A Novel Based Synthesis of Silver/Silver Chloride Nanoparticles from Stachys emodi Efficiently Controls Erwinia carotovora, the Causal Agent of Blackleg and Soft Rot of Potato

In recent years, the biological synthesis of silver nanoparticles has captured researchers’ attention due to their unique chemical, physical and biological properties. In this study, we report an efficient, nonhazardous, and eco-friendly method for the production of antibacterial silver/silver chloride nanoparticles utilizing the leaf extract of Stachys emodi. The synthesis of se-Ag/AgClNPs was confirmed using UV-visible spectroscopy, DPPH free radical scavenging activity, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and X-ray diffraction (XRD). An intense peak absorbance was observed at 437 nm from the UV-visible analysis. The Stachys emodi extract showed the highest DPPH scavenging activity (89.4%). FTIR analysis detected various bands that indicated the presence of important functional groups. The SEM morphological study revealed spherical-shaped nanoparticles having a size ranging from 20 to 70 nm. The XRD pattern showed the formation of a spherical crystal of NPs. The antibacterial activity performed against Erwinia carotovora showed the maximum inhibition by centrifuged silver nanoparticles alone (se-Ag/AgClNPs) and in combination with leaf extract (se-Ag/AgClNPs + LE) and leaf extract (LE) of 98%, 93%, and 62% respectively. These findings suggested that biosynthesized NPs can be used to control plant pathogens effectively.


Introduction
Plant growth and development are affected by various biotic and abiotic factors affecting its yield and quality [1]. It is estimated that the world population is going to increase up to 10 billion by 2050 which will pressurize farmers for nutritious and safe food production in near future. The present food production systems are mainly threatened by diseases, pests, microorganisms, drought, and sudden climate changes [2].
Among these, phytopathogens are causing serious diseases in agricultural crops, resulting in global food insecurity [3]. The increased demand for vegetables and fruits such as potatoes, tomatoes, and eggplants has employed about 800 million individuals and contributes more than 33% of the world's agricultural production [4]. The agricultural productivity of vegetables and fruits decreases due to the diseases caused by phytopathogens, which in turn increase the market price of such products [5]. Potato (Solanum tuberosum L.) is considered to be an attractive crop in the agricultural sector due to its high nutritional value as it is a good source of vitamins, proteins, energy, minerals, and carbohydrates [6]. Globally, potato is one of the most consumed foods, occupying fourth position after corn, rice, and wheat [7]. Potato is an important crop; it is not only utilized as a source of food, but as a feedstock for other industrial products. Potato crop yield is not only affected by

DPPH Assay
The widely used method for determining free radical scavenging uses DPPH, a free radical with great stability. The S. emodi plant extracts showed strong antioxidant activity when they were assayed through DPPH free radical scavenging activity. The results showed that the S. emodi plant extract exhibited the highest DPPH scavenging activity (89.4) for the 1000 µg/mL concentration. Similarly, the obtained results of the plant sample concentration were compared with those of standard ascorbic acid ( Figure 1).

Characterization of Silver/Silver Chloride Nanoparticles
The color of the solution started turning brown immediately after placing the solution in sunlight and turned completely dark brown after 20 min. This was due to the reduction of silver ion to silver/silver chloride nanoparticles in the reaction mixture [38]. The silver nanomaterial synthesis was achieved by using varying volumetric ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 v/v) of the S. emodi extract and silver nitrate solution. The UV-visible spectrum of the reaction mixture was recorded after 24 h. Nine various peaks were obtained for different ratios. The maximum absorbance was observed at 437 nm ( Figure 2) and pH 11 for the 6:4 (v/v) ratio, where its pointed peak indicated the formation of spherical-shape (se-Ag/AgClNPs) silver/silver chloride nanoparticles [39].
The FTIR pattern was used to study the various functional groups which might be involved in se-Ag/AgClNP synthesis and could play an important role as a stabilizing agent. The FTIR spectral analysis showed various peaks for different functional groups. A small broad peak at 2035 cm −1 was observed due to C=C=N stretching, which could be a ketenimine-like compound. A small peak at 1975 cm −1 was observed due to C-H bending of the possible aromatic compound. A broad peak was observed at 1576 cm −1 for the N-H bending of the amine compound ( Figure 3).

