Characterization and fungistatic activity of Eucalyptus globulus leaf extract mediated phytosynthesized silver nanoparticles

In the present study silver nanoparticles were synthesized through green approach using leaf extract of Eucalyptus globules and confirmed by change in colour of reaction mixture, the peak obtained at 425nm in UV-visible spectroscopic study and characterized by XRD, DLS, SEM, EDAX and FTIR for their structure, size, shape and possible biomolecules responsible for the reduction of silver ion to silver nanoparticles. XRD spectrum confirmed the particles were face centered cubic in nature with an average particle size of 25nm which is in agreement with DLS data (25.31nm). SEM images of nanoparticles revealed the spherical shape of particles and the Energy Dispersive X-ray analysis (EDAX) confirmed the significant presence of elemental silver. Synthesized silver nanoparticle showed good fungicidal activity against Didymella bryoniae, Fusarium oxysporum, Aspergillus flavus and Rhizoctonia solani. KeywordsFungistatic, activity, Phytosynthesis, Silver nanoparticles, Eucalyptus globulus


I. INTRODUCTION
The scientific community has oriented towards the field of nanotechnology especially in the research of metal nanoparticles from last few years. The synthesis and applications of nanoparticles gained more importance in the field of chemistry, electronics, cosmetics, textiles industries, drug delivery systems, as biosensors, diagnostics, therapeutics, antimicrobial and environmental remediation [1][2][3][4].
Due to high incidence of microbial infections and the growing resistance of pathogens to conventional antimicrobial drugs has created the need for novel antimicrobials. Nanoparticles provide a better alternative and also an interesting tool for material science as well as number of novel applications in biological sciences [5]. Among the metal nanoparticles, silver nanoparticles have gained more importance due to their electrical conductivity, chemical stability, catalytic, optical and antimicrobial properties [6].
Silver is a safe and nontoxic antibacterial agent and has the ability of killing about 650 types of microorganisms which cause diseases in human [7]. Due to their account of increasing antimicrobial property displayed by silver nanoparticles [8], they are projected as future generation antimicrobials [9].
Though the chemical and physical method are available for the synthesis of nanoparticles the green synthesis approach will be nontoxic, ecofriendly, cost effective and single step method which would be suitable for large scale production [10,11]. In this method organisms like algae, fungi, bacteria and plants can be utilized in the synthesis silver nanoparticles. Among these plants can be utilized effectively as they are blessed with magical phenomenon of producing bioactive secondary metabolites [1] which act as reducing as well as capping agents in the silver nanoparticles synthesis process [12].
The aim of the experiment was to synthesize silver nanoparticles from the leaf extract of Eucalyptus globulus which is an important tree species easily available and well known for its medicinal uses and test the fungicidal efficacy of the synthesized silver nanoparticles against four (4) fungal pathogens known to cause plant diseases.

Plants and chemicals
AR-grade silver nitrate (AgNO3) was purchased from HIMEDIA laboratories and fresh Eucalyptus globulus Labill. Leaves were collected from the University of Mysore Manasagangotri campus, Mysuru, Karnataka State, India. Double-distilled water was used for the experiments.

Test fungal pathogens
Phytopathogens used in the present study namely, Didymella bryoniae, Fusarium oxysporum, Rhizoctania solani and Aspergillus flavus were procured from Department of Studies in Botany, University of Mysore, Manasagangotri, Mysuru, Karnataka State, India.

Preparation of Leaf extract
Ten grams of fresh leaves were washed with tap water for about two-three times to remove the surface dust particles then with double distilled water. Excess water on the leaf surface was drained out and leaves were cut into small pieces of 0.5 to 1cm and boiled with 100ml of double distilled water for about 10-15 minutes. Then it is cooled, filtered with muslin cloth followed by filtration in Whatman No. 1 filter paper. Obtained extract was stored at 4°C for further use.

Synthesis of Silver nanoparticles
For the synthesis of silver nanoparticles, Silver nitrate solution of 1 mM concentration was treated with leaf extract in different proportion viz. 9:1, 3:1, 1:1, 1:3 and 1:9 ratios to standardize the metal precursor to plant extract ratio [13,14] and the reaction mixture was kept in dark condition at room temperature. Change of color from yellowish to brown indicated the formation of silver nanoparticles which was further confirmed by UV-visible spectroscopic study. After the completion of reaction, the solution was centrifuged at 12000 rpm for 20 minutes, the obtained silver nanoparticles were dried and stored for further characterization.

