Antimicrobial potential of Ag-doped ZnO nanostructure synthesized by the green method using Moringa oleifera extract

Journal Pre-proof Antimicrobial potential of Ag-doped ZnO nanostructure synthesized by the green method using Moringa oleifera extract Swati, Ritesh Verma, Ankush Chauhan, Mamta Shandilya, Xiangkai Li, Rajesh Kumar, Saurabh Kulshrestha

PII:

S2213-3437(20)30078-6

DOI:

https://doi.org/10.1016/j.jece.2020.103730

Reference:

JECE 103730

To appear in:

Journal of Environmental Chemical Engineering

Received Date:

4 November 2019

Revised Date:

23 January 2020

Accepted Date:

27 January 2020

Please cite this article as: Swati, Verma R, Chauhan A, Shandilya M, Li X, Kumar R, Kulshrestha S, Antimicrobial potential of Ag-doped ZnO nanostructure synthesized by the green method using Moringa oleifera extract, Journal of Environmental Chemical Engineering (2020), doi: https://doi.org/10.1016/j.jece.2020.103730

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Antimicrobial potential of Ag-doped ZnO nanostructure synthesized by the green method using Moringa oleifera extract Swatia, Ritesh Vermab, Ankush Chauhanb, Mamta Shandilyab, Xiangkai Li c, Rajesh Kumarb,d* and Saurabh Kulshresthaa* a

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Faculty of Applied Science and Biotechnology, Shoolini University of Biotechnology & Management Sciences, Bajhol-Solan (HP)-173212 b School of Physics and Materials Science, Shoolini University of Biotechnology & Management Sciences, Bajhol-Solan (HP)-173212 c Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Science, Lanzhou University, Tianshuinanlu #222, Lanzhou 730000, Gansu Province, P.R. China d

Himalayan Centre of Excellence for Renewable Energy, Shoolini University of Biotechnology &

*Corresponding authors:

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Dr Rajesh [email protected]

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Management Sciences, Bajhol-Solan (HP)-173212

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Graphical abstract

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Dr Saurabh [email protected]

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Abstract

Novel properties of green synthesis have paved a new area of research among the scientific

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community. In the present study, a systematic investigation has been carried out to synthesize highly oriented and uniform Ag-doped ZnO nanostructures using the extract of Moringa oleifera (MO). The structural and morphological characteristics of synthesized nanostructures were investigated using XRD and FESEM. The crystallite size was calculated to be 54.1 nm and 36.187 nm with the Scherrer method and Williamson-Hall method, respectively. FESEM confirms the flower-like structure of the nanostructures and EDX analysis confirmed the presence of Silver 2|Page

(Ag), Zinc (Zn), and Oxygen in the synthesized sample. TEM images reveal the continuous planes of the crystal and confirm the high crystallinity of the sample. Antimicrobial activities were analyzed against different human pathogenic bacteria, (i.e., Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, MRSA, Salmonella typhii, Klebsiella pneumonia), yeast (Candida albicans) and plant pathogenic fungus (i.e., Fusarium spp., Sclerotinia sclerotiorum, and Rosellinia necatrix). Nanostructures showed maximum inhibition zone against Staphylococcus aureus (17mm) as compared to other bacteria and showed 18 mm inhibition zone against C.

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albicans. Nanostructures showed growth inhibition of 56.8%, 34.78 % and 48.9 % Rosellinia necatrix, Fusarium spp. and Sclerotinia sclerotiorum, respectively. The antimicrobial activity of Ag-doped ZnO was observed to be effective. The present work gave a conceivable method to

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develop nanostructures with desirable properties to be applied in antimicrobial activities.

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antifungal property.

