Endophytic Mycoflora of Mirabilis jalapa L. and Studies on Antimicrobial Activity of its Endophytic Fusarium sp.

Transcription

1 ASIAN J. EXP. BIOL. SCI. VOL 2(1) 2011: Society of Applied Sciences ORIGINAL ARTICLE Endophytic Mycoflora of Mirabilis jalapa L. and Studies on Antimicrobial Activity of its Endophytic Fusarium sp. Rakshith Devaraju and Sreedharamurthy Satish* Department of Studies in Microbiology, Manasagangothri, University of Mysore, Mysore , Karnataka, India. ABSTRACT Antimicrobial activity of extra-cellular antimicrobial secondary metabolites of endophytic Fusarium sp. isolated from the stem of Mirabilis jalapa L. was evaluated by an agar disc diffusion technique against selected groups of Gram-positive and Gram-negative human and phytopathogenic bacteria, yeasts and filamentous fungi. Aspergillus species were more found to be sensitive followed by Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis). Highest inhibitory activity was reported against Gram-positive bacteria, where as reduced, but similar effects, were shown against Aspergillus species and Gram-negative bacteria (E.coli, Salmonella typhi, Xanthomonas campestris, Xanthomonas axonopodis malvacearum, Xanthomonas campestris PV oryzae and X anthomonas campestris pv vesicatoria). KEY WORDS: Endophytes, Antimicrobial secondary metabolite, Fusarium sp, biotope INTRODUCTION In the continual search by both pharmaceutical and agricultural industries for new products, natural selection has been found to be superior to combinatorial chemistry for discovering novel substances that have the potential to be developed into new industrial products. Since natural products are adapted to a specific function in nature, the search for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes. Endophytic fungi inhabit such a biotope [1]. Endophytes may be defined as microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects [2]. Endophytic microorganisms are to be found in virtually every plant on earth. These organisms reside in the living tissues of the host plant and do so in a variety of relationships ranging from symbiotic to pathogenic. In culture, outside of their host tissue, endophytic fungi are also known to produce a number of important secondary metabolites including anti-cancer, anti-fungal, anti-bacterial anti-diabetic and immunosuppressant compounds [3]. SubglutinolA, an immunosuppressant produced by an endophytic Fusarium subglutinans strain isolated from T. wilfordi [4]. Two antibacterial and xanthine oxidase inhibitory cerebrosides, one of which is chemically new, were characterized from the chloroform methanol (1:1) extract of Fusarium sp. IFB-121, an endophytic fungus in Quercus variabilis [5]. Over 50 antibiotic substances were detected in Fusarium spp. and Trichoderma spp. alone [6]. An endophytic fungus having antimicrobial properties against Bacillus subtilis, Staphylococcus aureus, Mycobacterium smegmatis, M.phlei, E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans, Fusarium oxysporum and F. semitectum with MIC range between 20-30μg/ml was isolated from Taxus wallichiana of Arunachal Pradesh, India [7]. Occasionally, these compounds are the same as those produced by the respective host plants, thus triggering the expectation that endophytic fungi can serve as an alternative source of important plant secondary metabolites. The current study was conducted to isolate and screen for endophytic fungi with antimicrobial activities from Mirabilis jalapa L. and to optimize the medium for the isolation and characterization of antimicrobial active principles. ASIAN J. EXP. BIOL. SCI. VOl 2 (1)