Characterization of Silver/Silver Chloride Nanoparticles
The color of the solution started turning brown immediately after placing the so tion in sunlight and turned completely dark brown after 20 min. This was due to the duction of silver ion to silver/silver chloride nanoparticles in the reaction mixture [3 The silver nanomaterial synthesis was achieved by using varying volumetric ratios (1 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 v/v) of the S. emodi extract and silver nitrate soluti The UV-visible spectrum of the reaction mixture was recorded after 24 h. Nine vario peaks were obtained for different ratios. The maximum absorbance was observed at 4 nm ( Figure 2) and pH 11 for the 6:4 (v/v) ratio, where its pointed peak indicated the f mation of spherical-shape (se-Ag/AgClNPs) silver/silver chloride nanoparticles [39].

Characterization of Silver/Silver Chloride Nanoparticles
The color of the solution started turning brown immediately after placing the solution in sunlight and turned completely dark brown after 20 min. This was due to the reduction of silver ion to silver/silver chloride nanoparticles in the reaction mixture [38]. The silver nanomaterial synthesis was achieved by using varying volumetric ratios (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 v/v) of the S. emodi extract and silver nitrate solution. The UV-visible spectrum of the reaction mixture was recorded after 24 h. Nine various peaks were obtained for different ratios. The maximum absorbance was observed at 437 nm ( Figure 2) and pH 11 for the 6:4 (v/v) ratio, where its pointed peak indicated the formation of spherical-shape (se-Ag/AgClNPs) silver/silver chloride nanoparticles [39].   agent. The FTIR spectral analysis showed various peaks for different func A small broad peak at 2035 cm −1 was observed due to C=C=N stretching, w a ketenimine-like compound. A small peak at 1975 cm −1 was observed due to of the possible aromatic compound. A broad peak was observed at 1576 cm bending of the amine compound ( Figure 3). Scanning electron microscopic analysis was used to verify the morpho of the synthesized silver/silver chloride nanoparticles. The obtained SEM re that the produced nanoparticles had random morphology with sphericaltures detected in the micrograph. The obtained results showed the size of th nanoparticles was in the range of 20 to 70 nm ( Figure 4). Scanning electron microscopic analysis was used to verify the morphology and size of the synthesized silver/silver chloride nanoparticles. The obtained SEM results showed that the produced nanoparticles had random morphology with spherical-shaped structures detected in the micrograph. The obtained results showed the size of the synthesized nanoparticles was in the range of 20 to 70 nm ( Figure 4).
Molecules 2023, 28, x FOR PEER REVIEW 5 of 14 The FTIR pattern was used to study the various functional groups which might be involved in se-Ag/AgClNP synthesis and could play an important role as a stabilizing agent. The FTIR spectral analysis showed various peaks for different functional groups. A small broad peak at 2035 cm −1 was observed due to C=C=N stretching, which could be a ketenimine-like compound. A small peak at 1975 cm −1 was observed due to C-H bending of the possible aromatic compound. A broad peak was observed at 1576 cm −1 for the N-H bending of the amine compound ( Figure 3). Scanning electron microscopic analysis was used to verify the morphology and size of the synthesized silver/silver chloride nanoparticles. The obtained SEM results showed that the produced nanoparticles had random morphology with spherical-shaped structures detected in the micrograph. The obtained results showed the size of the synthesized nanoparticles was in the range of 20 to 70 nm ( Figure 4). The crystalline nature of the sliver/silver chloride nanoparticles was confirmed by using XRD analysis at a 2θ angle ranging from 10 • to 80 • . The XRD diffraction peaks situated at 38.  The crystalline nature of the sliver/silver chloride nanoparticles was confirmed b using XRD analysis at a 2θ angle ranging from 10° to 80°. The XRD diffraction peaks si uated at 38

Antibacterial Activity
The antibacterial activity against E. carotovora resulted in significant inhibition by various concentrations (500 µg mL −1 , 250 µg mL −1 , 100 µg mL −1 , 80 µg mL −1 , 50 µg mL −1 , 20 µg mL −1 , and 10 µg mL −1 ). The centrifuged nanoparticles in combination with the leaf extract (se-Ag/AgClNPs + LE) at a concentration of 500 µg mL −1 showed a maximum inhibition of 98%. The centrifuged nanoparticles alone (se-Ag/AgClNPs) inhibited the growth of the bacteria by 93%, while the leaf extract alone (LE) showed an optimal inhibition of 62%. The control treatment showed no inhibition of the cell growth of E. carotovora. The inhibition patterns of the various concentrations of se-Ag/AgClNPs + PE, se-Ag/AgClNPs, and PE are shown in Figure 6.

