Nanosulfur: A Potent Fungicide Against Food Pathogen, Aspergillus niger

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Nanosulfur: A Potent Fungicide Against Food Pathogen, Aspergillus niger Samrat Roy Choudhury, Kishore K. Nair, Rajesh Kumar, Robin Gogoi, Chitra Srivastava et al. Citation: AIP Conf. Proc. 1276, 154 (2010); doi: 10.1063/1.3504287 View online: http://dx.doi.org/10.1063/1.3504287 View Table of Contents: http://proceedings.aip.org/dbt/dbt.jsp?KEY=APCPCS&Volume=1276&Issue=1 Published by the American Institute of Physics.

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Nanosulfur: A Potent Fungicide Against Food Pathogen, Aspergillus niger Samrat Roy Choudhurya, Kishore K. Nairb, Rajesh Kumarb, Robin Gogoic, Chitra Srivastavad, Madhuban Gopalb, B. S. Subhramanyamd, C. devakumarb and Arunava Goswamia a

Agricultural and Ecological Research Unit, Biological Sciences Division, Indian Statistical Institute, 203 B. T. Road, Kolkata, West Bengal- 700108, India. b Department of Agricultural Chemicals ,cPlant Pathology and dEntomology, Indian Agricultural Research Institute, Pusa Campus, New Delhi, India. Abstract. Elemental sulfur (S0), man’s oldest eco-friendly fungicide for curing fungal infections in plants and animals, is registered in India as a non-systemic and contact fungicide. However due to its high volume requirement, Indian agrochemical industry and farmers could not effectively use this product till date. We hypothesize that intelligent nanoscience applications might increase the visibility of nanosulfur in Indian agriculture as a potent and eco-safe fungicide. Sulfur nanoparticles (NPs) were synthesized bottom-up via a liquid synthesis method with average particle size in the range of 50-80 nm and the shapes of the NPs were spherical. A comparative study of elemental and nano-sulfur produced has been tested against facultative fungal food pathogen, Aspergillus niger. Results showed that nanosulfur is more efficacious than its elemental form. Keywords: Elemental sulfur, Nanosulfur, Fungicide, Aspergillus niger. PACS: 81.16.Be, 87.85.Rs

INTRODUCTION 0

Sulfur (S ) represents the ninth and least abundant essential macronutrient in plants, however it is a prerequisite for L-methionine biosynthesis; the most essential amino acid for initiating protein synthesis in living organisms. It is the only inorganic phytoalexin produced by species of different plant family viz. solanaceae, Malvaceae Sterculiaceae, and Leguminosae to combat fungal invasions. [3]. Elemental sulfur is widely used as a fungitoxic agent in agricultural fields against a wide range of powdery mildews, certain smut and rust fungi and some other common fungal pathogens including Cladosporium fulvum, and Fusarium spp. Sulfur is believed to be enzymatically reduced in living cells to form hydrogen sulfide and this reduction proceeds by a single electron transfer from the donor in the cell to sulfur [4]. Production of excess hydrogen sulfide within the fungal body spells enhanced toxicity and leads to the inhibition of fungal growth. Despite the antifungal efficacy of sulfur, its use is now restricted among farmers and agrochemical industries mainly for two reasons, firstly sulfur is required in bulk quantities for application in agricultural fields and secondly it is also likely to induce resistance in the target species. It is desirable to CREDIT LINE (BELOW) BEisINSERTED ONat the same time, the considerably reduce the amount, so that theTO cost cut down and THE FIRST PAGE OF EACH PAPER build up of resistance in target pathogens is minimized. Intelligent bottom up nanoscience application is the most appropriate tool to resolve the aforesaid problems The present study emphasizes the efficacy of CP1276, International Conference on Advanced Nanomaterials and Nanotechnology (ICANN-2009) edited by P. K. Giri, D. K. Goswami, A. Perumal, and A. Chattopadhyay © 2010 American Institute of Physics 978-0-7354-0825-8/10/$30.00

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nanosulfur, a new fungitoxic agent against Aspergillus niger which has never before been subjected to sulfur action. Aspergillus niger is a pervasive fungi which has both clinical and agricultural importance. This ubiquitous fungus is responsible for black mould disease of certain vegetables, aspergillosis in animals and is a common food contaminant. Some medically important strains are known to possess nephrotoxic mycotoxin called ochratoxin. Sulfur nanoparticles can be synthesized by many techniques like water in oil microemulsion technique [2], cystine modified nanosulfur formulation [5] and liquid synthesis method [1]. Among them, application of liquid synthesis technique is the simplest of all. The process can be carried out easily at room temperature and with minimal facility.

Materials and Methods Particle Synthesis Sublimed sulfur powder of 100 mesh size (149 micron) was thoroughly pulverized in a high energy ball mill for 1hour. 1gm of sublimed sulfur powder was then incorporated into 100ml of sodium sulfide (2mol/L) solution under continuous stirring until the color of the solution turned reddish black which ensures the formation of sodium polysulfide solution. Polyethylene glycol-400(PEG-400) was selected as the surfactant. Aqueous PEG-400 solution was prepared mixing PEG-400 and ddH20 in 5:1 ratio. Appropriate amount of sodium hydroxide (0.1 mol/L) was then added to the aqueous PEG-400 solution to enhance the pH of the surfactant solution above 8. Around 12.5ml of sodium polysulfide solution was next incorporated into the surfactant solution in the reaction beaker. 200ml of formic acid solution (formic acid: ddH20 in 4:1 ratio) was dripped into the reaction beaker containing surfactant solution plus sodium polysulfide under continuous stirring (1400 rpm or more) and at ambient temperature. Resultant product was then centrifuged at 5000rpm for 20mins at ambient temperature. PEG-400 supernatant was discarded and the precipitate was repeatedly washed with absolute ethanol to get rid excess PEG-400. The product was finally subjected to vacuum rotary evaporator [EYELA N-N series] to remove residual alcohol from the nanosulfur formulation.