2 MATERIALSAND METHODS Isolation and cultivation of fungal endophytes Endophytic Fusarium sp. isolated from stem of Mirabilis jalapa by surface sterilized dipping in 75% ethanol for 1 minute, immersed in 4% NaOCl for 3 minutes and then rinsed with 75% ethanol for 1 minute, then rinsed 3 times with sterile distilled water and sample segments were allowed dry on sterilized blotter in an clean laminar air flow [8]. The sterilized tissue samples were plated on potato dextrose agar medium contained in Petri dishes, amended with chloremphenicol and streptomycin sulphate (150 mg/l). The Petri dishes were incubated at 26º C for 3 weeks in a light chamber. The light regimen was 12 hours dark: 12 hours light cycles. Fungal colonies were transferred to potato dextrose agar, Malt extract agar and czapek's dox agar plates without antibiotic and identified using standard monographs. Data analysis The frequency of colonization of fungi is calculated as the number of segments yielding a given fungus divided by the total number segments incubated x 100 [9] and dominant endophytes were calculated as percentage colony frequency divided by sum of percentage of colony frequency of all endophytes 100 [10]. Bacteria Test human pathogenic bacteria, E.coli (MTCC 7410), Staphylococcus aureus (MTCC 7443), Staphylococcus epidermidis (MTCC 435), Bacillus subtilis (MTCC 121), Salmonella typhi (MTCC 733) Authentic pure culutures of phytopathogenic Xanthomonas axonopodis PV malvacearum ( X.a. pv. m) from cotton ( Gossypium herbaceum L. ), Xanthomonas oryzae pv oryze ( X.o. pv. o) from rice ( Oryza sativa L), Xanthomonas campestris pv vesicatoria ( X.c.pv.v) from tomato ( Lycopersicon esculentum Mill.) were obtained from DANIDA Lab, DOS in applied Botany and Biotechnology, University of Mysore, India. Screening assay forantimicrobialactivity An eight-day-old culture of endophytic Fusarium species grown on potato dextrose agar, malt extract agar and czapek dox agar without antibiotic was used to test for antimicrobial activity using an agar disc diffusion technique on solid media. The procedure was modified from Rois et al. (11). 100μl of bacterial suspension adjusted 0.5 McFarland 4 standard, fungal spore and yeast suspension (10 C.F.U/ml) were aseptically surface spread on 20 ml solidified nutrient agar and Sabouraud's agar repectively. Plates were allowed to stand for 10 min at room temperature and then inoculated with 6 mm agar discs containing the endophytic Fusarium species. The plates were then incubated for 24 h at 37 C for bacteria and yeast for 48 h at 28 C for fungi. Each experiment was repeated at least three times and measured diameters of the inhibition zone surrounding the agar disc was averaged and expressed in mm. The mean data of the inhibition zone diameter were recorded. RESULTSAND DISCUSSION A total of 17 endophytic fungal isolates belonging to 10 genera were isolated from the Mirabilis jalapa. The colonization frequency was 17% (Table 1). The fungal composition included 70.2% of hyphomycetes, 17.5% of coelomycetes, 11.6% of ascomycetes and 11.6% of sterile mycelia. In the present investigation we screened only Fusarium sp because its colonization frequency is more and it is dominant fungi. Data on antimicrobial activity for endophytic Fusarium species, using the whole agar diffusion disc technique against selected Gram-positive ( Bacillus subtilis, Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative human and phytopathogenic bacteria ( E-coli, Salmonella typhi, Xanthomonas campestris, Xanthomonas axonopodis malvecearum, Xanthomonas campestris pv oryzae and xanthomonas campestris pv vesicatoria ) and fungi ( A. flavus, A. niger, A. nidulans,a. flavus oryzae, A. flavus