Journal of Food Science and Engineering 1 (2011) 386-389
Endophytic Fungi Isolated from Mangrove (Rhyzopora mucronata) and Its Antibacterial Activity on Staphylococcus aureus and Escherichia coli
A. A. Prihanto1, M. Firdaus2 and R. Nurdiani1
1. Laboratory of Microbiology, Faculty of Fisheries and Marine Science, Brawijaya University, Veteran St, Malang 65145, Indonesia
2. Laboratory of Biochemistry, Faculty of Fisheries and Marine Science, Brawijaya University, Veteran St, Malang 65145, Indonesia Received: August 14, 2011 / Published: October 20, 2011.
Abstract: Disc diffusion method was ud to investigate the antibacterial activity of methanol extract of five endophytic fungi isolated from mangrove (Rhizopora mucronata) on Staphylococcus aureus ATCC 9144 and Escherichia coli ATCC 8739. Fungi were isolated from leaf, stem and root of R. mucronata. Potato dextro agar and potato dextro broth were ud for fungi purification and fermentation. Fungal metabolites were extracted with methanol (1:1 v/v) for 24 h before vacuum evaporated to yield the crude extracts. Antimicrobial activity test was done according to the Kirby-Bauer test. The inhibition
zone around each disc was considered to measure antibacterial activity of endophytic fungi. Only three endophytic fungi showed inhibition activity on S. aureus ATCC 9144 and two endophytic fungi showed inhibition on E. coli ATCC 8739. The results suggested that the extracts have a high potential for enteric dias treatment caud by foodborne pathogen.
Key words: Antibacterial, endophytic fungi, mangrove, Rhyzopora mucronata.
陈慥1. Introduction
The emerging of foodborne dias are a phenomenon in recent years. Safety and food control have been recognized as important issues in many countries for decades. The recent outbreaks of foodborne dia have shaken the consumers’ trust in the food safety systems. Most foodborne dia from fishery product is caud by Staphylococcus aureus and Escherichia coli. The major impact of microbiological foodborne is gastroenteritis which may result in hospitalize and death.
In developing countries, numerous communities have been using local plants in different ways to treat various dias including gastroenteritis. Medicinal plants constitute an effective source of both traditional and modern medicine. Though some plants are assumed
Corresponding author:A. A. Prihanto, rearcher, rearch field:marinemicrobiology.E-mail:**************.id.to produce metabolites with biological activities, it has been reported recently that fungal endophytes residing within the plants could also produce metabolites similar to or with more activity than that of their respective hosts [1]. Therefore, it is believed that arch for novel compounds should be directed towards plants that commonly rve indigenous populations for medicinal purpos and plants growing in unique environmental tting or interesting endemic locations as they are expected to harbor novel microbial that may produce unique metabolites [2].
Mangrove is a host for groups of fungi known as “manglicolous fungi” which play important roles in nutrient cycle of mangrove habitat. Previous study on condary metabolites of endophytic fungi from mangrove revealed that the fungi produced antibiotic, including griofulvin, which commonly found in Penicillium griofulvum
. With this knowledge, we
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isolated endophytic fungi from mangrove Rhizopora mucronata on Porong river estuary to investigate its antimicrobial activity against Staphylococcus aureus and Escherichia coli.
2. Materials and Methods
2.1 Location and Collection of Plant
Plant materials were collected from Porong River banks. Porong River is assumed to have a high content
of heavy metals as it is the estuary of “Sidoarjo Mud Volcano”. Samples were collected during August-October 2010. The mangrove species chon for the prent study is Rhizopora mucronata.
Three parts of plant (leaf, stem, and root) were cut off with ethanol-disinfected cutter. Each part was placed parately in sterile polythene bags to avoid moisture loss. The materials were transported to laboratory within 12 h and stored at 4 °C until isolation procedures were completed.
2.2 Isolation of Endophytic Fungi
The samples were washed thoroughly with sterile distilled water. The materials were then surfaces sterilized using ethanol 75% (1 min), 0.5% sodium hydrochloride (3 min), and ethanol 75% (30 s) and rind thoroughly with sterile distilled water. The samples were grinded using sterile mortar. 1 g of samples was put in 9 mL NaFis and vortexed. Furthermore, 1 mL of aliquot was plated onto potato dextro agar (PDA; 12 g Difco potato dextro broth, 20 g agar/L, with streptomycin 100 mg/L) using spread plate technique. The plates were then incubated at room temperature until fungal growth appeared (1-3 week). Each fungal colony was transferred into PDA for purification of fungal strain. The fungal isolates were identified bad on their morphological and reproductive characters.
