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Activity of Biosynthesized Silver Nanoparticles in Combination with. Synthetic and Natural Fungicide Against Some Pathogenic Fungi†. S. ROY. * and T.K. DAS.

Asian Journal of Chemistry; Vol. 25, Supplementary Issue (2013), S315-S317

Activity of Biosynthesized Silver Nanoparticles in Combination with Synthetic and Natural Fungicide Against Some Pathogenic Fungi† S. ROY* and T.K. DAS Department of Biochemistry and Biophysics, University of Kalyani, Kalyani-741 235, India *Corresponding author: E-mail: [email protected] AJC-12863

Silver nanoparticles have been synthesized biologically using Aspergillus foetidus MTCC8876 fungi and then characterized the same by various biophysical and biochemical techniques. It is observed that this synthesized nanoparticle have very potential antifungal activity against some pathogenic fungi. In this experiment we used some synthetic and natural fungicide tebuconazole, trifloxystrobin and hexaconazole, as well as plant extract of ginger and teak. These fungicides are well known and have been used to control the fungal infection of plant. We observed the effect of silver nanoparticles in combination with systematic as well as natural fungicide against some Aspergillus sp. and Fusarium sp. It was observed that the diameter of inhibitory zone of fungicide + silver nanoparticles were higher compare to only fungicide or only silver nanoparticles against pathogenic fungi. So, it is assumed that the silver nanoparticles are very useful antifungal substance in-combination with systematic and natural fungicide to control the pathogenic fungi. Key Words: Silver nanoparticles, Fungicides, Aspergillus sp. and Antifungal activity.

INTRODUCTION Since last decade it is found that infections created by resistant microorganisms could not respond to conventional treatment, resulting in prolonged illness and greater risk of death. Recently it is reported that pathogenic bacteria and fungi resistant to commercially available synthetic antimicrobial agents have been gradually increasing and arising a serious problem in medical treatment for cure of microbial infection and diseases1. Among all inorganic antimicrobial agents, silver has been used mostly since ancient times to cure the infectious diseases developed in human body. In high concentration, silver is toxic to living being including human, whereas in low concentrations it is found to be nontoxic2. In recent study it is found that microorganisms have been used in synthesis of nanoparticles eco-friendly, e.g., fungi used for the synthesis of silver and gold nanoparticles3,4. It is reported that silver nanoparticles (AgNPs) could show antimicrobial specially antibacterial activities' but report in antifungal activities of metal nanoparticles or antifungal activities of the combined effect of metal nanoparticles and synthetic compounds are very scanty3,5,6. The combined effects of silver nanoparticle and antibiotics or commercial fungicides have been reported5,6. In the present study an attempt has been taken to study the antifungal activities of the biosynthesized silver nanoparticle also

to evaluate the efficacy of the antifungal activities of silver nanoparticle in presence of the synthetic fungicide like tebuconazole, trifloxistorbin and hexaconazole and also in presence of identified natural fungicidal extracts like teak and ginger, while plant and human pathogenic fungi have been considered as the testing fungal strains.

EXPERIMENTAL Synthesis of silver nanoparticles: The fungus A. foetidus MTCC8876 was grown in 250 mL Borosil flasks containing 100 mL Czapek-Dox (CD) broth medium as described by Raper and Thom (1949) at 25 ºC for 72 h in a shaker (120 rpm). After incubation, mycelial biomass was separated by filtration, washed repeatedly with sterile distilled water to remove the traces of media components and re-suspended in 100 mL distilled water and it was further incubated at 25 ºC for 72 h. The suspension was finally filtered through Whatmann filter paper no. 41. The cell filtrate were challenged with AgNO3 solution (1 mM final conc.) and shaking incubated at room temperature in dark. Characterization of synthesized silver nanoparticles: The silver nanoparticles formed have been characterized by the biophysical and biochemical methods like UV-visible spectroscopy analysis, FTIR spectroscopy analysis, particle size (DLS) analysis, zeta potential measurement, AFM analysis,

†International Conference on Nanoscience & Nanotechnology, (ICONN 2013), 18-20 March 2013, SRM University, Kattankulathur, Chennai, India

S316 Roy et al.