Fungal Isolation, Subculture and Antifungal Assay Strains of fungi have been identified from rotten potatoes from commercial market of Calcutta, India. Fungal isolates were washed with 1% sodium hypochlorite treatment and stained with Safranin O after Farrokhi et al., [6] prior to their preliminary identification. Morphological characterization of the strains was carried out by Indian Agricultural Research Institute (IARI). Strains were regularly sub cultured in Potato dextrose agar [PDA] at 30°C so as to ensure their viability.

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FIGURE 1. Characterization of Sulfur nanoparticle. (i) DLS size distribution of the sulfur nanoparticles, (ii) UV-VIS characterization of elemental and nanosulfur, (iii) SEM image for sulfur nanopaticles

Comparative antifungal assay of elemental and nanosulfur was carried out after Dhingra et.al, [7] with four different doses (2000ppm, 1000ppm, 500ppm and 125ppm) and mixed with PDA to prepare the poisoned food. An appropriate amount of spore was taken from the 5 days old fungal culture, and an aliquot of fungal suspension was prepared in 1 ml of triple distilled water. Triplicate of each treatment plate was made. Growth and sporulation of the fungi were examined after 48hrs of incubation at 30°C.

Results and Discussion

Polyethylene glycol-400 was selected as the surfactant for the synthesis as it is a nonionic hydrophilic surfactant with high HLB (hydrophilic lipophilic balance), rendering facilitated absorption by the living organisms and lowers the risk of opsonization from the living cell surface. Its higher biocompatibility and rapid biodegradability makes it suitable for the agricultural applications.

Analysis of DLS and UV-VIS Dynamic light scattering (DLS) [Nano S (red badge), Ze-1600- Malvern] was used to measure the average size range of the nanoparticles [Fig-1(i)]. Response of nanosulfur to ultraviolet and visible range of electromagnetic radiations was determined through UV-VIS [Perkin Elmer; Lambda 25]. For UV-VIS spectroscopy nanosulfur formulation was scanned for 5 times in order to confirm the mean absorbance spectrum [Fig-1(ii)].

Analysis of SEM, TEM and EDAX Surface topology of sulfur nanoparticles were determined by scanning electron microscope [FEI Quanta-200 MK-2] at 50,000 magnifications under 15.00kV. SEM image revealed spherical surface of the particles [Fig-1(iii)]. Transmission electron microscope (TEM) [JEOL 2010F] was used to analyze the nanostructured materials with atomic scale resolution, which revealed average particle size was around 50nm [Fig-2(i). Purity and chemical composition of the fabricated nanomaterials thus obtained were analyzed with Energy-dispersive X-ray spectroscopy (EDAX) [FEI Quanta-200 MK-2; Fig-2(ii)] 156

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FIGURE 2. (i) TEM image of sulfur nanoparticles, (ii)EDAX spectrum of sulfur nanoparticles, (iii) Particle distribution in bar diagram, (iv) Antifungal assay with elemental and nanosulfur, (v) Comparative study for the efficacy of elemental and nano sulfur against Aspergillus niger; Disk-1: Control, Disk-2: Elemental Sulfur treated fungus, Disk-3 & 4: Nanosulfur treated fungus.

Analysis of antifungal Assay Nanosulfur proved to be efficacious as a fungitoxic agent over the commercially available elemental sulfur. The synthesized nanoformulation retards the growth of fungi but also inhibits sporulation. In comparison elemental sulfur retards fungal growth in a dose dependent manner but fails to inhibit spore formation. Figure 2(iv) and 2(v) represents the comparative antifungal assay and zone diameter reduction for each dose with elemental and nanoform of sulfur.

ACKNOWLEDGMENTS We are grateful to NAIP-ICAR-World Bank (Comp-4/C3004/2008-09; PI: A. Goswami, ISI and CCPI: C. Devakumar, IARI) & Department of Biotechnology (DBT),Govt. of India (BT/PR9050/NNT/28/21/2007 & BT/PR8931/NNT/28/07/2007) and ISI plan project for 2008-2011 for their generous financial support.

REFERENCES 1. Y. Guo et.al, “Synthesis and Characterization of Sulfur Nanoparticles by Liquid Phase Precipitation Method”, ACTA CHIMICA SINICA -CHINESE EDITION, 63; Part 4,337-340(2005) 2. Y. Guo et. Al, “Preparation and Characterization of monoclinic sulfur nanoparticles by water-in-oil microemulsion technique”, Powder Technology, 162, 83-86(2006) 3. J. S. Williams and R. M. Cooper, Plant Pathology, 53, 263-279(2004) 4. R. G. Owens,“Chemistry and Physiology of Fungicidal Action”, Annu. Rev. Phytopathol, 1963.1:77100. 5. X. Y. Xie et.al, “Cystine modified nano-sulfur and its spectral properties”, Materials letters, 63, 1374-1376(2009) 6. R. F. Nejad et.al, Pakistan Journal of Biological Sciences 10(21); 3793(2007) 7. O. D. Dhingra & J.B Sinclair, Basic Plant Pathology Methods (2nd Edn)CRC Press,Inc,1995,pp. 272

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