columnaris, A. tamarii, A. candidus, A. clavatus. A. terreus. and F. verticilloides, F. oxysporum and C. albicans) are shown in tables 2 and 3, figures 1 & 2 for endophytic Fusarium species, respectively. The extent of antimicrobial activity is expressed in diameter of inhibition zones (mm). Standard antibiotic discs (Gentamycin 10μg/disc; Nystatin 10 μg/disc and Streptocycline-K 10μg/disc for phytopathogenic bacteria) were included to monitor the experimental conditions and to facilitate better comparative analysis. Data in tables and figures clearly showed the production of antimicrobial secondary metabolites from endophytic Fusarium sp. isolated from Mirabilis jalapa to selected species of microbes, representing both prokaryotic and eukaryotic systems. Our results are based on testing secondary metabolites produced during the stationary phase (10- day-old-cultures) and directly diffused through agar discs. In general, fungi ( Aspergillus species) were more sensitive to secondary antimicrobial metabolites produced from endophytic Fusarium sp. than were bacteria (Table 2). Moreover, Gram-positive bacteria were more susceptible to fungal inhibitory compounds when compared to Gramnegative human and phytopathogenic bacteria (Table 1). It is found that there was no antifungal activity exhibited 76 ASIAN J. EXP. BIOL. SCI. VOl 2 (1) 2011

3 against other phytopathogenic Fusarium species and yeasts ( Candida albicans). Production of secondary metabolic products are not only influenced by the genetic base of the organism, physicochemical environment in which the microbe is growing, species, or race, rather than organism group or type but also depends on specific media or kind of nutrients available, i.e., production of antimicrobial secondary metabolites was quantitatively more prominent in endophytic Fusarium species grown on potato dextrose agar medium which is depicted by zone of inhibition than in malt extract agar and czapek dox agar medium respectively. This method can be employed for (1) differentiating the isolates screening for bioactives (2) preliminary screening to reduce the cost of fermentation and laborious work of screening for bioactives. This study helps to optimize conditions of endophyte fermentation that has been found to show antimicrobial activity in order to enhance the yield of active substances synthesized by endophytes and also support that endophytes contribute to their host plant by producing a plethora of substances that provide protection and ultimately survival value of the plant. *Based on 100 segments plated Table 1 List of endophytic Fungi isolated from Mirabilis jalapa L. Endophytic Fungi No. of Endophytes Colonization Frequency* Table 2 Antibacterial activity of endophytic Fusarium sp. grown on different mycological media. (Zone of inhibition in mm) Test PDA CDA MEA Gentamycin Streptocycline- Microorganisms 10μg/disc K 10μg/disc Bacillus subtilis ± ± ± ± ND- Staphylococcus ±1.0 -NS- -NS ± NDaureus Staphylococcus ±1.54 -NS ± ±1.63 -NDepidermidis E.coli ± NS- -NS ± ND- Salmonella typhi ±1.15 -NS ± ± ND- Xanthomonas ± NS- -NS- -ND ± 2.64 axonopodis pv malvacearum Xanthomonas ± NS- -NS- -ND ± 1.52 oryzae pv oryzae Xanthomonas ± NS- -NS- -ND ± 2.00 campestris pv vesicatoria Values are mean inhibition zone (mm) ± S.D of three replicates NS- Not sensitive ND-Not determined Dominant fungi Ascomycetes Chaetomium sp Sporormia sp Coelomycetes Phoma Phomopsis sp Hyphomycetes sp. Acremonium sp Aspergillus niger Cladosporium sp Curvularia sp Fusarium sp Trichoderma sp Sterile mycelia No. of isolates 17 17% ASIAN J. EXP. BIOL. SCI. VOl 2 (1)