2.3 Fungal Cultivation and Extraction of Metabolites
Potato dextro broth was ud for fermentation of fungal endophytes. A fungal strain that covered the surface of the inoculated Petri dish was cut into small pieces of 1.5 cm × 1.5 cm and transferred
with a sterile loop into 500 mL Erlenmeyer flask containing either 200 mL PDB. The flasks were incubated at room temperature for 3 weeks with static condition. After the incubation period, the cultures were taken out and filtered to remove the mycelia. The fungal metabolites were extracted in methanol (1:1 v/v) and macerated for 24 hour. Furthermore, they were evaporated in vacuum evaporator to yield the crude extracts. The crude extracts were then dissolved in Dimethyl sulphoxide (DMSO) for antimicrobial bioassay.
2.4 Antimicrobial Activity Test
Antimicrobial activity test was done according to the Kirby–Bauer test. Susceptibility disks (Oxoid) are impregnated with 500 µg of crude extracts and placed on agar plates that have been inoculated with either the standard gram positive bacteria (Staphylococcus aureus ATCC 9144) or gram negative bacteria (Escherichia coli ATCC 8739). The plates were incubated for 24 h in 37 °C. After 24 hour the clear zone was measured in millimeter. The negative control ud was DMSO while the positive control was streptomycin.
3. Result and Discussion
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3.1 Endophytic Fungi
The endophytes were isolated using mycological medium namely potato dextro agar (PDA). A total of five strains were obtained from the leaves, stems and roots of Rhizophora mucronata. Endophytic fungi can be found in almost all plant part. Each part of plant generally is found at least two colonized with different endophytic fungi species (Table 1). The total numbers of colonies of endophytic fungal leaf is fewer than stem or root. Root showed in great quantities of endopytic fungal colony. The fact that endogenous bacterial population is higher in roots may reflect the fact that the root is the major site where microorganism access to the plants [3]. The colony numbers in part of the plant
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Endophytic Fungi Isolated from Mangrove (Rhyzopora mucronata ) and Its Antibacterial
Activity on Staphylococcus aureus and Escherichia coli
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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 S
1 Fusarium sp S2 Penicillium sp. S3 Aspergillus sp. S4 Acremonium sp Root R1 Penicillium sp. R2 Aspergillus sp. R3 Ampelomyces sp. R4 Fusarium sp R5 Acremonium sp .
em to be correlated with distance to soil. The existence of Penicillium sp. in root indicated that the soil fungal could distribute to new habitat such as plant
becau the origin habitat of Penicillium is the soil. Root is part of plant that showed transition of microorganism as it root possd enormous fungal species. We assume that this result was associated with plant protection from dias caud the extreme
environment. Endophytes with mangrove roots confer
protection from a diver environmental conditions and allows them to successfully compete with saprophytic fungi that decompos nescent roots [4, 5]. The study from K. Ananda et al. [6] revealed that the occurrence
of mainly soil fungi from mangrove roots is 88.5%.
Another result using riparian plant species also showed that endophytic fungi on roots have veral similarities with terrestrial fungi species [7].
3.2 The Antibacterial Activities of Endophytic Fungi Different genera of fungi showed different antimicrobial spectrum. A broad spectrum of antibacterial activity was showed only by Penicillium sp. Penicillium sp inhibit both S.aureus ATCC 9144 and E. coli ATCC 8739 with diameter zone 12.13 ± 1.01 mm and 11.82 ± 1.66 mm, respectively (Fig. 1). The Penicillium sp. metabolite was very effective and showed the highest zone of inhibition against S. aureus ATCC 9144 and less effective with the lowest zone against E. coli ATCC 8739.
Fungal endophytic that hinder gram positive bacteria (S.aureus ATCC 9144) were Aspergillus sp. and Fusarium sp. Aspergillus, Fusarium, Penicillium and Trichoderma exhibit a wide range of biological activities, such as antibiotic, antifungal, cytotoxic, neurotoxic and phytotoxic [8]. Fusarium is known to have various bioactivities [9, 10]. Even our result
showed only Penicillium sp. that has a wide range of antibacterial, we presumed it was becau the bioactive was still in a crude extract form.
Fig. 1 Inhibition zone of S. aureus ATCC 9144 and E . Coli ATCC 8739 by endophytic fungi extract (A = Penicillium sp., B = Aspergilus sp, C = Acremonium sp., D = Fusarium sp., E = Ampelomyces sp.).
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Endophytic Fungi Isolated from Mangrove (Rhyzopora mucronata) and Its Antibacterial
可爱机器人拉肚子吃什么好Activity on Staphylococcus aureus and Escherichia coli
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In this study, Penicillium sp. and Ampelomyces sp. exhibited potential bioactivity against gram negative bacteria (E. coli ATCC 8739). It ems that gram negative bacteria were slight resistant in compare to gram positive bacteria (S. aureus ATCC 9144). Our investigation supports the claims that bioactive fungal constituents produced by tropical endophytic fungi are rich source of novel metabolites exhibiting a wide range of important biological activities [1]. Fungal endophytes have been recognized as repository of novel condary metabolites for potential therapeutic u [11].
Acknowledgment
This rearch was supported by World Class University Grant, Brawijaya University, Directorate General of Higher Education, Ministry of Education, Indonesia.
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