Discussion
Plants produce different types of secondary metabolites which are potentially against various insects and phytopathogens. As compared to commercial fungicide pesticides, medicinal plants have more antifungal and antibacterial properties due presence of secondary metabolites which are more active in controlling plant disease are eco-friendly with fewer side effects [40]. As compared to commercial fungicide pesticides, medicinal plants have more antifungal and antibacterial properties due presence of secondary metabolites which are more active in controlling plant disease are eco-friendly with fewer side effects [41]. The spectrophotometer peak is depende

Discussion
Plants produce different types of secondary metabolites which are potentially active against various insects and phytopathogens. As compared to commercial fungicides and pesticides, medicinal plants have more antifungal and antibacterial properties due to the presence of secondary metabolites which are more active in controlling plant diseases and are eco-friendly with fewer side effects [40]. As compared to commercial fungicides and pesticides, medicinal plants have more antifungal and antibacterial properties due to the presence of secondary metabolites which are more active in controlling plant diseases and are eco-friendly with fewer side effects [41]. The spectrophotometer peak is dependent on the size of the nanoparticles. A smaller particle size represents peaks at a shorter wave-length while a larger particle size indicates a longer wavelength peak [42]. Our findings regarding UV-visible analysis were in compliance with those of previously described studies [43] in which silver/silver chloride nanoparticle peaks were observed at around 420 nm. Similar results were also reported by Patra et al. [44] using Pisum sativum plant extract and Kup et al. [45] using a plant extract of Aesculus hippocastanum. They used various techniques to characterize their synthesized nanomaterials. According to the UV-visible analysis, the formation of Ag-NPs was observed at a wavelength above 420 nm.
The color change in the mixture from violet blue to yellow proved the reduction of DPPH radical by the antioxidant compounds in plants [46]. This is due to the potential of methanolic extract in S. emodi plants as antioxidants. The highest DPPH free radical scavenging activity was shown by the plant extract at a concentration of 1000 µg/mL, which was 89.4%. Similar results were shown by the previous studies by Tatarczak et al. [47] where the DPPH radical was reduced by the phytochemicals in plants, proving its strong antioxidant activity. Khan et al. [48] synthesized Au/MgO nanomaterial by using Tagetes minuta which exhibit excellent antioxidant activity with 82% scavenging capability.
FT-IR revealed that stretching in the band from 3000 to 2000 cm −1 revealed good bonding between the functional groups and the Ag. The observed FTIR spectrum of the synthesized nanoparticles was in complete agreement with previous studies [49]. The FTIR pattern showed the presence of biological groups in the S. emodi extracts which could be involved in reducing and capping the biosynthesized nanoparticles (se-Ag/AgClNPs). The agents which could be responsible for the bioreduction and stabilization of silver ions into silver NPs present in S. emodi extract were confirmed by the FTIR pattern. The obtained bands of FTIR could be attributed mainly to the phenols, terpenoids, and flavonoids present in S. emodi plant extracts. The present study agreed with the study by Mohamed et al. [50] which also suggested that flavonoids, phenols, and proteins could be the reducing and stabilizing agents of silver/silver chloride nanoparticles.
Our SEM observation of the synthesized nanoparticles was in complete accordance with that previously observed by Khan et al. [51], who prepared silver nanoparticles by using Mentha spicata. Their SEM results at different magnifications showed sphericalshaped particles with size ranges from 21 to 82 nm. According to Yousaf et al. [52], silver nanoparticles were synthesized from the extracts of Achillea millefolium L. Their SEM results had an average diameter of 14.27, 18.49, and 20.77 nm with spherical, cubical, and rectangular morphology which positively correlates with the present study. This study suggested that the obtained se-Ag/AgClNPs were capped by biomolecules present in the S.emodi plant extracts and these metabolites may be manipulated by metallic silver to biogenically synthesize silver/silver chloride nanoparticles. The S. emodi NPs' size could also be detected from the sharpness and broadness of the XRD peaks. The Figure 5 peaks show that se-Ag/AgClNPs were in the nanosize range. Our XRD results were generally in accordance with those XRD patterns previously described by Hashemi et al. [53] which had the same peaks. Our results are in positive agreement with Sing et al. [54]; their XRD peaks were very strong and revealed that the synthesized Ag/AgClNPs were in the nanosize range and had a crystalline nature.
