Antimicrobial Activity of Endophytic Fungi from Eleocharis Dulcis

(Burm. f.) Henschell

Elisa Nurnawati, Juliana Hutajulu, Hary Widjajanti and Muharni

Department of Biology, Faculty of Mathematics and Natural Sciences, Sriwijaya University, Palembang, Indonesia

Keywords: Eleocharis Dulcis, Antibacterial Activity, Penicillium Citrinum, Alkaloid, Terpenoid, Phenol.

Abstract: Extracts of endophytic fungi isolated from Eleocharis dulcis were tested for antibacterial ability. Two

isolates showed the strongest activity toward Staphylococcus aureus, Escherichia coli and Salmonella typhi

than another isolates. The results of diffussion methods for antibacterial test of extract of DP1J1 isolate

against Staphylococcus aureus, Escherichia coli and Salmonella typhi were 86.31%, 77.4%, and 83.15%

respectively. MIC extract of DP1J1 isolate against Staphylococcus aureus, Escherichia coli and Salmonella

typhi were 0,03%, 0,02% and 005%, respectively. Based on bioautography test, extract of DP1J1 isolate was

contained alkaloid, phenol and terpenoid. DP1J1 identified as Penicillium citrinum.

1 INTRODUCTION

The search for antibacterial secondary metabolites

from natural materials has been a lot of done.

Relevant to the higher level of resistance of

pathogenic bacteria to commercial antibacterial

compounds. Bioactive plant compounds are

potential source of medicinal ingredients. The use of

plant as raw material for the production of

antibacterial compounds generated new problems.

Utilization of large amounts of plant biomass caused

reduced plant biological resource (Allurappa, et al,

2018).

Water chesnut (Eleocharis dulcis (Burm.f.)

Henschell) are often found in South Sumatra

swamps, especially in Ogan Ilir district. This plant

adapted to acid swamp land conditions. The content

of secondary metabolites is very interesting to know.

The juice of the water chesnut tuber contains

antibiotic compounds which are effective in

inhibiting Staphylococcus aureus, Escherichia coli,

and Aerobacter aerogenes (Asikin dan Thamrin,

2012). Ethanol, ethyl acetate and n-hexane extracts

of water chestnut leaves have been known to contain

triterpenoid compounds, tannins and flavonoids.

These compounds are antibacterial activity (Baehaki

et al., 2018).

Endophytic fungi can be found in plants.

Endophytic fungi was penetrate and live in the plant

tissues which innoxious to the host. The fungi are

able to produce the same bioactive compounds as

plants due to genetic recombination between plants

and fungi followed by coevolution. The ability of

microbes to produce the same bioactive compounds

as plants can be used as a substitute for taking up

very large plant biomass. Endophytic fungi can

produce functional compounds in the form of

anticancer, antiviral, antibacterial, antifungal and

growth hormone for plants (Tan and Zou, 2001;

Noverita et al., 2009)

Plants which adapted to live in unique

environments such as in swampy areas potentially

produce bioactive compounds that are used to

survive. Based on this, endophytic fungi from water

chestnut have not been studied yet, so it is necessary

to explore endophytic fungi from water chestnut that

potentially produce antibacterial compounds against

Staphylococcus aureus, Escherichia coli and

Salmonella typhi.

Nurnawati, E., Hutajulu, J., Widjajanti, H. and Muharni, .

Antimicrobial Activity of Endophytic Fungi from Eleocharis Dulcis (Burm.f.) Henschell).