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Keywords: - Green synthesis; Moringa oleifera; nanostructures; antibacterial property;

1. Introduction

Many physio-chemical methods have been used so far to synthesize nanoparticles that are

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tedious and possess hazardous solvents or reagents as the expensive substrate with potentially adverse effects, as well as requiring specific instrumentation [1, 2]. Synthesis of nanoparticles via eco-friendly, i.e., green synthesis, provides simplicity, low cost, ambient atmosphere synthesis, non-toxicity, and environmental compatibility [3, 4]. Due to these novel properties, green synthesis has attracted various researchers amongst the scientific community from around the world. Green synthesis of metal nanoparticles is an exciting subject of nanoscience as it provides the most stable nanoparticles with varying shapes [5]. Plant extracts have been found an 3|Page

up-and-coming candidate for facile synthesis of nanoparticles [3]. Zinc Oxide (ZnO) is an essential inorganic semiconductor material due to its high photostability, nontoxicity, thermal stability, oxidation resistivity, high electron mobility [6]. ZnO nanoparticles, in particular, are environment-friendly, offer easy fabrication, and are non-toxic, bio-safe, and biocompatible [7]. The biological applications of ZnO nanoparticles make them ideal candidates for biological sensing, gene delivery, drug delivery, wound dressing material, antifungal, antibacterial activities [8-10]. Besides, ZnO nanoparticles are inexpensive and exhibit

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morphological versatility such as nanorods, nanoflowers, nanospheres, nanotubes, etc. and was more influential on rheological parameters [11-12, 13]. According to Fattahi et al., 2014, the organic components played an important role in the morphology and texture of the final products

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[14]. Photocatalytic processes driven by ZnO nanostructure semiconductors have great potential for decontamination of organic compounds in water due to ease, complete mineralization of

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pollutants and environmentally friendly [15]. Semiconductor photocatalysts have recently

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demonstrated high photocatalytic activity for decontamination and Silver nanoparticles combine with semiconductor, which promotes the separation of charges, produces more photo-generated

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charges [16, 17]. Heterogeneous hybrid systems also have developed as compelling methodologies for decontamination of aqueous solutions containing non-biodegradable compounds [18].

In this study, we are reporting the structural, anti-microbial and anti-fungal properties of

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Ag doped-ZnO prepared via green synthesis approach. Ag exhibits intriguing properties like nontoxicity, good electrical conductivity, and high thermal conductivity as compared to other noble metals [19]. It also acts as an electron sink, traps photogenerated electrons, and increases the yield of charge separation state [20]. Ag is relatively non-toxic, good oxygen adsorption behavior and Ag decorated materials have shown high efficiency against the infections caused by pathogens [21, 22, 23]. 4|Page

In particular, Ag-ZnO has been frequently explored for biomedical applications such as dye degradation reagent, toxic dye absorber, wound treatment, cancer treatment, drug delivery, etc. due to its high antimicrobial, antifungal activities [24-29]. The seeds of MO contain a variety of properties that are useful in the medicinal field [30-32]. Seeds are edible in fresh, dried, or with the seed pods and are considered to be antipyretic, acrid, bitter [33]. The seed's medicinal properties are well documented and supported by the experience of traditional Ayurvedic practitioners [31]. MO seed extracts showed action against hepatic carcinogen metabolizing

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enzymes and are known to have anticancer activity [34]. Medicinal mediated synthesis of metal or metal-based nanoparticles has been found as a promising area of research. It encouraged us to synthesize Ag-ZnO nanostructures via green method. The approach is green because the main

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reducing agent Moringa oleifera was used which is one of the best-known medicinal plants in the world. As a reducing agent, Moringa oleifera negates the requirement of other synthetic reducing

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2. Materials and methodology

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agents. It is also called “needy” or the tree that never dies.

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2.1 Moringa oleifera seeds collection

Moringa oleifera seeds were collected from the Mandi district of Himachal Pradesh. The seeds were separated from the membranes, and then kernels were crushed to make a fine powder

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using pestle-mortar.

2.2 Sample preparation Silver doped zinc oxide nanostructures were synthesized using green combustion method, according to Khan et al., 2018, with slight modifications [35]. Zinc acetate (Himedia), Silver Nitrate (Himedia), and Sodium Hydroxide pellets (Himedia) were used as the precursor. 0.5 M Zinc Acetate aqueous solution was prepared in 100 ml of distilled water and stirred for 30 minutes at

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room temperature using the magnetic stirrer and 0.01 molar solution of AgNO3 in 10 ml distilled water was added to the solution at continuous stirring (Say A). Then 40 ml of aqueous MO seed extract was added to solution A and stirred for 20 minutes. 2 M Sodium Hydroxide aqueous solution was added slowly to the solution until the pH reaches 12, and the formation of white precipitates can be observed. The precipitates were allowed to settle down overnight and washed adequately with distilled water, and particles were obtained after centrifuged dried at room temperature.