Plants belonging to the Stachys genus are very medicinal and have been used since early eras as traditional medicine to cure many problems such as gout, cough, fever, asthma, earaches, genital tumor, abdominal cramps, menstrual disorder, and dizziness. Advanced research shows that Stachys genus plant extracts have strong antifungal, antibacterial, antinephritic, antioxidant, and anti-inflammatory activities [31]. Previously, Shakeri et al. [55] reported the strong antibacterial efficacy of Stachys against Staphylococcus aureus, Bacillus cereus, Staphylococcus epidermidis, Escherichia coli, Salmonella typhi, and Pseudomonas aeruginosa. Jan et al. [56] also observed the antibacterial effects of Stachys against various bacteria. They showed that ethyl acetate, aqueous, n-hexane, and ethanolic extracts of the Stachys parviflora plant showed strong antimicrobial activity against six bacteria (Bacillus atrophaeus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli, and Bacillus subtilis) and one fungi (Candida albicans). The antibacterial activity of the study showed similar results to those previously reported studies that suggested that biosynthesized Ag/AgClNPs (by using grape pomace aqueous extract) have potential in controlling the growth of E. carotovora [57]. E. carotovora is a Gram-negative bacterium which affects most vegetables and fruits in the field and during shipment or in storage [58]. E. carotovora infect the host plant through some injuries and degrade its cell wall, followed by tissue maceration leading to the soft rotting of stems and fruits [9,59].
In the current study, the antibacterial effect of the S. emodi leaf extracts (500 µg/mL-10 µg/mL) was tested against E. carotovora. High concentrations (500 µg/mL and 250 µg/mL) showed promising results against the bacteria while the lowest activity was observed for the lowest (10 µg/mL) concentration of the leaf extract. Similarly, high concentrations (500 µg /mL, 250 µg/mL, and 100 µg/mL) of plantcoated silver/silver chloride nanoparticles (se-Ag/AgClNPs + PE) had high antibacterial activity as compared to the lowest concentrations (80 µg/mL to 10 µg/mL). The activity of the centrifuged nanoparticles (se-Ag/AgClNPs) was high for the 500 µg/mL and 250 µg/mL concentrations. Overall, the activity of the plant-coated nanoparticles was superior to the centrifuged nanoparticles and plant extracts alone. This might be due to the synergism of secondary metabolites with silver ions which makes its activity more efficient. The antibacterial activities of the present study agreed with those of Arif et al.'s study [38], in which silver nanoparticles synthesized from Euphorbia wallichii were tested against phytopathogens. Our study is in positive correlation with the previously reported study by Balachandar et al. [60] who studied the activities of biologically synthesized nanoparticles against various phytopathogens, and noticed a strong growth inhibition of the plant pathogens. NPs synthesized by using Eucalyptus camaldulensis were tested against various bacteria and were found to significantly reduce the Gram-negative bacteria growth. The antibacterial character of Ag/AgClNPs prepared from E. camaldulensis could be ascribed to the small particle size and high surface-to-volume ratio, which let the nanoparticles interact with bacterial membranes [61]. According to a proposed mechanism which describes how silver particles act, due to their small size and spherical shape, they can penetrate bacterial cell walls and can increase their permeability by bringing some structural changes; these changes include the generation of pores in the bacterial cell wall through reactive oxygen species production. Silver ions can also damage important cell enzymes, proteins, and nucleic acids of the bacteria, resulting in bacterial cell death [62].

