Biodiversity of Endophytic Fungi in Sembilang National Park of

South Sumatera

Rifka Rimbi Anggraini

, Uun Yanuhar

, Yenny Risjani

Master Program in Faculty of Fisheries and Marine Science, Brawijaya University, Jalan Veteran Kota Malang 65145,

Indonesia

Departement of Aquatic Resource Management, Faculty of Fisheries and Marine Science, Brawijaya University, Jalan

Veteran Kota Malang 65145, Indonesia

*corresponding author: Rifka Rimbi Anggraini

Keyword: Bruguiera gymnorrhiza, Endophytic fungi, Sembilang National Park

Abstract: Endophytic fungi originating from areas affected by tidal water are microbes that are rich in natural bioactive

products and secondary metabolites. The purpose of this study was to determine endophytic fungi that live in

symbiosis with mangrove plants from Bruguiera gymnorrhiza species taken from the Sembilang National

Park, South Sumatra. The research method in isolating symbiont mushrooms was carried out using the Direct

Planting method with PDA (Potato Dextrose Agar) media. The results in this study indicate that there are

three types of endophytic fungi, namely Aspergillus flavus, Penicillium sp., and Aspergillus niger.

1 INTRODUCTION

The potential possessed by the diversity of natural

resources, especially plants, still needs to be studied.

According to Prihatiningtias (2005), sources of

bioactive compounds are obtained from plants,

animals, microbes and marine organisms which are

continuously being explored as more and more new

diseases emerge. These endophytic microbes were

first discovered by Darnel et al on 1904 and from then

on, the definition of endophytic microbes was agreed

as microorganisms that live in plant tissue systems

and symbiotic mutualism (Stone et al., 2000).

Endophytic fungi are one of the endophytic

microbial organisms (Strobel, 2003). In-plant tissues

that have endophytic fungi can produce compounds

that have the same properties as the host plant,

although the types of compounds are different. The

activity of compounds produced by endophytic fungi

is usually greater than that of the host compound

(Strobel et al., 2004).

One of the plants that contain a lot of bioactive

compounds produced by endophytic fungi in

mangrove plants. According to several researchers in

Noor et al. (2012) mangroves are plants that live

between sea and land, in the form of shrubs and trees

and at high tide, the roots of these mangroves will be

flooded by water and the receding time of the roots

will be seen. Bruguiera gymnorrhiza is a species of

mangrove that grows on muddy soils, is flooded

during high tide and does not like hard substrates such

as sand.

So far, many researchers have succeeded in

isolating endophytic fungi and secondary metabolite

compounds from various types of plants. However,

researchers who isolate endophytic fungi from B.

gymnorrhiza mangroves and information on

endophytic fungi in mangroves as producers of

natural ingredients are still limited in Indonesia,

especially in the Mangrove Ecosystem in South

Sumatra. Limited information, the authors have

conducted research on the isolation of endophytic

fungi in mangrove B. gymnorrhiza plants taken from

the mangrove area of the Sembilang National Park in

South Sumatera.

2 MATERIALS AND METHODS

The research sample was taken using a purposive

sampling method which is located in Sembilang

National Park, South Sumatera (Fig. 1).

Anggraini, R., Yanuhar, U. and Risjani, Y.

Biodiversity of Endophytic Fungi in Sembilang National Park of South Sumatera.

DOI: 10.5220/0009588001050111

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 105-111

ISBN: 978-989-758-483-1

105

Figure 1. Research Location

The tools used in this research are aluminum

foil, autoclave, identification book, bunsen, petri dish,

cover glass, cutter, erlenmeyer, freezer, measuring

cup, hot plate, incubator, inoculating loop, cotton,

filter paper, laminar air flaw, masks, microscopes,

analytical balances, glass objects, tweezers, and

plastic wrap. The materials used in this study were

apart, sterile seawater, distilled water, 70% alcohol,

dextrose, 75% ethanol, seawater, chloramphenicol,

lactofenol blue cotton, potato dextrose broth (PDB),

potato dextrose agar (PDA), Bruguiera gymnorrhiza

and spiritus.

2.1 Collection and Preparation of

Bruguiera Gymnorrhiza Samples

B. gymnorrhiza mangrove samples were randomly

selected from one of the tree representatives in the

mangrove zoning of the Sembilang National Park

area, South Sumatra. Mangrove samples taken are the

roots, stems and leaves as much as ± 500 gram each

part (Fig. 2). The sample taken is put into a sterile

plastic sample so that the sample is not contaminated,

then put in a cool box. In handling in the laboratory,

samples that have been taken are washed using sterile

seawater 3 times to remove impurities. Furthermore,

soaked using 70% alcohol for 1-2 minutes to kill the

epiphytic fungus that sticks to the surface. After that,

the samples were rinsed again using sterile seawater

(Kjer et al., 2010).