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2.3 Characterization 2.3.1 Structure and Morphology

The formation and superiority of compounds were verified with X-ray diffraction (XRD)

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technique. X-ray powder diffractometer (Rigaku Minifiex 600, Japan) with CuKα radiation (λ =

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1.5405 Å) in an extensive range of Bragg angles 2θ (20° ≤ 2θ ≤ 60°) at a scanning rate of 2° min−1 was used to study the XRD patterns of the compounds at room temperature using. FESEM images

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were taken using the Hitachi SU 8010 series field emission scanning electron microscope at Panjab University, Chandigarh, India. The samples were investigated under 5 kV beam energy in

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order to obtain the excitation of all the elements. Analysis of elements was carried out by energydispersive X-ray (EDX) analysis using a BRUKER system attached to the FESEM mentioned above at Panjab University, Chandigarh, India. TEM images were taken using a high-resolution Transmission electron microscope of FEI make of USA, FP 5022/22-Technai G2 20 S-TWIN, at

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Indian Institute of Technology, Mandi.

2.3.2 Organisms collection For antibacterial studies, the microbial strains Staphylococcus aureus (MTCC 96), Salmonella typhi (MTCC 734), Pseudomonas aeruginosa (MTCC 2453), Klebsiella pneumonia (MTCC-39), Escherichia coli (MTCC 82), MRSA (Methicillin-resistant Staphylococcus aureus 6|Page

Standard strain-CA 05 SCCmec Type IV) were obtained from parasitology laboratory; Candida albicans (ATCC-90028) was obtained from yeast biology laboratory, and for antifungal studies, the strains Fusarium spp., Sclerotinia sclerotiorum and Rosellinia necatrix were obtained from Molecular plant-microbe interaction laboratory of Shoolini University of Biotechnology and Management Sciences, Solan, India.

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2.3.3 Agar well diffusion method for antimicrobial activity: The nanostructure prepared using MO seeds was tested by the agar well diffusion method. To perform the antimicrobial assay, nutrient agar, as well as ampicillin, was used. Bacterial and yeast suspensions were prepared from cultures by the direct colony method. In Bacteria, these

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pre-culture broths were allowed to stand overnight in a rotary shaker at 37°C for 16-18 h and

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incase of yeast, 30°C for 24-48 hours after which these cultures were maintained on broth in the refrigerator for further use [36].

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The test materials having antimicrobial activity inhibited the growth of the microorganisms, and a bright, distinct zone of inhibition was visualized surrounding the medium.

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The antimicrobial activity of the test was determined by measuring the diameter of the zone of inhibition expressed in millimeters. The appearance of growth around all the bacteria was considered as bacteriostatic, whereas no growth was considered as a bactericidal effect.

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2.3.4 Antifungal Assay:

Nanostructure solution was prepared at concentrations of 50mg/ml (prepared in 10%

DMSO), were added in sterilized potato dextrose agar. A 6mm diameter of the actively growing mycelium disc of the pathogen of 6-7 days old culture (Fusarium spp., Rosellinia necatrix) and 3-4 days old culture (Sclerotinia sclerotiorum) were placed in the center of the Petri dish. Plates without extract served as negative control and 5mg/ml of Hygromycin as the positive control. 7|Page

Plates without extract served as negative control. Fusarium spp., Rosellinia necatrix were incubated at 25 ± 2°C for 7-8 days for and Sclerotinia sclerotiorum was incubated at 25 ± 2°C for 3-4 days. The circular growth of mycelium was measured after 3-4 days (Sclerotinia sclerotiorum) and 6-7 days (Fusarium spp., Rosellinia necatrix) of incubation. After incubation, the circular growth of mycelium was measured. The growth results were compared with the negative control. The experiment was repeated three times, and the mean of the readings was taken for further calculations. The percent inhibition of the fungus in the experiment was

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calculated using the following formula; C  T   100    C 

(1)

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Where L is the percent inhibition; C is the colony radius in the control plate, and T is the radial

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growth of the pathogen in the presence of nanoparticle extracts [37].

