A. A. Prihanto , M. Firdaus, and R. Nurdiani

Endophytic Fungi Isolated from Mangrove Rhizophora mucronata and its Antibacterial Activity on Staphylococcus aureus and Escherichia coli

Objectives1. Isolate endophytic fungi from R.

mucronata

2. Test the isolated endophytic fungi for antibacterial activity

IntroductionoStaphylococcus

aureus and Escherichia coli are prevalent in fish products that causes food borne diseases to consumers

Escherichia coli

o Gram negative

o Rod-shaped

o commonly found in the lower intestine of warm-blooded organisms

o can cause serious food poisoning in humans

Staphylococcus aureus

o Gram positive

o Grape-like clusters

o commonly found in the human respiratory tract and on the skin

o cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis), and food poisoning

Endophytic Fungi

Rhizophora mucronataLN: Bakauan-babaeEN: Stilt Mangrove

o Stipules: yellowish, greenisho Bark: rough, brown to dark

greyo Flowers and Fruit: white to

cream in color with short styleo 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

o Pathogenic attacksHigh heavy metal contentHigh salinityDroughtInadequate nutrients

o AnginaHemorrhageDiarrheaHematuriaDiabetes

Medicinal plant

Stressful environment

Manglicolous fungi

(decomposition of organic matter in

the mangrove ecosystem)

Griseofulvin(Anti-fungal

drug)

Why choose mangroves in prospecting endophytes?

Methodology

I. Collection

o Plant materials were collected from Porong River banks.

o estuary of “Sidoarjo Mud Volcano”

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

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

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

Table 1. Endophytic fungi isolated from Rhizophora mucronata Part of plant Strain no. Species

Leaf L1 Aspergillus sp.

  L2 Acremonium sp.

  L3 Fusarium sp.

Stem S1 Fusarium sp.

  S2 Penicillium sp.

  S3 Aspergillus sp.

  S4 Acremonium sp.

Root R1 Penicillium sp.

  R2 Ampelomyces sp.

  R3 Fusarium sp.

  R4 Acremonium sp.

  R5 Aspergillus sp.

Results & Discussion 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.

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.

Penicillium sp. Aspergillus sp. Acremonium sp. Fusarium sp. Ampelomyces sp.0

2

4

6

8

10

12

14 Staphylococcus aureus Escherichia coli

Extracts of endophytic fungus

Inh

ibit

ion

zon

e (m

m)

Results & Discussiono 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.

Conclusiono 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.