UV-visible spectroscopy
The small amount of reaction mixture (9:1 ratio) was taken out at regular interval of time after 5min, 1hr, 2hr, 3hr, 4hr, 24hr and 48hrs to study the synthesis of silver nanoparticles by measuring the optical density at the wavelength range of 300-700nm using UV-Vis spectrophotometer Beckman Coulter DU730.

Dynamic Light Scattering (DLS) Analysis and Zeta potential
The average particle size, the distribution and zeta potential were analyzed by Microtrac (USA) particle size analyzer.

XRD analysis
The crystalline nature of phytosynthesized silver nanoparticles was determined by X-Ray Diffraction (XRD) analysis using Rigaku Destop Miniflex II X-Ray Diffractometer with 2θ angle 10 to 80°. Further the average particles size was calculated using Debye-Scherrer formula [15][16][17].

Fourier Transform Infra-red Spectroscopy (FTIR)
The FTIR investigation of synthesized silver nanoparticles was carried out using Perkin Elmer Spectrum Two Spectrophotometer in the range of 600 to 4000 cm-1 for the analysis of possible bio-reducing and capping agent present in the leaf extract which are responsible for the reduction of the silver ions to silver nanoparticles.

Scanning Electron Microscopy and EDAX analysis
The morphological characteristics of Eucalyptus globulus leaf extract mediated phytosynthesized silver nanoparticles were studied using Hitachi S-3400N Scanning Electron Microscope (SEM). Images were captured by placing minute amount of nanoparticles on the carbon tape coated over copper grid and sputter coated with gold. Elemental composition and the presence of elemental silver was studied by Energy Dispersive X-ray Spectroscopy (EDAX) using HITACHI (Noran System 7, USA) system attached to SEM.

Fungicidal efficacy of synthesized silver nanoparticles
Fungicidal efficacy of Eucalyptus globulus leaf extract mediated synthesized silver nanoparticles were tested by agar well diffusion method against Didymella bryoniae, Fusarium oxysporum, Rhizoctania solani and Aspergillus flavus . About 20ml of Potato dextrose agar medium was poured and allowed to solidify. 100µl of Seven days old fungal inoculums were uniformly spread over petriplate containing PDA media and 8mm wells were punched on the petriplates using sterile cork borer to load the test samples. Then, 100μl of Silver nanoparticles(1mg/ml), plant extract, Silvernitrate, DMSO and standard fungicide(1mg/ml) with commercial name Sectin (Fenamidone 10% + Mancozeb 50%) were loaded to respective wells. Where, standard Sectin and DMSO was used as positive and negative control respectively. The plates were incubated at room temperature for 3-4 days and zone of inhibition was measured in mm. All tests were carried out in triplicate and repeated thrice.

Statistical analysis
All the data was subjected to one-way analysis of variance (ANOVA) using graph pad prism 5 software and represented at P ˂ 0.05 level of significance.

Synthesis of silver nanoparticles
When 1mM silver nitrate was added to leaf extract in varying proportions (9:1, 3:1, 1:1, 1:3 and 1:9) and observed for change in color. The change in color from pale yellow to dark brown indicated the reduction of silver ion to silver nanonparticles [18-20] due to excitation of surface Plasmon vibration of metal electrons [21,22] in the reaction mixture with 9:1 ratio and the intensity of color increased with increase in the incubation time. Whereas, no such changes were found in reaction mixtures with other proportions (Figure: 1A) suggesting that 9:1 ratio of metal precursor to leaf extract could be a better proportion for synthesis of silver nanoparticles (Figure: 1B). The phytochemicals present in the leaf extract act both as reducing and stabilizing agent in the synthesis process [23][24][25].