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3. Results and discussion:

The present study reports the use of MO seeds for the synthesis of nanostructures, which are prepared by green synthesis and free from chemicals.

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The appearance of the light brown precipitate obtained after the synthesis process is a clear indication of the formation of Ag-doped ZnO nanostructures formed in the reaction mixture.

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3.1 X-Ray Diffraction:

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Figure 1(A) showed the XRD patterns at room temperature for Ag-doped ZnO prepared by green synthesis. However, the XRD spectrum exhibits sharp diffraction peaks at 2θ of 31.7º, 34.4º, 36.3º, 47.5º and 56.6º corresponding to (100), (002), (101), (102), (110) plane matched

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with JCPDS file 751526. The crystallite size was calculated using Scherrer formula as given below,

(2)

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The average size was found to be 54.1nm. The crystal structure was identified by calculating the unit cell parameters a = b = 3.220 Å and c = 5.200 Å, and the c/a ratio was found about 1.614, which is very near to the standard value 1.66 for hexagonal structure. Also, the appearance of the ZnO peaks along with the absence of PVA peaks confirms that the high purity of the ZnO nanofiber. All peaks showed considerable broadening, which was the cause of nanophase formation with less internal stress. No impurity peaks were observed, which confirms the purityof the sample. The absence of an impurity peak is also because silver was not incorporated in the crystallite ZnO.

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The crystallite size was calculated from the Scherrer method. However, this method does not give an accurate size because it does not account for the lattice strain due to the presence of dopants. Thus, to calculate the size accurately Williamson-Hall's plot was constructed (Figure

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1B) of Ag-doped ZnO nanostructures. In this method, the relation of crystallite size, lattice strain, and peak broadening is calculated using the following formula,  k    ( 4  S in  )  D 

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 hkl C o s   

(3)

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Williamson-Hall method is essential because it shows the line broadening in XRD peaks is substantially isotropic, and the least square fitting of data points gives positive value slope and non-zero intercept [38]. The results obtained from the Scherrer method and Williamson-Hall plot are

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summarized in Table 1. The difference between the two calculated sizes is because the Williamson-Hall method includes the strain contribution in size, but the Scherrer method excludes it. FTIR

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FTIR is a remarkable technique to study the stretching and bending vibrations of specific material. It was observed that Ag doped-ZnO contains specific vibration modes as shown in Figure 1(C). The bands between 400 and 750 cm-1 corresponds to the metal oxide bonds and band from 900-1500 cm-1 are due to the stretching and bending frequency of oxygen [39-41]. The occurrence of peak around 708.79 cm-1 could be attributed to Ag-ZnO. The wider peak can be correlated to the organic capping of Ag-ZnO [42]. The presence of band near 1656.35 cm-1 might be due to the H-O-H bending [43]. This might be due to the adsorption of a small amount of moisture by the sample. However, there were no

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prominent peaks observed around 1400 and 1500 cm-1 which shows the absence of C=O and O-H bending vibrations, respectively [43]. A minor peak around 1656.79 cm-1 shows that there is a small presence of O-H bending vibrations. Thus, it is evident from FTIR that all the modes of Ag-

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doped ZnO were present in the synthesized sample.

3.2 FESEM and EDX Study

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The flower-like grains are developed with less agglomeration, as shown for the FESEM images of Ag-doped ZnO as shown in Figure 1(D). The doping percentage of Ag can also be responsible for the apparent crystal formation and larger particles in these samples. In green- synthesis,

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plant extract performed as additives from solvent media. They act as binders between the particles and facilitate the self-assembly process that generates mesocrystals. This is an essential characteristic in the formation of crystals, which leads to structure formation. In general, in a mesoscale

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transformation, the nanostructures are interspaced by organic additives but may involve orientation order and self-assembly of faceted microstructures, even in the absence of additives. EDX analysis was performed to confirm the presence of Ag in the ZnO crystal. It is shown in Figure 1(E) that along with zinc and oxygen, silver is also present. This data supports our XRD pattern, which has shown the shift in peaks due to the presence of Ag in ZnO crystal. 3.3 TEM Study

TEM image taken for the sample shows the continuous pattern of all the planes, as mentioned in an image with two planes, plane 1, and

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plane 2 as shown in Figure 2(A). This TEM image gives the side view of the sample and confirms the FESEM result which shows that the flowerlike structure is being developed by small thin films. The SAED pattern in Figure 2(B) clearly shows that the nanostructures formed are crystalline.

