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Antibacterial Activity of Endophytic fungi isolated from Rhizophora mucronata
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
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
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. 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
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. 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. 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. 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. 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. 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.
Notas do Editor
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.