(a)

(b)

(c)

Figure 2: (a) Leave, (b) Root and (c) Stems of Bruguiera

gymnorrhiza (Personal Docummentation)

2.2 The Manufacture of Media Growth

Endophytic Fungi

In this research, there are two media used, namely

Potato Dextrose Broth (PDB) as liquid media and

Potato Dextrose Agar (PDA) as solid media. The way

of making these media is as follows

2.2.1 Potato Dextrose Broth (PDB)

12 gram PDB dissolved with 500 ml of seawater in an

erlenmeyer tube. The erlenmeyer tube is closed using

a cotton swab that is coated with aluminium foil.

Seawater and media are homogeneous using a hot

plate. The media is waited until completely

homogeneous. Then the media is sterilized by

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

106

autoclave for 15 minutes with a temperature of 121

and a pressure of 1 atm (Ariyono et al., 2014).

2.2.2 Potato Dextrose Agar (PDA)

PDA media as much as 19.5 gram in 500 ml of

seawater was dissolved in an erlenmeyer tube. The

erlenmeyer tube is closed using a cotton swab that is

coated with aluminium foil. Seawater and media are

homogeneous using a hot plate. The media is waited

until completely homogeneous. Then the media is

sterilized by autoclave for 15 minutes with a

temperature of 121

C and a pressure of 1 atm. Then

chill the media a few moments then put

Chloramfenicol as much as 0.1 gram. Modifications

from Ariyono et al. (2014).

2.3 Growth of Endophytic Fungi

Isolates

Samples (roots, leaves and stems) were cut to size ±

1x1 cm. The sample was put into the GDP medium

with a ratio of 1: 9 (g/v) where 10 gram of sample was

added 90 mL of PDB media. Samples are stirred

using a shaker for 4-7 days until the colour of the

water turns brownish turbid at a speed of 150 rpm at

room temperature (Kjer et al., 2010).

Samples on PDB media are put into test tubes with

the principle of multilevel dilution. The last three

dilutions (10

-4

,10

-5

and 10

-6

) were taken for planting

by the pouring method on 1 mL petri dishes. PDA

media that have been made are taken, poured into a

petri dish while homogenized until the media

becomes solid and incubated for 7 days at 25

(Benson, 2002).

2.4 Purification of Endophytic Fungi in

Potato Dextrose Agar (PDA) Media

Fungal colonies that have grown on PDA media were

previously regrown on sterile PDA media based on

morphological differences from each growing colony

to obtain endophytic fungal colonies according to

their respective morphology. Fungal colonies on

PDA media were taken using a sterile round

inoculating loop then etched on aseptic sterile PDA

media in laminar airflow. If one mushroom colony is

still mixed with other colonies, then it is refined

repeatedly until a pure mushroom colony is obtained

(Ariyono et al., 2014).

2.5 Characterization of Endophytic

Fungi

Characterization was carried out on each fungal

colony macroscopically and microscopically. The

results of observations are used as ingredients for

identification of endophytic fungi. Gandjar (1999)

mentions macroscopic observations include the

colour and surface of the colony, radial lines from the

centre of the colony towards the edge of the colony,

and concentric circles in concentric or non-concentric

Petri dishes and colony growth (cm/day).

Microscopic observations include hyphae bulkhead,

hyphae growth, hyphae colour, presence or absence

of conidia and conidia form. Microscopic

observations were made on the last day observations

(5-7 days) using a microscope.

This microscope observation was carried out

using the slide culture method. A sterile petri dish is

provided, a buffer ring is placed inside and 5 mL of

distilled water is added to maintain moisture. The top

of the ring is placed with glass preparations/object

glass and sterile PDA media pieces on it. Fungi

culture is taken and applied to the entire surface and

closed using a glass cover. Fungi cultures were

incubated for 5-7 days at 25

C. Cultures that have

grown on the cover glass are placed at the top of the

glass preparation which is dripped with lactofenol

blue cotton to increase the transparent effect on the

fungi to be more easily observed under a microscope

at magnifications of 10X and 40X (BKIPM, 2014).