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This confirms the XRD analysis showing the high crystallinity of the sample. 3.4 Antimicrobial activity

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The antimicrobial activity of Ag-doped ZnO was checked against gram-negative and gram-positive bacteria shown in Figure 3(A), where 10% DMSO and ampicillin were used as the negative and positive control, respectively.

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The inhibition zones (in mm) of varying sizes were obtained, as mentioned in Table 2 against Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, MRSA, Klebsiella pneumoniae, Salmonella typhi and yeast, i.e., C. albicans. It has been observed that antimicrobial activity against

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candida albicans and Staphylococcus aureus is substantially good as compared to other, as shown in Figure 3(B). The inhibition zones were measured by taking the nanostructure solution. Ampicillin showed the appearance of inhibition zones of different sizes in different bacteria and

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Fluconazole showed inhibition zone against C. albicans. Maximum inhibition zone was found in the AgZnO nanostructures against C. albicans (18mm), Staphylococcus aureus (17mm) and followed by the other microorganism as shown in all the tested organisms Table 2. The presence of zone of inhibition clearly demonstrates that the mechanism of the activities of ZnO nanoparticles, which includes interruption of the membrane with high rate of multiplication of surface oxygen species and at last go to the death of pathogens. Interestingly, the size of the inhibition zone was different according to the type of pathogens, synthesis method and the concentrations of nanoparticles. The appearance of no growth around all the inhibition zones of bacteria was considered as a bactericidal.

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Different scientists revealed the antimicrobial activity of Moringa oleifera against a variety of pathogens [44, 45]. ZnO nanoparticles from the leaves of M. oleifera exhibit antibacterial activity against gram-positive and gram-negative bacteria at the concentration of 200 µg/ml

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[46]. AgNPs derived from M. oleifera leaf powder showed no activity against K. pneumoniae [47]. In 2015, Sujitha et al. reported that the AgNPs from the seeds of M. oleifera are reported to control primary dengue vector Aedes aegypti and against dengue serotype DEN-2 [48].

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3.4.1 Anti-fungal activity

These dopped nanostructures were also found to be effective against all tested fungal isolates. It is essential to mention that nanostructures

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were able to inhibit the growth of fungus (Fusarium spp., Sclerotinia sclerotiorum, and Rosellinia necatrix) in the present study (Figure 4 A). Variable percent inhibition was obtained, as mentioned in Table 3 against Fusarium species, Rosellinia necatrix, and Sclerotinia

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sclerotiorum. Percent inhibition of different fungi is also represented in graphical form as shown in Figure 4 (B) It is clearly shown that Ag-doped ZnO has indicated the maximum inhibition against the Rosellinia necatrix which causes significant root rot disease in Apple. The growth of 20.3

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mm was observed for the Rosellinia necatrix when inoculated on the media containing nanostructures and 47 mm around the negative control. 56.8% of growth inhibition was recorded. Similarly, growth inhibition of 34.78 % and 48.9 percent was obtained for Fusarium spp. and Sclerotinia sclerotiorum, respectively. Kasprowicz et al. reported the antimicrobial activity of silver NPs against plant pathogens Fusarium culmorum [49]. In 2011, He et al. reported that ZnO NPs could be used as an effective fungicide against B. cinerea and Penicillium expansum in agriculture and for food safety [50]. 4. Conclusion: 13 | P a g e

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Moringa oleifera is one of the best-known medicinal plants. In this study, a systematic procedure has been carried out to synthesize highly oriented and uniform Ag-doped ZnO nanostructures by an eco-friendly method using the Moringa oleifera seeds extract. M. oleifera seeds

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extract has been used as a reducing agent for the synthesis of nanostructures. XRD pattern shows that the sample preparation is crystalline with no impurity phases. The crystallite size was found about 54.1 nm and 36.187 nm from the Scherrer method and William Hall method,

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respectively. The flower-like shape of the sample was observed using FESEM. TEM shows the continuous plane of the crystal, and the SEAD pattern confirms the high crystallinity of the sample. FTIR studies revealed that the presence of phytoconstituents which were the surface-active

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molecule stabilized the nanoparticles. The antimicrobial analysis of the green synthesized AgZnO nanostructures was observed that it inhibits the growth of different pathogenic bacteria (both gram positive and gram negative). It is important to mention that the synthesized nanostructures

sclerotiorum).