DOI: 10.5220/0010138000002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 155-161

ISBN: 978-989-758-556-2

155

2 MATERIAL AND METHODS

2.1 Sampling

Water chestnut was obtained from the swamps along

the road between Palembang and Indralaya by

random sampling method. Sampling was carried out

in two locations and the coordinate were S 03 °

08'04.1 " E 104 ° 41 '57.3 "and S 03 ° 08 '39.2" E

104 ° 41 '36.5 ". The samples used in this study

were old roots, stems and leaves with good quality

as a source of fungi isolates

2.2 Isolation of Endophytic Fungi

The roots, stems and leaves are washed in running

water to remove dirt. The part of plants were surface

sterilized gradually by means of samples soaked in

70% alcohol and 0.1% NaOCl alternately then

rinsed with sterile distilled water and dried on sterile

tissue. The part of plants were split and placed on a

PDA medium with the position of the surface of the

sample attached to the agar and then incubated at

room temperature (27°C). Pure fungal isolates were

made into work culture and stock culture on slant

tube medium.

2.3 Cultivation of Endophytic Fungi

Suspended endophytic fungal propagules of ± 10

propagules / mL were inoculated in PDB medium

placed in a 1 liter cultivation bottle with two

repetitions. Culture was incubated at room

temperature until the medium undergoes a color

change which indicates that secondary metabolite

compounds have formed. The medium without the

addition of endophytic fungi is used as a control.

After the secondary metabolites are formed, the

fungi biomass is separated from the medium. The

liquid-liquid fractionation medium (partition) with

ethyl acetate solvent and concentrated with a rotary

evaporator.

2.4 Antibacterial Assay (Kirby Bauer

Methods)

The test bacteria that have been equivalent to Mc

Farland 0.5 were spread into the MHA medium.

Disc paper that containing 2% antibacterial extract

and 50 µl of them is placed on the surface of the

MHA media. Tetracyclines with a 0.1%

concentration of 50 µl were used as positive

controls. The culture was incubated at 37 ° C for

1x24 hours (Rosyidah et al., 2010). Determination of

the antibacterial activity of secondary metabolite

extracts was carried out by measuring the diameter

of inhibitory zones formed in secondary metabolite

extracts of endophytic fungi compared diameter of

inhibitory zones on standard antibiotics, tetracycline

(Noverita et al., 2009; Balouiri et al, 2016). The

activity of secondary metabolite extracts tested

against standard antibiotics is determined by the

following equation.

Percentage of ntibacterial activity =

஺

஻

x 100%

A= inhibition zone (mm) of extract

B= inhibition zone (mm) tetracycline (Chan,

2007).

2.5 Minimum Inhibition Concentration

(MIC)

Extracts of secondary metabolites of endophytic

fungi were made in different concentrations of 2%,

1%, 0.5%, 0.25%, 0.125%, 0.06% and control. The

extract was dripped on disc paper and placed on a

bacterial inoculated MHA medium and then

incubated at 37 ° C for 24 hours. The diameter of

inhibition zone formed is observed and measured. If

the lowest concentration was still capable to inhibit

bacterial growth so the extract concentration is

reduced and tested again to the lowest concentration

that was unable to inhibit bacterial growth

(Ruangpan, 2004; Fatisa, 2013).

2.6 Bioautografi Assay

The secondary metabolite extract of endophytic

fungi which showed the highest antibacterial ability

was dissolved with ethyl acetate solvent. Extracts of

secondary metabolites were dripped on TLC plates

and developed with n-hexane: ethyl acetate (8: 2).

TLC plates were sprayed with 10% H2SO4. The

color formed indicates the group of secondary

metabolites.

TLC plates that have been developed are placed

on top of a dense bacterial culture. The spots/stains

on the chromatogram are attached to the medium so

that the compound can diffuse into the agar medium.

The culture was incubated for 24 hours and then a

clear zone was observed which was due to inhibition

of the active compound (Salni et al., 2011; Balouiri

et al, 2016).

2.7 Characterization and Identification

of Endophytic Fungi

Endophytic fungi of water chestnut with highest

antibacterial ability are characterized based on

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macroscopic and microscopic morphology.

Macroscopic morphological characters observed

included colony growth, colony diameter, color of

colony and media and reverse colony. The fungi is

grown in different media (PDA, MEA and Czapex

Dox Agar). Preparation of fungi was observed under

a microscope. Morphological characters of

microscopic fungi were hyphae, hyphae colors,

conidial shapes, conidia colors, and conidial sizes.

