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Evaluation of Antimicrobial Potential of Aspergillus Ibericus Isolated from Rhizospheric Soil of Ficus Religiosa

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Evaluation of Antimicrobial Potential of Aspergillus Ibericus Isolated from Rhizospheric Soil of Ficus Religiosa International Journal of Pharmacy and Biological Sciences TM ISSN: 2321-3272 (Print), ISSN: 2230-7605 (Online) IJPBSTM | Volume 8 | Issue 4 | OCT-DEC | 2018 | 320-331 Research Article | Biological Sciences | Open Access | MCI Approved| |UGC Approved Journal | EVALUATION OF ANTIMICROBIAL POTENTIAL OF ASPERGILLUS IBERICUS ISOLATED FROM RHIZOSPHERIC SOIL OF FICUS RELIGIOSA Geetanjali Geetanjali1, Pranay Jain1* and Ram Kumar Pundir2 *1Department of Biotechnology, University Institute of Engineering & Technology, Kurukshetra University, Kurukshetra 2Department of Biotechnology Engineering, Ambala College of Engineering & Applied Research, Devsthali, Mithapur, Ambala, Haryna *Corresponding Author Email: [email protected] ABSTRACT Inappropriate and irrational use of antimicrobial medicines has prompted the resistant microbes to emerge, spread and persist. Antimicrobial resistance may be intrinsic or acquired as it can develop through the spontaneous mutation of existing genes or through the transfer of genes from other resistant species or strains. Therefore, antimicrobial resistance has resulted research and development for the discovery of new antibiotics against pathogens at all times. Natural products with industrial applications can be produced from secondary metabolism of living organisms including plants, animals and microorganisms. The present study highlights the bioactivity of rhizosphere soil fungi Aspergillus ibericus from Ficus religiosa against opportunistic pathogens, through the production of secondary metabolite exhibiting antimicrobial activity. Antimicrobial metabolite obtained from A. ibericus was found to be effective against test microbes gram-positive bacteria, Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans and Streptococcus pyogenes, gram-negative bacteria, Pseudomonas aeruginosa and Escherichia coli and yeasts such as Candida albicans and C. tropicalis. The antimicrobial metabolite was thus broad spectrum in nature. The effect of fermentation medium, temperature and pH was also observed on the production of antimicrobial metabolite. KEY WORDS Antimicrobial Resistance, Rhizosphere Soil, Oppurtunistic Pathogens, Aspergillus ibericus INTRODUCTION purposes. However, overconsumption of antibiotics Antibiotics are the most important pillar of current caused an enormous selective pressure on the bacteria medications [1]. The antimicrobial compounds have to gain resistance or die [5]. The increasing frequency of been found to cure various kind of bacterial and fungal multi-resistant pathogenic bacteria has compromised infections, but the discovery of these agents has been the clinical treatment of an emerging infectious tempered by appearance of resistant microbial diseases. There is an urgent demand for new pathogens. Emergence of drug resistance in them has antimicrobial compounds active against current emphasized the need of research for new compounds resistant pathogens and emerging pathogens [6]. [2,3,4]. Antibiotics nowadays are taken for granted to Natural drug discovery involves the search for natural treat bacterial infections. The antibiotics are sources such as soil, bacteria, mold and plants for new successfully used for prophylactic or therapeutic chemical entities (NCE). Natural products provide a vast purposes regularly in clinical, veterinary and agricultural source of chemically diverse biologically active leads for International Journal of Pharmacy and Biological Sciences Pranay Jain* et al 320 www.ijpbs.com or www.ijpbsonline.com ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. therapeutic agents [7}. Most of the antibiotics used all and air dried) was suspended in 9ml sterilized distilled over the world are derived from natural compounds. water blank no. 1 and shaken vigorously to obtain Fungal antagonism has been reported against many uniform suspension. 1ml of suspension was transferred, pathogenic organisms by researchers [8]. Besides the while in motion, from stock suspension (No. 1) to sterile antimicrobial property, fungi isolated from soil are also water blank no. 2 with sterile pipette under aseptic the sources of other industrially important compounds conditions to make 1:100 (10-2) dilution. For fungi, 10-2 like enzyme inhibitors, antihelminthics, antitumour to 10-5 dilutions were used. Approximately 20-25ml agents, insecticides, vitamins, immunosuppressant and cooled (45°C) molten nutrient agar and potato dextrose immunomodulators [8]. Penicillium, Cephalosporium and agar was added to each petri plate. After solidification Aspergillus species have gained importance as produced of agar media, 100µl aliquots of