Screening of Antibacterial Potency and Molecular Identification of

Endophytic Bacteria from Soursop Leaf (Annona muricata L.)

Fitri Yuniarti, Wahyu Hidayati, Lulu Shofaya

Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. DR. Hamka (UHAMKA)

Jl. Delima II/IV, Klender, Jakarta Timur 13460 Indonesia

Keyword: endophytic bacteria, Annona muricata L., antibacterial, PCR

Abstract: Soursop leaves (Annona muricata L.) is one of the medicinal plants identified as a source of endophytic

bacteria producing secondary metabolites. Several studies have reported that secondary metabolite

compounds extracted from soursop leaves have inhibitory activity against pathogenic bacteria and fungi and

have anticancer activity. This study aims to isolate endophytic bacteria of soursop leaf and identify

molecular isolates producing antibacterial metabolites by PCR method. This study began with endophytic

bacterial isolation of soursop leaves, followed by screening for antibacterial potency using disc diffusion

method and identification of molecular isolates which had the highest antibacterial activity. After isolation,

three isolates were obtained: BW-1LM, BW-2LP, and BW-3LK. The result of antibacterial activity test

showed that BW-1LM isolate had the highest activity against bacterium of Bacillus subtilis and Salmonella

typhi test. Molecular identification was obtained by BW-1LW isolate having 99% similarity level to

Bacillus licheniformis DSM 13 strain. The conclusion is soursop leaves contains endophytic bacteria which

have antibacterial activity against Bacillus subtilis and Salmonella typhi.

1 INTRODUCTION

Indonesia is a country rich in natural resources and

one of them is medicinal plants. The different types

of plants that exist are crucial natural resources in

producing various bioactive compounds that are

potential to be developed. The plants produce

secondary metabolites with molecular structures and

diverse biological activities, and has good potential

to be developed into a cure for various diseases

(Radji, 2005). The resulting secondary metabolites

are thought to be the result of coevolution or genetic

transfer of host plants into endophytic microbes (Tan

and Zou, 2001).

Endophytic microbes are microorganisms whose

habitat is within the plant organs over a period and

can produce secondary metabolites that have

bioactivity, such as enzymes, antimicrobial agents,

anti-fungus, anticancer, and plant growth regulators

(Kumala, 2014). One of the medicinal plants that

can be used as a source of endophytic bacteria is

soursop leaf (Annona muricata L.). Soursop is a

plant that has medical properties and is widely used

in Indonesia. The part of soursop plants that are

widely used for medicinal purpose is the leaves.

Soursop leaf contains saponin compounds,

flavonoids, coumarins, alkaloids, and tannins. Haro

et al. (2014) reported soursop leaf extract methanol

might inhibit Escherichia coli and Staphylococcus

aureus bacteria. The information about the ability of

endophytic bacteria in producing bioactivity

compounds encourage the research on endophytic

bacteria in soursop leaf.

The polymerase chain reaction known as

Polymerase Chain Reaction (PCR) is an enzymatic

synthesis process to amplify nucleotides in vitro

(Fatchiyah, 2011). The PCR process is a recurrent

cycle process, including denaturation, annealing, and

extension by DNA polymerase enzymes. The

amplification process was continued by the analysis

of electrophoresis and identification using the

marker gene of 16S rRNA. Genes 16S are genes

specific to prokaryotic species (Clarridge, 2004).

Resti et al. (2013) reported results of endophytic

bacteria from onion plants obtained 82 isolates

endophytic bacteria, and six isolates of them have

potential ability to bacterial leaf blight disease.

This study is conducted to obtain endophytic

bacteria from soursop leaves that have antibacterial

activity and molecularly identifies endophytic

Yuniarti, F., Hidayati, W. and Shofaya, L.

Screening of Antibacterial Potency and Molecular Identiﬁcation of Endophytic Bacteria from Soursop Leaf (Annona muricata L.).

DOI: 10.5220/0008241201690175

In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 169-175

ISBN: 978-989-758-349-0

169

bacteria of soursop leaf (Annona muricata L.)

producer of antibacterial secondary metabolites. The

antibacterial activity test was performed by disc

diffusion method. Isolates that had the greatest

antibacterial activity were continued by identifying

endophytic bacteria molecularly using 16S rRNA

gene.