Preparation of Leaf Extract
Healthy plant specimens were collected, washed thoroughly, and dried up at room temperature. The dried specimens were ground into a fine powder and used for the synthesis of silver/silver chloride nanoparticles. For the preparation of leaf extract, 1 g of the ground powder was mixed in 100 mL of distilled water and the solution was heated at 40-50 • C for 15 min on a hot plate. The solution was left to cool down and then it was filtered with the help of Whatman No. 1 filter paper (pore size of 11 µm) and was stored in a refrigerator at 4 • C for further use.

Antioxidant Activity
The DPPH free radical scavenging capacity of the sample plant was determined according to the protocol of Govindappa et al. [63] with a minor modification. The plant solution was prepared by taking 10 mg of the powdered plant in 10 mL of methanol. Through the two-fold dilution of the plant stock solution, 5 different concentrations (1000 µg/mL, 500 µg/mL, 250 µg/mL, 125 µg/mL, and 62.5 µg/mL) were formed. The DPPH solution was already prepared and stored at room temperature in the dark. A total of 1 mL of the DPPH solution was added to 2 mL of these diluted samples of the plant extract and kept for incubation in the dark for 30 min. The absorbance of all concentrations was measured with a Multiskan TM Sky Microplate Spectrophotometer (MAN0018930, Santa Clara, CA, USA) at 517 nm while ascorbic acid was used as standard. The percent activity was calculated with the given formula. % antioxidant activity = (OD of the control − OD of the sample × 100)/control OD (1)

Biosynthesis of Silver/Silver Chloride Nanoparticles
Following the procedures of Arif et al. [63] and Ul Haq et al. [64] with certain modifications, the green synthesis of nanoparticles was accomplished. The diluted leaf extract (2.5 mg/mL) solution was mixed appropriately with silver nitrate (4 mM) solution at equal volume (1:1) and was kept under sunlight for 20 min. The mixture was adjusted at different pHs ranging from 6 to 12. For the separation of the synthesized Ag/AgClNPs, the solution was centrifuged (Centrifuge 5425, Eppendorf, Hamburg, Germany) at 15,000 rpm for 15 min. The residual settled material was collected in a distinct tube and was then dissolved in deionized water followed by centrifugation again at 12,000 rpm for 10 min. This was repeated multiple times and the obtained pure nanoparticles were subjected to various characterizations.

Characterization of Synthesized Particles
The biosynthesis of se-Ag/AgClNPs was confirmed through various characterization techniques, which were as follows: Fourier transform infrared spectrophotometric analysis was carried out using an FTIR (Spectrum two-103385; Waltham, MA, USA) spectrophotometer equipped with ATR. The FTIR spectroscopic analysis was performed between the ranges of 4000 and 400 cm −1 . The various functional groups were identified by comparing the observed peaks with an IR spectrum table.
Scanning electron microscopy (JSM-5910, JEOL, Tokyo, Japan) was used to find out the morphology and distribution of the nanoparticles.
The X-ray diffraction (XRD) analysis of the silver/silver chloride nanoparticles was carried out using an X-ray diffractometer (Model: X-3532, JEOL, Tokyo, Japan). The XRD patterns were evaluated to find out the peak intensity, position, and width. The mean crystallite size was measured using Debye-Scherrer's formula.

Antibacterial Activity
The antibacterial activity of the green synthesized se-Ag/AgClNPs against E. carotovora was accomplished using the methods of Ahmad et al. [65] with certain modifications. The freshly grown culture of E. carotovora was acquired from (FCBP-PB-421) First Fungal Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences (IAGS) University of Punjab, Lahore, Pakistan and inoculated in nutrient broth and placed overnight at 28 • C in an incubator (FTC-90E Velp Scientifica, Lombardia, Italy). The activity was implemented with a microtiter plate (96-well) assay with various concentrations (500 µg mL −1 , 250 µg mL −1 , 100 µg mL −1 , 80 µg mL −1 , 50 µg mL −1 , 20 µg mL −1 , and 10 µg mL −1 ) of centrifuged silver nanoparticles alone (se-Ag/AgClNPs) and in combination with leaf extract (se-Ag/AgClNPs + LE) and leaf extract (LE) alone. 150 µL concentration of each treatment and 150 µL of E. carotovora suspension were poured into each well of the microtiter plate. The control well was adjusted with bacterial suspension. The optical density (OD) at 600 nm was recorded immediately and placed in a shaking incubator for 24 h. After 24 h, the OD was again read at 600 nm and the bacterial growth inhibition was calculated using the given formula: Bacterial growth inhibition = Control − Treatment/Control × 100 (2)

Conclusions
In the present study, we showed the efficient biosynthesis of silver/silver chloride nanoparticles and their antibacterial screening against E. carotovora using S. emodi. The characterizations of the prepared nanoparticles showed a significant biosynthesis of stable silver/silver chloride nanoparticles. Our study showed that the size of the nanoparticles ranged from 20 to 70 nm with an average diameter of 38 nm. Moreover, the antibacterial activity resulted in the significant growth inhibition of E. carotovora by the biogenically synthesized silver/silver chloride nanoparticles. The study concluded that biosynthesized Ag/AgClNPs have the potential to control the growth of E. carotovora through in vitro activities. It is recommended to evaluate the potential of se-Ag/AgClNPs through in planta means. However, further studies should confirm the effectiveness of these nanoparticles against other plant pathogens to protect important crops.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All authors confirm that the generated data are available in the manuscript.