suspension of dilutions commercial antibiotics, penicillin G, V, cephalosporin, were added to labeled and sterilized petri plates and griseofulvin, fumagillin, variotin, fusidic acid, siccanin spread with spreader. Inoculated plates were incubated and xanthocillin [9]. Antibacterial, antifungal and at 30°C for 4-5 days for fungi. antitumour compounds are found to be produced by Purification and maintenance of isolates many Aspergillus species isolated from various soils Fungal colonies appearing on their respective media such as asterric acid, asterriquinone, aspulvinone, were transferred to potato dextrose agar plates (one citrinin, emodin, butyrolactone I, geodin, terrecyclic colony on each plate) at 30°C for 4-5 days. The colonies acid, questrin, itaconate, sulochrin and lovastatin [10]. were then transferred on potato dextrose agar slants Many antimicrobial and antitumour quinones have and incubated at 30°C for 4-5 days for fungi and were been reported from Aspergillus species [11]. In keeping maintained at 4°C in a refrigerator for further studies. view of the above justifications, for the continuous Morphological and Molecular identification of fungal search of new isolates from rhizosphere soil of isolate medicinal plants, having antimicrobial activity, the Fungal isolate was identified as Aspergillus ibericus present study highlights the bioactivity of rhizosphere based on colony morphology, pigmentation, growth soil fungus Aspergillus ibericus from Ficus religiosa pattern and sporulating structure by lactophenol cotton against opportunistic pathogens, through the blue staining by following various manuals and production of secondary metabolite exhibiting monographs [15,16,17]. Molecular identification was antimicrobial activity. The isolated PIF2 strain performed by National Fungal Culture Collection of (Aspergillus ibericus) belongs to Order Eurotiales, Class India (NFCCI), Agharkar Research Institute, Pune Eurotiomycetes and Family Trichocomaceae. Serra et al. Screening for antimicrobial activity of soil microbes by [12] for the first time isolated a new species of section agar well diffusion method Nigri, A. ibericus from wine grapes and dried vine fruit in The antimicrobial activity of pure fungal isolate Portugal and Spain and reported that strains of A. Aspergillus ibericus was evaluated by using agar well ibericus did not produce detectable ochratoxin A diffusion assay [18] against gram-positive bacteria, (carcinogenic) in culture media, therefore could be Bacillus subtilis (MTCC 441), Staphylococcus aureus exploited for biotechnological applications. (MTCC 87), Streptococcus mutans (MTCC 497), Streptococcus pyogenes (MTCC 1924); gram-negative MATERIALS AND METHODS bacteria, Pseudomonas aeruginosa (MTCC 424), Collection of soil samples Escherichia coli (MTCC 40) and yeasts namely Candida Soil samples were collected from rhizosphere of albicans (MTCC 227) and Candida tropicalis (MTCC medicinal plant Peepal (Ficus religiosa) from Botanical 3421) which were procured from CSIR- Institute of garden, Kurukshetra University, Kurukshetra. by Microbial Technology, Chandigarh, India. All test removing 1-1.5 inch of top soil with sterilized spatula. microbes were grown in their specific media, nutrient Isolation of Aspergillus ibericus from soil agar, malt extract agar and brain heart infusion agar. The serial dilution agar plate method was used for Nutrient agar plates were inoculated with 100µl of isolation of Aspergillus ibericus from soil sample [13,14]. standardized inoculum (0.5 McFarland Standard) of Potato dextrose agar (PDA) (CDH) for fungi was used as each test microbe (in triplicates) and was spread with isolation medium. One gram of soil (finely pulverized sterile swabs. Wells were made into agar plates International Journal of Pharmacy and Biological Sciences Pranay Jain* et al 321 www.ijpbs.com or www.ijpbsonline.com ISSN: 2230-7605 (Online); ISSN: 2321-3272 (Print) Int J Pharm Biol Sci. containing the test microbe inoculum. 200µl volume of flasks. These flasks were autoclaved at 121°C for 15 extract was poured into a well of inoculated plates. minutes. For each value, three replicates were used. Uninoculated potato dextrose broth (Hi-Media) was Three disks cut from four days old colony of selected used as negative control. Antibiotics ciprofloxacin fungus were added as inoculum in each flask. The (antibacterial) and fluconazole (antifungal) were used as inoculated flasks were incubated at 30°C for 12-14 days positive control. Then plates were left at room under stationary condition. The extracts after filtration temperature for ten minutes allowing the diffusion of with filter paper were assayed for antimicrobial activity extract into agar. After incubation for 24 hours at 37°C, against test microbes by using agar well diffusion the plates were observed for inhibition zone method [10]. surrounding the well containing
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