2 MATERIALS AND METHODS

2.1 Materials

Microcentrifuge refrigerator (PerfectSpin 24 Plus),

PCR thermo cycler (Tanach RAY-MG48),

electrophoresis (Mupid EXU), UV transilluminator

(Genesys 20), Rotary shaker (Eyela). Soursop leaf

samples (Annona muricata L.) NaOCl 5.3%,

medium Nutrient Agar (NA) and Nutrient Broth

medium (NB), EDTA, Lysozyme, Isopropanol,

Ethanol 70%, and Wizard Genomic DNA

Purification Kit from Promega, Forward primary 27f

and reverse primary 1492r (Rosita 2012). Nuclease-

free water, GoTaq Green PCR Master Mix of

Promega, Gel agarose, ethidium bromide (EtBr),

Tris Acetate EDTA (TAE) buffer solution, 1kb

ladder DNA (Promega) and loading dyes (Thermo

Scientific), Medium Muller Hinton Agar MHA),

bacterial cultures of Bacillus subtilis and Salmonella

typhi test.

2.2 Sample Preparation and Plant

Identification

Plant samples were taken from Tangerang area and

then identified in Herbarium Bogoriense, Botanical

Field of LIPI Research Center, Cibinong-Bogor.

2.3 Isolation of Endophytic Bacteria

This stage applied the method used by Kumala

(2014) and began with samples of fresh soursop

leaves. The leaves were washed with water and then

cut. The sample pieces were then sterilized by being

immersed in 75% ethanol for 1 minute and then

soaked with 5.3% sodium hypochlorite for 5

minutes. Then the sample was rinsed three times

with 75% ethanol. The sterile samples were then

grown on Nutrient Agar (NA) media which had been

added with nystatin and incubated in dark spaces at

room temperature and observed until growing

colony appeared (Pratiwi, 2008).

2.4 Characterization and Gram

Staining

This study used microscopic and macroscopic

observations. Macroscopic observations include

colony pigmentation, colony form, colony elevation,

colony surface, and colony consistency. Microscopic

observations include the shape and colour of cells

using Gram staining.

Gram staining begins by doing the bacterial

scraping on the glass object then sprayed with

crystals violet for 1 minute as much as one drop,

then washed with running water and dried again.

After that, one drop of Lugol solution is added and

let stand for 1 minute, then washed with water and

dried again. As decolorizing agent, drops of 96%

alcohol is added to the object glass until the dye is

faded, and then rinsed with water and allowed to

dry. The last stage, one drop of safranin is given as a

counterstain, and let stand for 30 seconds (Pratiwi,

2008).

2.5 Fermentation of Endophytic

Bacteria

The pure cultured bacteria obtained are inoculated in

a test tube containing medium nutrient broth (NB),

then incubated for 7x24 hours with a shaker speed of

145 rpm. After that, every 1x24 hours as much as 1

ml of the mixture was taken and centrifuged for 3

minutes at a speed of 5000 rpm. The centrifugation

supernatant was transferred to a new micro tube and

stored at 40°C. (Kumala et al., 2007)

2.6 Screening Antibacterial Activity of

Endophytic Bacterial

Screening by disc diffusion method used Salmonella

typhi and Bacillus subtilis. The endophytic bacterial

isolates from tilted agar were regenerated into

nutrient agar medium (NA), whereas the test

bacteria were regenerated into five mL nutrient broth

medium (NB). Suspension of test bacteria that has

been fulfilled the transmittance value of 25% is

taken as much as 10% liquid culture of the test

bacteria is fed into MHA media with a temperature

of 37 ℃.

Paper discs that have been soaked fermented

supernatant then placed on a medium that has been

inoculated pathogenic bacteria. Subsequently

incubated for 24-48 hours at 37 °C and was observed

antibacterial activity with the presence of an obstacle

zone around the disc paper (Simarmata et al., 2007).

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

170

2.7 Isolation of Genomic DNA

Bacterial Endophytes

The bacterial isolates to be used for the DNA

isolation process were first cultured in Nutrient

Broth medium (NB) and incubated at 37 °C for 24

hours. The isolation process is based on the protocol

contained in the Genomic DNA Purification Kit

(Promega, 2014). A total of 1.5 ml bacterial culture

was transferred into a sterile 2.0 ml micro centrifuge

(microtube) and centrifuged at 13000 times for 2

min. The forming supernatant was discarded, then

resuspended complete bacterial cell deposits with

480 μl EDTA 50Mm. A further 120 μl of lysozyme

was homogenized and incubated at 37 °C for 30

minutes, centrifuged for 2 min at a rate of 13000

times and disposed of the supernatant formed. The

next step was added 600 μl nuclei lysis solution then

homogenized and incubated at 80 °C for 5 minutes

to lyse the cells, the sample was allowed at room

temperature then added three μl RNase solution then

homogenized by reversing the tube. After that, it

was incubated at 37 °C for 30 minutes. The next step

was added 200 μl protein precipitation solution then

homogenized for 20 seconds with high vortex speed,

then incubated sample at the cold temperature for 5

minutes and centrifuged for 3 minutes at 13000

times.