UV-spectroscopic analysis
The synthesis of silver nanoparticles was further confirmed by the UV-visible spectroscopy. The reaction mixture with 9:1 proportion of metal precursor to leaf extract showed intense peak at the wavelength between 420nm to 440nm. Whereas no intense peaks were found at particular wavelength in reaction mixture with other proportions ( Figure: 2A). Therefore, 9:1 ratio proportion is considered as optimum concentration and further synthesis carried out with same proportion. The intensity of absorbance peak at 425nm due surface Plasmon resonance of conductive electrons on metal nanoparticles increased with increase in time from 5min, 1hr, 2hr, 3hr, 4hr, 24hr and 48hr with no changes in the wavelength ( Figure: 2B) [26]. Sharp peak and increase in the intensity indicated increase in the nanoparticles formation and intensity of the peak at 24hr is nearly close to that of 48hr indicating the completion of reaction [27]. The result corroborates the previous reports where the Surface Plasmon resonance band ranged between 420nm to 460nm [28][29][30].

Dynamic Light Scattering (DLS) Analysis and Zeta potential
DLS is an important and reliable technique for analyzing the velocity distribution of particle movement by measuring dynamic fluctuations of light scattering intensity caused by the Brownian motion of the particle [31,32]. The histogram of the particle size analysis (Figure: 3) revealed the average particle size of 25nm of phytosynthesized silver nanoparticles in colloidal solution. The synthesized nanoparticles showed negative zeta potential of -7.8mv which shows the repulsive nature and stability of the particles [33,34]. Figure: 3 Histogram of Dynamic light scattering spectroscopy

X-Ray Diffraction Studies
The peaks obtained by XRD studies (Figure: 4) [29,[35][36][37][38]. Along with above mention peaks some other peaks were also present which may be due to crystallization of organic materials of plant origin [17]. The average particle size of the synthesized nanoparticles calculated using Debye-Scherrer's formula was 25.74nm which is similar to the obtained DLS result.

Fourier Transform Infra-red Spectroscopy (FTIR)
The Fourier Transform Infra-red spectrum was shown in Figure: 5. The peaks in the spectrum attributed to the possible biomolecules in leaf extract which acts as reducing as well as capping agent [28,39,40]. The peak at 2916cm-1 corresponds to the symmetric stretch of methylene C-H group, peak at 2111cm-1 corresponds to C≡C of terminal alkyne group, peak at 1611 attributes to the N-H bond of primary amine. The peaks at 1036 and 659 may be due to CN stretch of primary amine and C-H bend of alkyne /OH out of plane of alcohol respectively [41,42].

Scanning Electron Microscopy and EDX analysis
The image (Figure: 6A, 6B) of Scanning Electron Microscope clearly showed that the particles are spherical in shape. Strong signals of Silver in the EDAX spectrum ( Figure: 7) of synthesized particles confirms 83.20% silver along with 16.8% of oxygen content, which may be due to the biomolecules and capping agents attached to the silver nanoparticles which correlates earlier reports [43,44].

Antifungal activity
The photosynthesized silver nanoparticles proved effective in agar well diffusion method against the tested phytopathogens namely, Didymella bryoniae, Fusarium oxysporum, Rhizoctania solani and Aspergillus flavus. Didymella bryoniae found to be more susceptible to the Silver nanoparticles with inhibition zone of 18.3 mm than the standard (Sectin) where it showed 10.33mm of inhibition ( Figure: 8). Aspergillus flavus, Fusarium oxysporum and Rhizoctania solani were also found to be susceptible to synthesized silver nanoparticles with inhibition ozone of 15.3mm, 14.6mm and 12.3mm respectively which was on par with the standard fungicide Sectin with inhibition zone of 17.3mm, 15.6mm and 14.6mm respectively (Table: 1).

IV. CONCLUSION
Green chemistry approach of synthesizing silver nanoparticles which is known as a green synthesis method is considered as a reliable method of nanoparticle synthesis. It is a simple, easy, economical and eco-friendly method which utilizes biomolecules from plant source as reducing as well as capping agent for the synthesis of nanoparticles. In the present study leaf extract of Eucalyptus globulus was used successfully for synthesis of silver nanoparticles with spherical shape and average particle size of 25nm. The fungicidal efficacy of synthesized nanoparticles proved to be effective in plant disease management as an alternative or along with conventional synthetic chemical compounds and hence can lead to the minimal use of chemical compounds which may be good sign of eco-friendly approach.