Isolation and Identification of Acanthus ilicifolius-Associated Fungi

and Investigation of Their Antibacterial Activity

Farras Daffa Imtiyaz

, Ferisa Lestari Nugrahayu

, Kawidian Putri Bayu Alam

, Insan Shiddiq

and

Hermin Pancasakti Kusumaningrum

Department of Biology, Faculty of Science and Mathematics, Diponegoro University, Semarang, Indonesia

Faculty of Public Health, Diponegoro University, Semarang, Indonesia

Keywords: Acanthus ilicifolius-Associated Fungi, Antibacterial, Bioactive compounds, Biopharmaceutical

Abstract: Antibacterial resistance is one of the biggest threats to the global health security today. Therefore, there is

an urgency to find alternative treatment to combat the bacteria. This paper aimed to identify and assess the

biological activity of Acanthus ilicifolius endophytic fungi, a medicinal plant originated from Asia used in

ancient medicine. In this experiment, two strains of bacterial were used, namely Staphylococcus aureus and

Pseudomonas aeruginosa. The tree samples were obtained from Jepara, Indonesia. Surface sterilization and

purification were applied to isolate the fungal. Moreover, their antibacterial ability was tested using agar

plug diffusion method. The study found that three isolates were broadly active against two types of

pathogenic bacteria; one of them was AB1-isolate. In the identification of the colony, AB1-fungal have both

black stain in the basal part and the outside layer, with rough structure and planted strongly to the basal

medium. The other fungal isolates, namely AD1 and AB2, showed a bacteriostatic effect against bacterial

test as well. Our results suggested that AB1-isolate, obtained from the stems, shows a good prospect to be a

pharmaceutical candidate to inhibit bacterial growth that normally present in the infection cases.

1 INTRODUCTION

Endophyte associates each other with the host plant

to protect them from pathogens (Vasundhara et al.,

2016); and the majorities are fungi (Strobel and

Daisy, 2003). Endophytic fungi belong to mitosporic

and meiosporic ascomycetes that reside beneath the

epidermal cell layer without causing damages. These

microorganisms are important sources of natural

products with pharmaceutical potential (Porras-

Alvaro and Bayman, 2011). They contain more than

40% of new chemical compounds from

microorganisms (Sieber and Marahiel, 2005) and

have wide-ranging applications (Zhao et al., 2011).

Acanthus ilicifolius is a type of mangrove plant

in Central Java, Indonesia, used as a traditional

medicine due to their abilities. Moreover, the plant

has anti-inflammatory, antioxidant and antimicrobial

activities. Those abilities are affected by

microorganisms beneath the plant tissues (Faeth and

Fagan, 2002). Since the plants are directly exploited,

the wild A. ilicifolius will quickly decrease,

therefore, the preferred technique to take the plant’s

pharmacological abilities is by exploring its fungal

endophyte beneath the tissues. Fungal can be grown

in the laboratory, enable us to utilize them without

exploiting the plant directly. In this paper, we were

exploring all the endophytic fungi ability to combat

two bacteria.

2 METHODS

2.1 Sample Area and Process of

Collecting

Acanthus ilicifolius samples were collected from the

growing areas named Mecok. The area is exposed to

ocean spray, mists and tides along Teluk Awur

coast, Jepara, Indonesia (S 6°37′7.86′′, W

110°38′41.16′′). Plant species found in the ground

were preferentially selected. Multiple numbers of

samples (n = 20) from Acanthus ilicifolius, locally

recognized as jeruju, were washed using tap running

water to remove the dust and dirt. Samples were

484

Imtiyaz, F., Nugrahayu, F., Alam, K., Shiddiq, I. and Sari, H.

Isolation and Identiﬁcation of Acanthus ilicifolius-Associated Fungi and Investigation of Their Antibacterial Activity.

DOI: 10.5220/0007545504840488

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 484-488

ISBN: 978-989-758-348-3

stored in the airless polythene bags within a cooler

bag until processing.