The supernatant containing the DNA is further

incorporated into a new microcentrifuge tube

containing 600 μl of isopropanol, homogenized

gently until visible strands of DNA thread. After

that, centrifuged at a rate of 13000 times for 2

minutes, then the supernatant formed was carefully

removed, and the tube was dried with absorbant

paper. A total of 600 μl of 70% ethanol was added to

the tube containing the DNA, homogenized the tube

gently to wash the DNA pellets. After that, it was

centrifuged at 13000 times for 2 minutes, and the

tube was dried for 15 minutes. The last stage was

added 100 μl DNA rehydration solution then

incubated at 65 °C for 1 hour. DNA was stored at 2-

8 °C (Promega, 2014).

2.8 DNA Amplification With PCR

The bacterial genomic amplification process was

based on the protocol contained in the Maxima Hot

Start Green PCR Master Mix (2X), using primer 27f

and primer 1492r. A total of 25 μl Maxima Hot Start

PCR master mix (2X) was inserted into a 0.5 ml

microtube. Nuclease-free water was added as much

as 9 μl, then the mixture was resolved until dissolved

completely by homogenized. Furthermore,

homogeneous mixtures were added by primer 27f

and primary 1492r, 2.5 μl of each. Then, it was

added with 3 μl of DNA and homogenized. The

amplification process was performed using Bartlett

and Stiling (2003) method, namely: Initial

denaturation at 95ºC for 5 minutes for 1 cycle,

Denaturation at 95°C for 1 minute for 30 cycles,

Annealing at 56ºC for 1 minute for 30 cycles,

Extension at 72ºC for 1 minute for 30 cycles, Final

extension at 72ºC for 10 minutes for 1 cycle. The

obtained amplicon was observed with

electrophoresis using a 0.8% agarose gel (Bartlett

and Stiling, 2003).

2.9 Sequencing of Gen 16S rRNA

The sample was put into a 0.2 ml dry and sterile

micro tube, then sent to Eijkman Molecular Biology

Institute, Jakarta Indonesia for further purification

and sequencing.

2. 10 Identification of 16S rRNA

Bacterial Endophytic Gene

DNA sequenced were analyzed with the bioedit

program and then compared with the sequence

database at the nBLAST site

(http:/www.blast.ncbi.nlm.nih.gov/). The result

obtained in this research was then compared to the

data on Gene Bank.

3 RESULTS AND DISCUSSION

3.1 Isolation and Characterization of

Endophytic Bacterial Morphology

The endophytic bacteria may be associated with the

host plant, the function is to help the metabolism

process of the host plant and to produce secondary

metabolites that are similar to the host plant

compounds (Kumala, 2014). Isolation of endophytic

bacteria was done by direct planting method on

Nutrient Agar (NA) medium. From the isolation

process, we obtained three endophytic bacterial

isolates which are labelled as BW-1LM, BW-2LP,

and BW-3LK. The macroscopic observation results

indicated that BW-1LM isolate is yellowish white,

irregularly shaped and has mucous consistency.

While microscopic results showed that bacterial

cells are identic with the Bacillus genus included in

Gram-positive bacteria. Macroscopic observation of

2LP isolate described that this isolate is BW-white,

Screening of Antibacterial Potency and Molecular Identiﬁcation of Endophytic Bacteria from Soursop Leaf (Annona muricata L.)

171

irregularly shaped and have slimy consistency, while

the results of microscopic bacteria cells are round

and a Gram-positive-bacteria. The macroscopic

observations of BW-3LK isolate are white,

irregularly shaped and slimy. Microscopic

observations of BW-3LK isolate showed that

bacterial cells are spherical and included in Gram

negative bacteria. Those observation results of the

endophytic bacteria characteristics can be seen in the

Table 1.

In Table 1, it can be seen that all of three isolates

have different characteristics. From the Gram

staining result, there are red colored bacteria that

have been known as Gram-negative bacteria, while

the purple colored bacteria are Gram-positive

bacteria (Radji, 2010). Gram staining can also show

the presence or absence of peptidoglycan content in

bacterial cell membranes. If the bacterial cell

membrane does not have a peptidoglycan

compound, the appearance of the resulting color

after being observed under a microscope is red

(Pratiwi, 2008).