Both macroscopic and microscopic characters were

compared with the books (Barnett & Hunter, 2006;

Houbraken et al., 2010).

3 RESULTS AND DISCUSSION

3.1 Isolation of Endophytic Fungi

A total of 9 endophytic fungal isolates were obtained

from the parts of water chestnut (Eleocharis dulcis

(Burm.f) Trinius ex. Henschell). The isolates had 2

isolates from the root, 3 isolates from the stem, and

4 isolates from the leaf. The highest number of

endophytic fungi isolates was found in the leaf tissue

of plants. Endophytic fungi that were isolated from

each of the rat purun tissue have different numbers

and types according to the adaptation of endophytic

fungi to their host plants. According to Noverita et

al., 2009) more than one type of endophytic fungi

can grow from one plant tissue due to the

physiological factors of the host plant, so that

various endophytic fungi are produced.

Table 1: Isolation and Purification of Endophytic Fungi

from Water Chestnut (Eleocharis dulcis (Burm.f) Trinius

ex. Henschell).

Sample Code Total

Root AP

, AP

Stem BP

, BP

Leaf DP

, DP

Total 9

3.2 Antibacterial Activity

Secondary metabolites extract from endophytic

fungi were tested for their antibacterial activity

towards S. aureus, E. coli dan S. typhi. Extracts of

secondary metabolites of endophytic fungi were

tested at 2%. The result of this study was inhibitory

zones formed around the paper disk. The diameter of

inhibition zone formed in testing antibacterial

activity can be seen in Figure 1.

Based on table 2, it is known that all extracts of

endophytic fungal isolates have different

antibacterial activity. This is evidenced by the

formation of inhibition zones around the paper disk.

Based on the criteria for antibacterial activity (Chan

et al., 2007) showed that the result of the percentage

of antibacterial activity of the secondary metabolite

extract of DP1J1 fungi isolates towards three test

bacteria was strongest than the other fungal isolates.

Based on Table 2, the result showed that the

antibacterial activity of DP1J1 isolates against

Escherichia coli, Staphylococcus aureus, and

Salmonella typhi were 77.4%, 86.31%, and 83.15% ,

respectively.

A large percentage of antibacterial activity of the

secondary metabolite extract was obtained from the

area of inhibition zone formed by each isolate. The

diameter of the inhibited zone measured was the

amount of antibacterial compounds that inhibit

bacteria (Balouiri et al., 2016). The higher

antibacterial activity of secondary metabolite extract

of endophytic fungi, the greater of inhibition zone.

Figure 1: Antibacterial Activity Test for Secondary

Metabolite Extracts (a) Escherichia coli (b)

Staphylococcus aureus and (c) Salmonella typhi.

Antimicrobial Activity of Endophytic Fungi from Eleocharis Dulcis (Burm.f.) Henschell)

157

Table 2: Antibacterial Activity of Metabolite Secondary Extract of Endophytic Fungi.

Isolate Origin

sample

Concentration

(%)

Antibacterial activity (%) Criteria

E. coli S. aureus S. typhi

Root

2 6,63 4,06 6,31 weak

2 8,84 5,07 12,63 weak

Stem

2 17,69 22,33 23,15 weak

2 11,06 13,20 13,68 weak

2 6,63 19,29 8,42 weak

Leaf

2 77,40 86,31 83,15 strong

2 13,27 10,15 18,94 weak

2 15,48 11,16 10,52 weak

2 11,06 18,27 13,68 weak

Tetracycline 0,1

Criteria of anticaterial activity : <50% (weak), 50-74% (medium), ≥75% (strong) (Chan et al., 2007).

