat plant showed elevation in the GR activity in all AgNPs treated conditions when compared with the control.
Similarly, a rapid elevation in GR content was observed by Yanik F and Vardar F, 2019, in their study on silver nanoparticle-induced morphological, physiological, and biochemical changes in the wheat plant [40]. An elevation in all antioxidant enzyme and phenol content was recorded.

Phenylalanine Ammonia-Lyase
The primary enzyme in the plant phenylpropanoid pathway, phenylalanine ammonialyase catalyzes the synthesis of secondary metabolites from L-phenylalanine, such as lignin, flavonoids, and phytoalexins. L-phenylalanine is non-oxidatively deaminated by the PAL enzyme, resulting in the formation of trans-cinnamic acid and a free ammonium ion. Phenylalanine, an amino acid, is converted to trans-cinnamic acid in plants, which is the first step in the transfer of carbon from primary metabolism to phenylpropanoid secondary metabolism.
As per the results obtained (Table 7) among all the treatments, T6 and T7 recorded the highest PAL activity, whereas the lowest PAL activity was observed in the control plant. Spot blotch infected plants also showed a small elevation in PAL activity. In the present study, biochemical changes in AgNPs treated wheat crops infected with spot blotch disease were analyzed. The wheat plant showed elevation in the peroxidase activity in all AgNPs treated conditions when compared with the control condition. As per the results obtained, all the AgNPs treated wheat plants showed elevation in PAL, but no reduction in disease severity was observed. With 30 ppm and 50 ppm silver nanoparticle treatment, crops showed a reduction in spot blotch severity and elevation in PAL activity was recorded. The present study indicates the crop showed more resistance towards disease at 30 ppm and 50 ppm, but all treatments showed elevation in all plant biochemical constituents.
It is possible that the rapid induction of PAL genes in resistant interactions between groundnut and its pathogen/elicitor is caused by the involvement of a signal transduction mechanism. This mechanism is triggered specifically as a result of the interaction between elicitor and receptor molecules, thereby showing differential transcriptional rates of PAL compatible and incompatible interactions. A mechanism quite similar to this one was postulated earlier in several plants [56,57]

Conclusions
Silver nanoparticles were successfully synthesized by using Mangifera indica leaf extract with a size below 100 nm. The formation of AgNPs was confirmed using various analytical techniques, such as UV-visible spectrophotometer, particle size analyzer (PSA), scanning electron microscopy (SEM), and X-ray diffraction. Biochemical analysis of silver nanoparticle treated wheat crop infected with spot blotch showed elevation in all biochemical constituents, e.g., reducing sugar, total phenol, superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), glutathione reductase (GR), and phenylalanine ammonia-lyase (PAL). Elevation in all biochemical constituents was recorded under all treatment conditions but a reduction in disease severity was recorded only in 30 ppm and 50 ppm concentration AgNPs treatments. The overall study revealed that the higher amount of reducing sugar, total phenols, antioxidant enzyme activity, and phytochemical precursor enzyme PAL plays an important role in the defense mechanism of plants against wheat spot blotch infection. Moreover, this study revealed that silver nanoparticles application on wheat crops causes the elevation in antioxidative enzyme activity and the amount of total phenol, reducing sugar content, and promising antibacterial activity against MDR strains. Data Availability Statement: All the data has been presented in this article.