3.2 Screening for Antibacterial Activity

The antibacterial activity test was performed by

inoculating the discs that had been immersed in the

supernatant of endophytic bacterial fermentation of

soursop leaf on Muller Hinton Agar (MHA)

medium. Those media were mixed with Bacillus

subtilis isolates (Gram-positive) and Salmonella

typhi isolates (Gram-negative). Three endophytic

bacterial isolates were inoculated on NB

fermentation medium, then fermented for 7x24

hours at 145 rpm. From the test results, only BW-1

LW isolate showed antibacterial activity against

both test bacteria.

The results of the activity test on B. subtilis

bacteria can be seen in Figure 1. Based on this

image, the inhibition zone can be seen around the

disc paper on fermentation products on the 5th and

6th days. Figure 2 is the result of the endophytic

bacteria secondary metabolite activity test against S.

typhi bacteria, on the 6th and 7th day of

fermentation products. There was an inhibition zone

around the disc paper, while the other two isolates

were unable to inhibit the growth of the two test

bacteria.

Table 2 shows the inhibitory zone diameter of

bacterial isolate BW-1 LM. This table shows the

activity produced by BW-1 LM isolate on 5th, 6th,

7th day fermentation. From the data, it can be seen

that BW-1LM endophytic bacterial isolate has

antibacterial activity against both test bacteria on

6th-day fermentation.

Table 1: Characteristics of endophytic bacterial isolates from soursop leaf (Annona muricata L.).

Isolate Code

Morphology

Morphology Pigmentation Consistency Elevation Edge

Cell

Shape

Color

BW-1 LM Irregular White

Yellowish

Slimy

Arise

Irre

ula

Basil Purple

BW-2 LP Irregular White Slimy Arise Irregular Circular Purple

BW-3 LK Irregular White Slimy Arise Irregular Circular Red

BW-1LM BW-2LP BW-3LK

Figure 1: Secondary Antibacterial Activity Test Results of Endophytic Bacteria from Soursop Leaves (Annona muricata

)

ainst

acillus subtilis Test Bacteria.

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

172

3.3 Amplification of BW-1LM Isolate

DNA by PCR

In prokaryotic organisms, there are three types of

rRNAs with 5S, 16S, and 23S sedimentation

coefficients. The 16S rRNA gene is a gene specific

to the prokaryotic species (Clarridge, 2004). The

16S rRNA gene region is a subunit of the 30S

prokaryotic ribosome that has 1542 nucleotide

components. The 16S rRNA gene region is

commonly used for bacterial identification and

phylogenetic studies (Kumala, 2014).

Amplification of the 16S rRNA gene was

performed on BW-1 LM endophytic bacterial isolate

by PCR method and using a 27f primer (5'-AGA

GTT TGA CTG GCT CAG-3 ') and 1492r primer

(5'-TAC GGC TTA CCT TGT TAC GA- 3 ') as used

by Rosita (2012). Primary 27f is a forward primer

attached to the 5 'end of the previously decomposed

target DNA strand, while the primary 1492r is the

reverse primer that will be attached to the other end

of the 5' single chain.

The amplification process successfully

performed was marked by the presence of DNA

fragments on the agarose gel, which can be seen in

Figure 3. The magnitude of the DNA fragment is in

the range of 1500 bp. This corresponds to a 16S

rRNA gene that has a magnitude of about 1500 bp

(Clarridge, 2004). The result of the amplicon that

has been in electrophoresis for the sequencing

process to know the order of nucleotide base.

Sequencing is a technique used to sequence

nucleotide bases in DNA fragments (Brown, 1995;

Sambrook et al., 1989). The results of sequence data

processing were analyzed using the nBLAST

program online on the NCBI website

(http://blast.ncbi.nlm.nih.gov/). The analysis was

conducted with the aim of comparing sequenced

data (query) from the results of research with DNA

sequences from various corners of the world that

were deposited and published on DNA banks or

Gene Bank.

Table 2: Measurement Results of Secondary Metabolite Constraint Zone Endophytic bacteria from Soursop Leaves

against Bacillus subtilis and Salmonella typhi.