4 Table 3 Antifungal activity of endophytic Fusarium Values are mean inhibition zone (mm) ± S.D of three replicates NS- Not sensitive ND-Not determiney sp. grown on different mycological media. (Zone of inhibition in mm) Test fungi PDA CDA MEA Nystatin 10μg/disc Aspergillus niger ± NS- -NS ± 1.15 Aspergillus ± ± ± ± 1.52 flavus A. nidulans ± NS- -NS ± 1.00 A. flavus oryzae ± NS ± ± 1.52 A.flavus ± NS- -NS ± 1.15 columnaris, A. tamarii ± ± NS ± 1.15 A. candidus, ± NS ± ± 1.52 A. clavatus ± NS- -NS- 18 A. terreus -NS- -NS- -NS ± 0.57 F. verticilloides -NS- -NS- NS ± 1.52 F. oxysporum -NS- -NS- NS ± 1.57 C. albicans -NS- -NS- NS ± 2.08 Fig-1 Antibacterial activity of endophyticfusarium species grown on different mycological media PDA CDA MEA Zoneofinhibitioninmm Gentamicin 10µg/disc Streptocycline-K 10µg/disc B. subtilis y S.aureus S.epidermidis E-coli S. typhi XAM XOO XCV Test bacteria Fig-2 Antifungal activity of endophytic Fusarium species grown on diffrent mycological media Zo ne o f in h ib itio n in m m PDA 5 CDA MEA Nystatin 10µg/disc 0 Aspergillus niger Aspergillus flavus A.nidulan s A.flavus oryzae A.flavus column aris, A.ta m a rii A.can did u s, A.cla va tu s A. terreus F.verticillo ides F.oxysporu m C. alb ican s Test fungi 78 ASIAN J. EXP. BIOL. SCI. VOl 2 (1) 2011

5 Acknowledgement: The Authors thank the university grant commission (UGC). Government of India, for providing the grant to carry out this work, and department of studies in Microbiology, University of Mysore for laboratory facilities. REFERENCES [1] Schulz, B., Boyle, C., Draeger, S. (2002). Endophytic fungi: a source of biologically active secondary metabolites. Mycological Research 106: [2] Bacon, C. W., Stone, J. K. and White, J. F. (2000).An overview of endophytic Microbial Endophytes 1: pp [3] Strobel, G. and Daisy, B. (2003). Bioprospecting for Microbial Endophytes and Their Natural Products. Microbiology and Molecular Biology Reviews 67(4): [4] Lee, J., E. Lobkovsky, N. B. Pliam, G. A. Strobel, and J. Clardy. (1995). Subglutinols A and B: immunosuppressive compounds from the endophytic fungus Fusarium subglutinans. J. Org. Chem. 60: [5] Shu, R.G., Wang, F.W., Yang, Y.M., Liu, Y.X and Tan, R.X. (2004). Antibacterial and Xanthine oxidase inhibitory cerebrosides from Fusarium sp.,ifb-121, an endophytic fungus in Quercus variabillis. Lipids 39(7): [6] Berdy, J. (1974): Recent development of antibiotic research and classification according to chemical structure. Adv App Microbiol 18: [7] Gogai, D. K., Hari, P., Boruah, D., Saikia, R and Bora, T. C. (2008). Optimization of process parameters for improved production of bioactive metabolite by a novel endophytic fungi Fusarium sp., DF2 isolated from Taxus wallichiana of North East India. World Journal of Microbiology & Biotechnology 24: [8] Suryanarayanan, T. S., Senthilarasu, G. and Muruganandam, V. (2000). Endophytic fungi from Cuscuta reflexa and its host plants. Fungal Diversity 4: [9] Krishnamurthy, Y. L., Naik, B. S and Jayalakshmi, S. (2008). Fungal communities in Herbaceous Medicinal plants from the Malnad Region, Southern India. Microbes and Enviroment 23(1): [10] Mahesh, B., Tejesvi, M. V., Nalini, M. S. Prakash, H. S., Kini, K. R., Ven Subbiah and Shetty, H. S. (2005). Endophytic mycoflora of inner bark ofazadirachta indicaa.juss. Current Science: 88(2) [11] Mandeel, Q., Al-Laith, A and Mohsen, L. (1999). Survey of Fusarium Species in an Arid Environment of Bahrain. V. Antimicrobial Activity of Some Local and International Fusarium Species. Pharmaceutical Biology: 37(3) Correspondence to Author: Dr. Sreedharamurthy Satish,Department of Studies in Microbiology Manasagangotri, University of Mysore, Mysore Karnataka. India. Tel.: satish.micro@gmail.com ASIAN J. EXP. BIOL. SCI. VOl 2 (1)