3.3 Minimum Inhibitory Concentration

(MIC)

The aimed of determination of Minimum Inhibitory

Concentration (MIC) was to know the lowest

secondary metabolite extract concentration value

capable to inhibit the growth of test bacteria

(Andrews, 2001). The extract concentration were

2%, 1%, 0.5%, 0.25%, 0.125%, and 0.06%. The

MIC value of secondary metabolite extract of DP1J1

isolates was determined because of strongest

antibacterial activity against the three test bacteria.

Table 3: Minimum Inhibitory Concentration (MIC) of

secondary metabolite extract towards E. coli, S aureus and

S. typhi.

Based on Table 3, the results showed that

inhibition of E. coli growth to ensue at 0.06% - 2%

of secondary metabolite extract DP1J1 isolates. In

0.06% of secondary metabolite extracts were still

capable to inhibit test bacteria, so the concentration

was reduced from 0.06% to 0.01%. Andrews (2001)

certify that antibacterial compounds at the smallest

levels with clear inhibition zone without the growth

of test bacteria are determined as MIC. The MIC

value obtained from the secondary metabolite

extract of DP1J1 isolates against E. coli was 0.03%.

The secondary metabolite extract of DP1J1 isolates

was still able to inhibit the S. aureus at a

concentration of 0.06%, so the concentration was

reduced from 0.06% to 0.01%. The MIC value of

DP1J1 secondary metabolite extract after

concentration reduction was obtained by 0.02%. The

secondary metabolite extract of DP1J1 isolates at a

concentration decrease of 0.06% was still able to

inhibit the S. typhi. Therefore the concentration of

DP1J1 isolates was reduced in concentration of

DP1J1 isolates from 0.06% to 0.01%. The results of

the MIC value of the secondary metabolite extract of

DP1J1 isolates were 0.05%.

The secondary metabolite extract of DP1J1

isolates had different MIC values for different types

of bacteria. The MIC value of metabolite extracts

secondary to the Staphylococcus aureus was lowest

than other bacteria because of differences in

sensitivity of gram-positive bacteria to gram-

negative bacteria against antibacterial. According to

Ullah & Ali (2017) differences in sensitivity of

gram-positive and gram-negative bacteria due to

differences in the structure of the cell wall where

gram-negative bacteria have a relatively more

complex cell wall structure, while gram-positive

bacteria that have a simpler cell wall structure so

that the test compounds which is active as an

antibacterial more easily enter the cell..

S. aureus is a Gram-positive bacterium with

thick peptidoglycan on cell wall. Based on the

results of the study, the MIC value of S. aureus was

lowest, so it can be assumed that the secondary

Isolate (%) Diameter of inhibition zone (mm)

E. coli S. aureus S. typhi

2 10 15 18

1 8,5 10 15

0,5 6,5 10 10

0,25 5 9 7

0,125 5 7 6

0,06 4 5 4

Control 0 0 0

0,06 3,5 2 3

0,05

3 2

0,04 2,5 1,5 0

0,03 2

1 0

0,02

0,01 0 0 0

Control 0 0 0

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metabolite content of DP1J1 fungi isolates was

easier to inhibit Gram-positive bacteria. The greatest

MIC value of secondary metabolite extract was S.

typhi compared to the other bacteria. It has been

known that S. typhi was pathogenic, so on the cells

contain a certain protein that interacts with

antibiotics (Lee et al., 2019)

3.4 Bioautography Test of Secondary

Metabolite Extracts

The result of bioautographic test of secondary

metabolite extracts using thin layer chromatography

(TLC) method showed that the secondary metabolite

extracts of endophytic fungi isolate DP1J1 contained

antibacterial compounds. Wardhani and Sulistyani

(2012) have determined the class of active

compounds which are potentially antibacterial using

thin layer chromatography (TLC). Based on the

research results obtained by the price of Rf

(retondation factor) antibacterial active compounds

in extracts of secondary metabolites of endophytic

fungi are presented in Table 4.