Figure 1: Map of the sampling area

2.2 Isolation and Identification

Endophytic fungi were isolated as described

previously (Ezra et al., 2004) with minor

modification. After sampling, samples were rinsed

by tap water to remove soil or dust in a beaker glass

containing distilled water. Stems, roots and leaves

surface were sterilized and impregnated by 90%

ethanol for 1 minute, 5% sodium hypochlorite for 1

minute, sterile double distilled water (DDW) for 1

minute, 70% ethanol for 1 minute, and a quick rinse

in sterile DDW. The Potato Dextrose Agar (PDA)

media (Becton, Dickinson & Co; Sparks, MD) was

diluted by seawater in the Erlenmeyer glass. The

media was autoclaved at 121

C for 15 minutes, and

then mixed with 50 mg of Chloramphenicol in order

to suppress the epiphyte bacteria.

The outer tissues of the plant samples were

removed after drying the tissues under sterile

laminar airflow and passing through the flame. The

internal tissues were cut into pieces of 0.5 inch and

plated on petri dishes containing PDA media that

had been made. Each petri dish was divided into

four segments, with each segment containing plant

samples from the same organ. The plates were

sealed and incubated at room temperature and

examined for emerging fungi every 2 - 3 days. As

fungi emerged, they were transferred to the new

PDA plates.

The fungi were identified based on

characteristics according to the methods described

by Kong and Qi (1985). Colony descriptions were

based on observations on PDA under ambient

daylight conditions after 4–7 days of incubation.

Identification was performed according to the

taxonomic key, including colony diameter, texture

and colour.

2.3 Agar Plug Diffusion Method

Strains of bacteria species used to test antibacterial

activity of isolated fungi were Staphylococcus

aureus and Pseudomonas aeruginosa. These

bacterial strains were taken from Kariyadi Hospital,

Indonesia. The bacterial inoculum was prepared by

mixing a few bacterial colonies (1 mL) with 9 mL of

sterile 1.5 % NaCl and compared the turbidity with

the standard 0.5 McFarland solution. The sterile

swab was dipped properly into the mix solutions to

adjust the inoculum. In order to decrease the liquid,

the swab was pressed with gentle rotation to the

inner surface of the test tube. To obtain a perfect

growth, the entire Muller Hinton Agar (MHA)

media (Merck) surface was swabbed uniformly. The

inoculated plates were stored at room temperature

for 3-5 minutes.

The antibacterial test used agar plug diffusion

method proposed by Elleuch et al. (2010). In this

method, the fungal inoculum was prepared as that of

disc diffusion method. The fungal mycelia were

cultured in PDA for 7 days at temperature of 30

After the fungal growth, the wells were prepared on

the plate by using sterile corks borer. The agar plugs

were transferred carefully to MHA media which

were already cultivated by bacterial test and allowed

to diffuse for 2 hours. The cultures were incubated at

C for 24-48 hours, and the plate was examined

for the presence of inhibition zones next to the

fungal plugs. After 24 hours of incubation, the

inhibition zone around each well was recorded.

3 RESULTS

Twelve segments from three parts of the plant were

used to obtain the endophyte. A total of eight fungi

isolates were obtained in the first screening, but only

four isolates were successfully re-cultivated. The

sampling type and the total number of isolates are

summarized in Table 1. Those fungi belong to four

genera of filamentous fungi. They are Fusarium sp.

(Putih), Ulocladium sp. (AB1), Cylindrocarpon sp.

(AD1), and Acremonium sp. (AB2).

Table 1: Quantity of fungi obtained from different part.

Sampling Types

Segments

Isolates

Leaves

Roots

Stems

Isolation and Identiﬁcation of Acanthus ilicifolius-Associated Fungi and Investigation of Their Antibacterial Activity

485

The isolated strains were cultivated in PDA for

eight days in the ambient of light. We performed

observation on aerial mycelium and substrate

mycelium, as mycelium serves as the key of fungi

identification, including the colours and the form of

it. Morphological observation revealed that both

aerial and vegetative hyphae were abundant, well

developed, and had a varied characteristics of

mycelium. The colour of aerial and substrate

mycelium of most strains varied from white to black.

Several isolated fungi inhibited at least one

bacterial test with agar plug diffusion method and

created inhibition zones (Table 2). Among the

fungal endophyte, “putih” strain did not exhibit any

inhibited zone to the bacteria in the MHA medium.

The greatest antibacterial activity was shown by

AB1 isolate.

