Antibacterial Activity of Endophytic fungi isolated from Rhizophora mucronata

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  • Food borne diseases do not only affect people's health and well-being, but they also have economic impacts on individuals and countries. In many developing countries, food borne disease outbreaks from bacteria, such as Escherichia coli and Salmonella spp., impose a substantial burden on health care systems and can markedly reduce the economic productivity of the countries.
  • Endophytes may be defined as microbes that colonize living internal tissues of plants without causing any immediate, overt negative effects. This particular definition implies a symbiotic or mutualistic relationship between the host plant and the endophytic microbe.Many are capable of synthesizing bio-active compounds that can be used by the plant for defense against pathogenic fungi and bacteria. It wasn't until the past decade or so, however, that endophytes have been studied for their potential as novel sources of effective new drugs. Microbes, both fungal and bacterial, have provided modern medicine with valuable new cures, including penicillin from the fungus Penicilliumnotatum, and bacitracin from the bacterium Bacillus subtilis. Additionally, taxol, an important chemotherapeutic agent, has been discovered to be synthesized by an endophyte of the Pacific Yew tree. Recurrent co-evolution of the endophyte with the host resulted to horizontal transfer of genes coding for metabolic pathways from the host to the endophyte, which enable the latter to biosynthesize compounds naturally produced by the host (Sachin, N. et al., 2013). Endophytes prevent pathogenic attack by producing secondary metabolites and by establishing endophytic niches through exhaustion of limited substrates and lignification of cell walls (Gao, S. et al., 2010). Other mechanisms for biological control of plant pathogens include production of antibiotics, lytic enzymes, unregulated waste products, physical/chemical interference and induction of host resistance (Pal, K. K. and Gardener, B. M., 2006).
  •  It is not known if all endophytes synthesize chemicals, but the argument has been strongly made that those plants in unique environments that struggle to compete with other living organisms, or that need as much assistance as possible to survive, are likely to host endophytes which can generate secondary metabolites that will, in turn, assist the plant. Manglocolous fungi play an important role in the decomposition of organic matter in the mangrove ecosystemGriseofulvin is used to treat skin infections such as jock itch, athlete's foot, and ringworm; and fungal infections of the scalp, fingernails, and toenails.
  • In a project by Vogel (2012) water samples of the river and the Sidoarjo Mud Flow have been taken and analyzed in order to see if the discharge of mud and water from Sidoarjo Mud Flow posses any potential risks to human health. The HPLC and ICP-OES analysis showed concentrations of several compounds like lead, strontium and phenol. The concentrations were high enough to posses a risk to human health in the Porong River.
  • Samples were washed with distilled water and surface sterilized using 75% ethanol (1 min), 5% sodium hypochlorite (3 min), 75% ethanol (30 s), and distilled water respectively. One gram of each part were was grinded using sterile mortar and placed into 9 ml testube and vortexed. One ml of the aliquot was plated onto Potato Dextrose Agar with 100 mg/L streptomycin using spread plate technique. Plates were then incubated at room temperature until fungal growth appeared. Distinct colonies were transferred into PDA plates to check purity. Pure cultures were identified.
  • Pure cultures of fungi were cut into small pieces (1.5 x 1.5 cm)and transferred into 500 ml Erlenmeyer flask containing 200 ml PDB using a sterile loop. The flasks were incubated at 25°C. After 3 weeks cultures were filtered to remove mycelia. Metabolites were extracted in methanol, macerated and evaporated in vacuum evaporator. The crude extracts were then dissolved in dimethyl sulphoxide for antimicrobial assay.
  • Antimicrobial activity of the extracts was tested using Kirby-Bauer test. Susceptibility disks (oxoid) were impregnated with 500 µg crude extracts and placed on agar plates inoculated with S. aureusand E. coli. The negative control used was DMSO while positive control was streptomycin. Plates were incubated for 24 hrs at 37°C and inhibition zones were measured.
  • Antibacterial Activity of Endophytic fungi isolated from Rhizophora mucronata