Determination of the class of active compound of

secondary metabolite extract was carried out on a

chromatogram sprayed with 10% H

. If the

cromatogram appeared purple stains which were

thought to be terpenoid group antibacterial

compounds. The active compound on the

chromatogram is attached to the agar medium, after

incubating it shows the results of the formation of a

clear zone around the chromatogram stain as in

Figure 2.

Figure 2: Bioautography test of secondary metabolite

extract. (a) Under visible light, (b) Under UV light, (c)

Bioautography test on agar medium

Table 4: Bioautographic Test Results and Rf Values of

Endophytic Fungal Secondary Metabolite Extracts.

Isolate Total

com

ounds

Rf color class of

com

ounds

0,14

rown Alkaloi

0,52 yellow Fenol

0,62 violet Ter

enoid

Based on the results of the study, it was found

that DP1J1 isolate had 3 active compounds according

to the number of stains formed on the chromatogram.

The clear zone formed in the bioautographic test was

found in the second stain which was suspected to be

phenol group with an Rf value of 0.52.

3.5 Characterization and Identification

of Endophytic Fungi

DP1J1 isolate was identified based on macroscopic

and microscopic morphological characters. DP1J1

isolate was grown on three fungi growth media,

namely Czapek Agar, MEA, and PDA. The colony

color on the PDA was different from the other

media. Colony color on PDA showed white colony

and slow growth with a colony diameter of ± 1 cm.

On the other hand, colony color on MEA showed

deep green color with a white edge, the opposite

color of the brownish yellow colony and a colony

diameter of ± 1.8 cm. On CDA medium, the color of

the colony formed was bluish green with a white

edge and a diameter of ± 1.5 cm (Figure 3).

Figure 3: Macroscopic morphology of DP1J1 isolate. (a)

PDA (top and reverse), (b) MEA (top and reverse), (c)

Czapek (top and reverse).

Antimicrobial Activity of Endophytic Fungi from Eleocharis Dulcis (Burm.f.) Henschell)

159

Microscopic morphology characters were

branched conidiophore, biverticillate, round conidia,

light brown conidia, and phialid flask-shaped

(Figure 4). Based on the microscopic character,

suspected DP1J1 isolate as Penicillium citrinum

(Barnett & Hunter, 2006; Houbraken et al., 2010).

Figure 4: Microscopic morphology of DP1J1 isolate.

The content of compound in the secondary

metabolite Penicilium citrinum were alkaloid,

phenol, and terpenoid compounds. Penicilium

citrinum have the ability to produce antibacterial

compounds which consist of three active

compounds. This study was compatible with the

research of Zhan et al (2014) which declare that the

result of chromatography test the Eleocharis dulcis

ethyl acetate fraction has been obtained bioactive

compounds in the form of flavonoids. Thus,

endophytic fungi isolates DP1J1, identified as

Penicilium citrinum, which were isolated from water

chestnut (Eleocharis dulcis) had the potential to

produce antibacterial compounds in all three test

bacteria. That is important to know is the group of

compounds that are active as antibacterial. Therefore

further examination of each class of these

compounds should be done.

4 CONCLUSION

Based on the results of the research, from 9

endophytic fungal isolates of water chestnut

(Eleocharis dulcis (Burm.f) Trinius ex. Henschell),

it was found that DP1J1 isolates showed strong

antibacterial activity against E. coli ATCC8739, S.

aureus ATCC6538, and S. typhi IPBCC B.11.669.

MIC value of DP

isolate were 30 µg/ml, 20 µg/ml

and 50 µg/ml towards E. coli ATCC8739, S. aureus

ATCC6538 and S. typhi IPBCC B.11.669,

respectively. DP

isolate has been identified as

Penicillium citrinum.

ACKNOWLEDGEMENTS

The authors thanks to Dr. Salni, MSi for the

discussion during the research and Universitas

Sriwijaya for granted Hibah Penelitian Sateks

funded via Anggaran DIPA Universitas Sriwijaya

No : SP DIPA-042.01.2.400953/2019, 5 Desember

2018.

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