2.6 Identification of Endophytic Fungi

Observations obtained from the characterization of

fungi will be used observation results obtained from

the characterization will be used for the identification

stage based on the guide book identification

Introduction to Food-Borne Fungi (Samson et al.,

1995), Introduction of General Tropical Molds

(Gandjar et al., 1999) and Identifying Filamentous

Fungi (St-Germain and Summerbell, 1996).

3 RESULT

3.1 Endophytic Fungi Pure Colony

In the growth of fungi in PDA media for 7 days at a

temperature of 25ºC found seven types of pure isolates

in mangrove B. gymorrhiza. Of the six types, there are

three types of isolates that differ in shape, colour and

texture. There are several differences in visually

purified endophytic fungi. The macroscopic

Biodiversity of Endophytic Fungi in Sembilang National Park of South Sumatera

107

observation of endophytic fungi can be seen in Table

Table 1. Macroscopic Characteristics of Endophytic Fungi

Organ Shape Colour Texture Figure

Stem

Round Brownish Smooth

Powder

Round Black As if

cotton

Root

Round Green Velvety

Round White Smooth

Powder

Leaf

Round White Smooth

Powder

Round Black Velvety

Fungi that have been made macroscopic

observations with visual observations, then the

microscopic observation stage is carried out to make

it easier to identify the results that have been

obtained. Microscopic characterization is a

continuation of the stage of identifying fungi. This

observation was carried out by observing hyphae,

spores, and conidia formed under a microscope lens

with a magnification of 400X. Macroscopic

observations can be seen in Table 2.

Table 2. Microscopic Characteristics of Endophytic Fungi

Organ Figure Expla-

nation

Sporae Hyphae

Stem

The

hyphae

form has

a divider

and is

branched

hyaline

Conidia

are

round

and

black

Root

The

hyphae

form has

a divider

and

branched

hyaline

Conidia

are round

and

black

Leaf

The

hyphae

form has

a divider

and is

branched

hyaline

Conidia

are round

and

black

4 DISCUSSION

In general, the fungi has been found to grow clearly.

Pure colonies were obtained based on differences in

the visual appearance of each isolate. The fungi

obtained at the stems has a brownish to black colour,

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smooth texture like cotton. The diameter size of fungi

colonies taken from stems tends to be smaller because

the growth rate is relatively slow. The fungi obtained

at the root looks even more different from other types

of fungi because of the difference in colour. This

fungi has a green colour with a larger colony diameter

than fungi obtained from the leaves. The speed of

growth of this fungi is also relatively faster than the

fungi from the isolation of the leaves.

The fungi obtained from the leaves has a black

spore colour. The size of the diameter of this fungi

colony is the biggest compared to the fungi isolated

from the stem and roots. The speed of growth of this

fungi is much faster compared to other fungi.

Therefore, the type of fungi obtained from these

leaves grows to meet the entire surface of the petri

dish.

The purification stage of the leaf part is more

abundant with endophytic fungi compared to roots

and stems. This is consistent with what was stated by

Noverita et al. (2009); Sinaga et al. (2009) where

more endophytic fungi isolates were obtained from

the leaves. This phenomenon is suspected because the

nutrients present in the leaves are more supportive of

the growth of endophytic fungi. Endophytic fungi

isolated from one host plant contain different types of

isolates, even from one living tissue obtained from a

plant can be isolated more than 1 type of endophytic

fungi. This is an adaptation mechanism of endophytic

fungi to the microecology and specific physiological

conditions of each host plant.

Macroscopically, the fungi of Aspergillus niger is

found in the leaves. This fungi is black and has white

hyphae. Based on Summerbell (1996) that the

Aspergillus niger fungi grow rapidly until its diameter

fills the entire surface of the PDA media. The

Aspergillus niger fungi itself is black and fills the

entire surface of the media. This is also supported by

microscopic results in which the Aspergillus niger

fungi has the characteristics of large enough black

spores covering all spore bubbles where the conia

spreads tightly and has clear conidiospores. These

characteristics are also found in microscopic images

of the fungi of Aspergillus niger based on references

from the identification book Summerbell (1996).

The endophytic fungi of Aspergillus niger is a

fungus that is isolated from every organ of the B.

gymnorrhiza mangrove plant. The characteristics of

Aspergillus niger are having a large black conidia

head and a round shape. The hyphae were insulated

and branched mycelium. This fungi also have vesicles

(bubbles) at the ends of the conidiophores and

becomes a place of conidia to grow. The conidia

shaped chain and black. This fungi grows well at

room temperature (Wuryanti, 2008).

The fungi of Aspergillus niger is a type that

produces quite a lot of compounds and enzymes.