TEM and EDX analysis and nitrate reductase assay. The details of the experimental results & discussions were described in our earlier publication7. Antifungal activity: Well diffusion method was used to evaluate in vitro antifungal activity7. The antifungal activity of the synthesized silver nanoparticles and the combined effect of fungicide and silver nanoparticles were examined considering Aspergillus niger, Aspergillus foetidus, Aspergillus flavus, Fusarium oxysporum, Aspergillus oryzae and Aspergillus parasiticus as testing fungal strains. The commercial standard tebuconazole, trifloxistorbin and hexaconazole were collected from BCKV (Kalyani, Nadia) and the natural antifungal agent; leaf extract of teak in methanol and the extract of well known ginger extract in chloroform were also prepared and purified. To determine the combined effect, each standard were mixed with equal amount of the freshly prepared silver nanoparticles. 80 µL spore suspension of each of the testing fungal strains have uniformly been spreaded separately over the CD agar plates and the cavities having 3-4 mm in diameter were made in the middle of each agar plates and were filled up individually with the test solution. The Standard antifungal wells (synthetic agent and natural extract used as fungicides) were considered as the positive controls whereas each standard with silver nanoparticles were considered as testing sample and fungal cell filtrate used for the synthesis of silver nanoparticle was used as negative control. All the plates were then incubated at 28 ºC for 2 days. Similar experiments were carried out with the use of silver nanoparticles only. Assessment of increase in fold area: The increase in fold area was measured by calculating the mean surface area of the inhibition zone of each antifungal agent and antifungal agent +silver nanoparticles. The fold increase area of different test fungi for antifungal agent and for antifungal agent +silver nanoparticles was calculated by the equation8 (B2-A2)/A2, where A and B were zones of inhibition for antifungal agent and antifungal agent + silver nanoparticles, respectively.

RESULTS AND DISCUSSION Tebuconazole in methanol (10 ppm), trifloxistorbin in acetonitrile (10 ppm) and hexaconazole in hexane (100 ppm) were taken as the synthetic antifungal agents and extract of teak (3 % in methanol) and ginger (2 % in chloroform) considered as natural antifungal agents9,10 which have been used against many testing fungal strains developing infections,were entitled as positive controls with respect to the synthesized silver nanoparticles. The diameter of inhibition zones and increase in fold area for all the test fungi (Aspergillus sp., Fusarium sp.) was measured. The antifungal activities of the synthetic/natural extract identified as fungicides have been found to be increased significantly in presence of silver nanoparticles. Maximum antifungal activity was exhibited by tebuconazole against F. oxysporum whereas lowest activity shown by the same against A. parasiticus. When trifloxistorbin was considered as synthetic fungicide maximum antifungal activity was reflected against A. foetidus and lowest activity against F. oxysporum and in case of hexaconazole it was maximum against F. oxysporum and lowest against A. flavus (Fig. 1). Teak extract showed maximum antifungal activity against A. niger and lowest against A. oryzae whereas ginger

Asian J. Chem.

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Fig. 1. Graphical representation of combination effect of synthetic fungicides (tebuconazole (A), trifloxistorbin (B) and hexaconazole (C) respectively) and silver nanoparticles against pathogenic fungi

extract showed maximum against A. flavus and lowest against on A. parasiticus. However, no enhancement of the antifungal activities were recorded in the cases of F. oxysporum, A. niger and A. foetidus (Fig. 2). The antifungal activities of the synthetic, natural fungicidal agents have been assayed on the basis of increase of fold area. These findings corroborate the results obtained by Gajbhiye et al.6 who reported that nano-Ag showed a significant effect against C. albicans followed by Trichoderma sp. and P. glomerata when fluconazole was used as a positive control. Kim et al.11 also reported that nano-Ag showed a significant effect against growth inhibition of C. albicans when amphotericin B was used as a positive control.

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Vol. 25, Suppl. Issue (2013)

Activity of Biosynthesized Silver Nanoparticles Against Some Pathogenic Fungi S317

ACKNOWLEDGEMENTS (E)

The authors are thankful to the Department of Science and Technology (DST) of India, for their DST INSPIRE fellowship for financial support during this work. The authors are also thankful to Arindam Mandal & Dr. Ramen Kole for their help in preparing and purification of the plant extract and the collection of synthetic fungicides.

REFERENCES Fig. 2. Graphical representation of combination effect of natural fungicides [teak (D) & ginger (E) respectively] and silver nanoparticles against pathogenic fungi; *Mean surface area of the inhibition zone was calculated for each from the mean diameter.; # Increase in fold area was calculated as [(B2-A2)/ A2], where A & B are the inhibition zones for silver nanoparticle and synthetic fungicides +silver nanoparticle respectively

Conclusion The antifungal activities of silver nanoparticles in combination of a standard synthetic antifungal agent and also in combination of a natural extract identified as antifungal agent against some pathogenic fungi can be considered as a new finding. Silver nanoparticles alone or silver nanoparticles along with synthetic or natural extract identified as fungicides are found to be effective agents against the growth of plant and/or human fungal pathogens.

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