Figure 2: Antibacterial test to (a) Staphylococcus aureus

and (b) Pseudomonas aeruginosa.

Table 2: Antibacterial activity of endophytic fungi isolates

Isolates

P. aeruginosa

S. aureus

AB2

AB1

AD1

Putih

Chloramphinacol

Notes: + = Positive and - = Negative

4 DISCUSSION

Mangrove is one of the vital ecosystems in the

world. In addition to becoming a home to various

organisms, it plays an important role as the source of

medicinal plants. The bioactive compound of

Acanthus species has been studied expansively.

However, there is still limited information regarding

endophytic fungi derived from the plant. Two

previous studies limitedly explained about the ability

of Aspergillus flavipes from Acanthus (Bai et al.,

2014) and the endophyte diversity (Ananda and

Sridhar, 2002).

In this article, four endophytic fungi derived

from A. ilicifolius plant were isolated and

investigated for their antibacterial activity. However,

only three fungi showed antibacterial activity against

the bacterial test. AD1 did not show antibacterial

activity against Staphylococcus aureus, whereas

AB1 and AB2 show antibacterial activities. The

inhibitory activities of those two fungi were higher

than the chloramphenicol as the positive control.

The fungal plugs “Putih” did not show any

antibacterial activity. This observation was also

reported in several studies, in which endophytic

fungi showed activity against bacteria (Santos et al.,

2015). The potency of the fungi is attributed to

possible contribution of the type of media and

culture condition in the biosynthesis of active

metabolites (De Siqueira et al., 2011; Dos Santos et

al., 2015). The antibacterial activity exhibited by the

A. ilicifolius endophyte can be said to be relevant

with the leaf activity against S. aureus and P.

aeruginosa (Govindasamy and Arulpriya, 2013).

Acremonium sp. in this study showed

antibacterial activity against two bacterial test. It

was relevant to the other Acremonium species

obtained from marine sediment (Samuel et al., 2011)

and Antiaris toxicaria organism (Dai et al., 2009).

They have been reported to be a source of diverse

antibacterial compounds. Efrapeptin G and RHM1,

isolated from Acremonium sp. 021172C associating

with sponge Axinella sp, was reported to have a

potent antibacterial activity (Boot et al., 2006).

Acremonium sp. endophyte of Taxus baccata was

the source of leuesnostatin A which exhibited

effectiveness against Pythium ultimum (Yu et al.,

2010). Moreover, amides of the corresponding

amino alcohols and amino aldehydes of D-allo- and

L-isoleucine of an intertidal Acremonium furcatum

can inhibit B. subtilis, S. aureus and E. coli

(Gallardo et al., 2006). Moderate inhibitory activity

against S. aureus was exhibited by the 19-O-Methyl-

pyrrocidine B, some pyridine alkaloids isolated from

Cylindrocarpon, which is an endophytic fungus of

Sapium ellipticum, with 25 μM MIC (Ramsay et al.,

2018).

Ulocladium sp. is the typical pathogenic fungi

that may be responsible for Allergic fungal sinusitis

and nasal polyposis (Kaur et al., 2010). However,

ICPS 2018 - 2nd International Conference Postgraduate School

486

the fungal compounds exhibit various abilities for

medical benefits and form the inhibitory zone

around the fungal plates. Ophiobolin T, isolated

from Ulocladium sp. associating with Everniastrum

sp., showed moderate antibacterial activity against

B. subtilis and MRSA (Wang et al., 2013).

Ulocladium botrytis from Callyspongia vaginalis

sponge produces ulocladol, a new tyrosine kinase

inhibitor and antimicrobial, to inhibit Myxilla

incrustans (Holler et al., 1999). Surprisingly, 1-

hydroxy-6-methyl-8-(hydroxyl methyl) from the

fungi has antifungal activity (Konig et al., 2005).

5 CONCLUSIONS

Marine environment is proven to provide sources of

antibacterial compounds, particularly produced by

fungi. A total of 4 endophytic fungi isolated from

Acanthus ilicifolius showed different activities

against 2 bacteria. However, in order to find the

most effective compounds, further studies are

needed to obtain comprehensive data regarding

those antibacterial activities.

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