According to Handajani and Purwoko (2008), the

fungi of Aspergillus niger can produce ochratoxin

compounds and can produce lipase enzymes. Lipase

enzyme is an enzyme that plays an important role in

the world of modern biotechnology because it has

high activity in hydrolysis and synthesis and chemical

reactions.

Aspergillus niger fungi which belong to the group

of phosphate solvent fungi. Phosphate solvent fungi

are able to be used as biofertilizer which is the result

of biotechnology engineering in the field of soil

science. Aspergillus niger has the ability to dissolve

phosphate compounds that are difficult to dissolve

into a form available to plants by producing organic

acids so that availability becomes faster (Artha et al.

2013).

In addition to secondary metabolites produced by

Aspergillus niger, this fungi is also able to produce

cellulase enzymes (Sa'adah et al. 2010), proteases

(Ramdhani et al. 2015), and chitinase (Purkan et al.

2016). Protease enzymes produced by A. niger fungi

are able to be grouped in alkaline proteases which are

one of the groups of hydrolytic enzymes that is able

to catalyze the hydrolysis process or the proteins

damage into their constituent amino acids (Ramdhani

et al. 2015). The forms of commercial products in the

application of alkaline proteases in the industrial

sector include the detergent industry, the food

industry, the pharmaceutical industry, milk, skin, and

meat processing (Ramdhani et al. 2015).

Described in the book Gandjar et al. (1999)

Aspergillus flavus is a fungi commonly found in nuts

(especially peanuts), spices, oilseeds, cereals, and

sometimes in dried fruit. The fungi of Aspergillus

flavus is a fungi that is green and shaped like soft hair

because it has fairly long hyphae. This fungi has a

fairly large size because it almost fills the entire

surface of the PDA media. Microscopic observations

show that these fungi have quite large spores, with

conidial heads scattered throughout the bubble

surface and has rough conidiospores walls

(Summerbell, 1996).

A. flavus fungi is also a fungus that can produce

aflatoxin compounds. The main aflatoxin compounds

are produced by the fungi of A. flavus, which is one

of the causes of cancer in humans (Handajani and

Setyaningsih, 2006). In addition to aflatoxin

compounds, Setiarto (2011) also suggested that the

fungi of A. flavus can produce ochratoxin and

zearalenone compounds. In extreme conditions, this

Biodiversity of Endophytic Fungi in Sembilang National Park of South Sumatera

109

type of fungi can infect grains directly which can later

cause aflatoxin accumulation. This can cause health

problems in animals and even humans due to

contamination of feed ingredients by aflatoxins. A.

flavus fungi can also be used as antibacterial

metabolites. A. flavus can inhibit the growth of

Echercia coli which is a bacterium that causes

diarrhoea.

Based on the results of Hidayati's research (2010)

the selection of six endophytic fungi isolates

produced antibacterial metabolites using the Kirby-

Bauer test method where the results were all isolates

could form inhibitory zones against the test bacteria.

A. flavus fungi can inhibit the growth of E. coli

bacteria by 9.33 mm. Research Raharjo et al. (2007)

said that this fungus is able to dissolve phosphate

which cannot be dissolved so that plants can be used

in growth.

Gandjar et al. (1999) describe the fungi of

Penicillium sp. has a surface with a velvety texture

although sometimes like cotton. The colours in the

colonies are sometimes yellow to brownish, greyish-

green to yellowish-green and greyish green.

Conidiophores in fungi arise from the substrate and

generally has many branches and smooth-walled.

Habitat from this fungi is very common in various

food products, as well as food items that are low in

the water. Penicillium sp. used in industry to produce

antibiotics (Crystovel, 2017).

These fungi are known as fungi that produce

antibiotic metabolites. Amaria et al. (2013) said that

Penicillium, Trichoderma and Aspergillus are fungi

that can release antibiotic-like substances that can

inhibit the growth of pathogens so that these fungi are

antagonistic fungi that can be used as biopesticide and

biofertilizer fungi. Subowo (2015) explains that the

fungi of Penicillium sp. able to decompose cellulose

and lignin compounds into simple carbon compounds

needed by soil microbes as an energy source so that

this fungi is very good for soil fertility.

5 CONCLUSION

The results of isolation and identification of

endophytic fungi from Bruguiera gymnorrhiza

mangroves taken from the Sembilang National Park,

South Sumatera are known that there are two types of

fungi from the Aspergillus genus namely Aspergillus

niger and Aspergillus flavus, and one of the

Penicillium genera namely Penicillium sp.

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