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have also shown antifungal efficacy against three different plant pathogenic fungal strains (Fusarium spp., Rosellinia necatrix and Sclerotinia

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Therefore, these nanostructures may be used as preventive or chemotherapeutic agents against the pathogens. Green based methods are more useful for production of stable and safer nanostructures as compare to chemicals methods, which are less stable and not environment friendly. The investigated eco-friendly AgZnO nanostructures prepared from M. oleifera seed extract expected to have more wide applications in the formulation of different antimicrobial products used in various fields such as medicine, agriculture, pharmaceutical industries. Declaration of Interest: None

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Authors Contribution Section Name of the authors Swati Ritesh Verma Ankush Chauhan Mamta Shandilya Xiangkai Li Rajesh Kumar Saurabh Kulshrestha

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Contributions Worked on material preparation, antimicrobial and antifungal activity of nanostructures Worked with Swati for the synthesis of nanostructures Worked with Swati for the synthesis of nanostructures Nanostructure characterization Critical analysis of results and guidance Conceptualize and execution of the nanostructure component of the paper, paper correction Team leader, overall conceptualization, execution and paper writing and final corrections

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5. Acknowledgment

All authors are thankful to the Vice-Chancellor, Shoolini University of Biotechnology and Management Sciences, Solan, for providing necessary facilities. The authors would also like to thank the support of the Scientific Writing Cell of Shoolini University for language editing

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and formatting of the manuscript.

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1(B)

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1(A)

1(C)

1(D)

1(E)

Figure 1. Figure showing the structural characteristics of Ag doped ZnO nanostructures. (A). XRD patterns of Ag doped ZnO nanostructures exhibiting sharp diffraction peaks. (B). Williamson-Hall plot of Ag-doped ZnO nanostructures, to calculate the size accurately. (C). FTIR spectra of Ag doped-ZnO prepared via green synthesis. (D). Flower like shape of Ag-doped ZnO nanostructures was observed using FESEM. The flower like grains is developed with less agglomeration. (E). EDX analysis was to confirm the presence of Ag in the ZnO crystal. 27 | P aperformed ge

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Figure 2. Figure showing Transmission Electron Microscopic (TEM) characterization of Ag-doped ZnO nanostructures. (A) TEM image of Ag-doped ZnO nanostructures shows the continuous pattern of all the planes as mentioned in image with two planes, plane 1 and plane 2. (B) The SAED pattern of Ag-doped ZnO nanostructures clearly shows that the nanostructures formed are crystalline in nature.

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Figure 3(A). Analysis of antimicrobial activity of Ag-dopped ZnO nanostructures against different pathogenic bacteria and yeast; (A) antimicrobial activity against S. aureus, (B) antimicrobial activity against E. coli, (C) antimicrobial activity against Pseudomonas aeruginosa,

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(D) antimicrobial activity against MRSA (Methicillin-resistant Staphylococcus aureus), (E) antimicrobial activity against Klebsiella pneumoniae, (F) antimicrobial activity against S. typhi and (G) antimicrobial activity against Candida albicans Where, 1-positive control, 2- Ag-dopped ZnO nanostructures, 3- negative control. 100 µl Ag-dopped ZnO nanostructures were used at final concentration of 100 mg/ml (prepared in 10% DMSO), 10 µl Positive control for bacteria (Ampicillin) was used at concentration of 100mg/ml, 10µl Positive control for yeast (Fluconazole) was used at concentration of 100mg/ml. Negative control (10% DMSO), produced no zone of inhibition and incubated at 37 ± 2°C for 18 hours. (B). Graphical representation (Mean ± SD) of antimicrobial activity (as zone of inhibition in mm) of Ag-dopped ZnO nanostructures and their comparison with the positive control against pathogenic bacteria and yeast i.e., S. aureus, E. coli, Pseudomonas, MRSA (Methicillin-resistant aureus), Klebsiella pneumoniae, S. typhi, Candida albicans. 29Staphylococcus |Page