    1. 1. Endophytic Fungi Isolated from Mangrove Rhizophora mucronata and its Antibacterial Activity on Staphylococcus aureus and Escherichia coli A. A. Prihanto , M. Firdaus, and R. Nurdiani
    2. 2. Objectives 1. Isolate endophytic fungi from R. mucronata 2. Test the isolated endophytic fungi for antibacterial activity
    3. 3. Staphylococcus aureus Escherichia coli Introduction o Gram positive negative o Staphylococcus Grape-like clusters Rod-shaped o aureus andfound in the o commonly found in commonly Escherichia coli are of human respiratory the lower intestine prevalent on fish skin tract and in the warm-blooded products skin causes that o organisms cause of food borne diseases o infections (e.g. boils), can cause to consumers respiratory disease serious food (e.g. sinusitis), poisoning in humans and food poisoning
    4. 4. Endophytic Fungi
    5. 5. Rhizophora mucronata LN: Bakauan-babae EN: Stilt Mangrove o Stipules: yellowish, greenish o Bark: rough, brown to dark grey o Flowers and Fruit: white to cream in color with short style o Leaves: broader leaves compared to R. apiculata and R. stylosa. Tiny black spots on the underside and pointed tip o Roots: stilt roots emerging in arches from the lower trunk, and prop roots may grow downwards from branches o Propagule: long and dull olive green color
    6. 6. Why choose mangroves in prospecting endophytes? Manglicolous fungi (decomposition of organic matter in the mangrove ecosystem) Griseofulvin (Anti-fungal drug) Medicinal plant o Angina Hemorrhage Diarrhea Hematuria Diabetes Stressful environment o Pathogenic attacks High heavy metal content High salinity Drought Inadequate nutrients
    7. 7. Methodology I. Collection o Plant materials were collected from Porong River banks. o estuary of “Sidoarjo Mud Volcano”
    8. 8. II. Isolation and Cultivation 1. Surface sterilization 75% ethanol (1 min), 5% sodium hypochlorite (3 min), 75% ethanol (30 s) 2. Grinding and Mixing 3. Spread plate technique 4. Incubation, checking for purity & identification
    9. 9. III. Extraction 1. Transfer of pure culture agar blocks (1.5 x 1.5 cm) for incubation 2. Filtration 3. Extraction of metabolites in methanol & Evaporation 4. Dissolution of crude extracts for antibacterial assay
    10. 10. IV. Test for Antimicrobial Activity 1. Kirby-Bauer Test a. 500 µg crude extracts b. Negative control used was DMSO c. Positive control was streptomycin 2. Incubation 3. Measurement of inhibition zones Kirby-Bauer Test
    11. 11. Results & Discussion Table 1. Endophytic fungi isolated from Rhizophora mucronata Part of plant Strain no. Species Leaf L1 Aspergillus sp. L2 Acremonium sp. L3 Fusarium sp. S1 Fusarium sp. S2 Penicillium sp. S3 Aspergillus sp. S4 Acremonium sp. R1 Penicillium sp. R2 Ampelomyces sp. R3 Fusarium sp. R4 Acremonium sp. R5 Aspergillus sp. Stem Root o A total of five strains of endophytic fungi were isolated from all plant parts showing that endophytes are present in almost all of the tissues of its host. o number of fungal colonies is highest in the root tissues which is consistent to the fact that the root is the major site where microorganisms gain access into the plant. o More endophytes in the roots is associated with plant protection from extreme stress since mangroves thrive in saline environment and the site of sampling has high heavy metal content.
    12. 12. Results & Discussion Staphylococcus aureus 14 Escherichia coli Inhibition zone (mm) 12 10 8 6 4 2 0 Penicillium sp. Aspergillus sp. Acremonium sp. Fusarium sp. Ampelomyces sp. Extracts of endophytic fungus Figure 1. Inhibition zones of S. aureus ATCC 9144 and E. coli ATCC 8739 by endophytic fungi extracts of Penicillium sp., Aspergillus sp., Acremonium sp., Fusarium sp., and Ampelomyces sp. o Results showed that Penicillium sp. produces the most effective metabolite since it exhibited a broad spectrum of antimicrobial activity against both Gram positive and negative bacteria.
    13. 13. Conclusion o Penicillium sp. isolated from R. mucronata can be used as a potent source of antibacterial drugs in fighting against food borne disease-causing bacteria such as E. coli and S. aureus. o Other plants that reside in unique biotopes may harbor a rich diversity of endophytic fungi that can also be a good source of novel secondary metabolites.

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