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Figure 4(A): Antifungal activity against Fusarium spp., Rosellinia necatrix and Sclerotinia sclerotiorum; (1A) Activity of Ag-dopped ZnO nanostructures against Fusarium spp., (1B) Negative Control against Fusarium spp., (1C) Positive Control against Fusarium spp., (2A)

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Activity of Ag-dopped ZnO nanostructures against Rosellinia necatrix, (2B) Negative Control against Rosellinia necatrix, (2C) Positive Control against Rosellinia necatrix, (3A) Activity of Ag-dopped ZnO against Sclerotinia sclerotiorum, (3B) Negative Control against Sclerotinia sclerotiorum, (3C) Positive Control against Sclerotinia sclerotiorum. A 6mm diameter of the actively growing mycelium disc of the pathogen of 6-7 days old culture (Fusarium spp., Rosellinia necatrix) and 3-4 days old culture (Sclerotinia sclerotiorum) were placed in the center of the Petri dish. 50mg/ml of Ag-dopped ZnO nanostructures were used (prepared in 10% DMSO), 5mg/ml (Hygromycin) was used as positive

control. Plates without extract served as negative control. Fusarium spp., Rosellinia necatrix were incubated at 25 ± 2°C for 7-8 days for and Sclerotinia sclerotiorum was incubated at 25 ± 2°C for 3-4 days. Results was compared with negative control. (B): Graphical representation (Mean ± SD) of percentage inhibition of nanostructures against different fungus i.e., Fusarium spp., Rosellinia 30necatrix | P a g e and Sclerotinia sclerotiorum.

Table 1: Table showing the Crystallite size of the Ag-dopped ZnO nanostructures calculated by Scherrer method and Williamson-Hall method Table 1. Crystallite size calculated by Scherrer method and Williamson-Hall method Scherrer method (D nm)

Williamson-Hall method (D nm)

54.1

36.187

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ro of

Ag- ZnO

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Table 2: Table showing antimicrobial activity of Ag-dopped ZnO nanostructures against pathogenic bacteria and yeast. Activity is shown as inhibition zones of Agdoped ZnO nanostructures Bacteria

Nanostructures Nanostructures Positive (mm)

Mean±SD

Positive

Negative

control

control

control

(mm)

Mean±SD

17.2 Staphylococcus

17.3

28.5 17 ± 0.436

27.6

28 ± 0.458

_

aureus 27.9

12.3

29.1

12.5

ro of

E. coli

16.5

12 ± 0.7

29.2

13.5 12.5

aeruginosa

13 ±0.5

lP

13

30.3

re

Pseudomonas

14.7

15 ± 0.264

30.4

29.6

13.9

26.3 14 ± 0.458

_

29 ± 0.360

_

27 ± 0.656

_

29 ± 0.458

_

24 ± 0.556

_

29.1

28.3

14.5

30 ± 0.608

29.3

15.1

ur

Klebsiella

15.2

na

MRSA

_

28.7

-p

11.2

29 ± 0.264

27.1

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pneumonia

Salmonella

13.6

27.6

15.9

28.6

15.2

16 ± 0.854

28.9

typhi

C. albicans

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16.9

29.5

17.3

24.5

18.2

18 ± 0.624

23.6

23.9

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18.5

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Table 3: Table showing antigungal activity of Ag-dopped ZnO nanostructures against plant pathogenic fungi. Activity is shown as percentage inhibition Fungus

Nanostructures

Negative

%age

Total

control

Inhibition

%age Inhibition

Fusarium spp.

30

46

34.783

31

48

35.417

29

44

34.091

20.0

47

57.447

21.5

48

19.4

46

24

45

46.666

23

47

51.106

49

48.979

sclerotiorum

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lP

25

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53.261

56.8±3.214

59.583

-p

Sclerotinia

re

Rosellinia Necatrix

ro of

34.78±0.66

48.9±2.22