No. Isolate Code Bacteria Antibacterial Activit

5Da

6Da

1 BW-1 LM B.subtilis 8,76mm 9,01mm -

2 BW-1 LM S.typhi - 9,11mm 9,21mm

Figure 3: Result of Amplicon Electrophoresis DNA

Isolate Bacteria Endophytes BW-1LM from Soursop

Leaf. Lane 1: Ladder DNA 1kb Lane 2: product PCR.

BW-1LM BW-2LP BW-3LK

Figure 2: Secondary Antibacterial Activity Test Results of Endophytic Bacteria from Soursop Leaves (Annona muricata

L.) against Salmonella typhi Test Bacteria.

Screening of Antibacterial Potency and Molecular Identiﬁcation of Endophytic Bacteria from Soursop Leaf (Annona muricata L.)

173

Criteria that meet the taxonomic requirements

are said to be the same species if the database

sequence has an identity percentage between 95% -

99% and can be defined at the species level if the

identity percentage is ≥99% (Clarridge, 2004).

Figure 4 is the result of sequence alignment analysis

with the nBLAST program. Result of the data shows

that the sample of BW-1LM isolate has zero E-value

and the same similarity percentage is 99% in

bacterial species of Bacillus licheniformis strain

DSM 13. The zero value expresses the higher level

of trust that the query sequence has high homology

level with database sequence. So the endophytic

bacterial isolates in this study can be named Bacillus

licheniformis strain BW-1LW.

4 CONCLUSION

It can be concluded that this research was

successfully obtained three endophytic bacterial

isolates from soursop leaf. However, only one of

them has the antibacterial activity against Bacillus

subtilis and Salmonella typhi. The result of

molecular identification with PCR technique showed

that BW-1LW isolate had 99% similarity to Bacillus

licheniformis DSM 13 strain.

ACKNOWLEDGEMENT

This research was funded by the UHAMKA

Research and Development Institute of the Year

Budget 2016-2017.

REFERENCES

Bartlett JMS, Stirling D. 2003. PCR Protocols. Humana

Press Inc. Totowa NJ. p. 90-91.

Brown TA. 1991. Pengantar Kloning Gen. Terjemahan:

Soemiati AM, Praseno. Yayasan Essentia Medica.

Yogyakarta. p. 182.

Clarridge JE. 2004. Impact of 16S rRNA Gene Sequence

Analysis for Identification of Bacteria on Clinical

Microbiology and Infectious Diseases. Journal

Clinical Microbiology Reviews. 17(4): 840-859.

Fatchiyah, Arumingtyas EL, Widyarti S, Rahayu. 2011.

Biologi Molekular (Prinsip Dasar Analisis).

Erlangga. Jakarta. p. 8, 18, 22-24, 35, 48-55, 112-113.

Figure 4: Description of Nucleotide Results BLAST Gen 16S rRNA Bacterial Isolate Endophytes from Soursop Leaf.

MICH-PhD 2018 - 1st Muhammadiyah International Conference on Health and Pharmaceutical Development

174

Haro G, Utami NP, Sitompul E. 2014. Study of The

Antibacterial Activities of Soursop (Annona muricata

L.) Leaves. International Journal of PharmTech

Research. 6: 575-581.

Kumala S. 2014. Mikroba Endofit: Pemanfaatan Mikroba

Endofit dalam Bidang Farmasi. PT. ISFI Penerbitan.

Jakarta. p. 15-47.

Pratiwi ST. 2008. Mikrobiologi Farmasi. Erlangga.

Jakarta. p. 129-130, 143-144, 188-191.

Promega Corporation. 2014. Wizard Genomic DNA

Purification Kit. USA. p. 1-19.

Promega Corporation. 2012. Protocols Go Taq Hot Start

Green PCR Master Mix (2X). USA. p. 1-2.

Radji M. 2005. Peranan Bioteknologi dan Mikroba

Endofit dalam Pengembangan Obat Herbal. Dalam:

Majalah Ilmu Kefarmasian. Laboratorium

Mikrobiologi Departemen Farmasi, FMIPA-UI.

Depok. p. 1-14.

Radji M. 2010. Buku Ajar Mikrobiologi: Panduan

Mahasiswa Farmasi dan Kedokteran. EGC. Jakarta. p.

99.

Radji M. 2011. Rekayasa Genetika Pengantar Untuk

Profesi Kesehatan. CV Sagung Seto. Jakarta. Hlm. 27-

28, 18-71.

Resti Z, Habazar T, Prima DP, Nasrun. 2013. Skrining dan

Identifikasi Isolat Bakteri Endofit Untuk

Mengendalikan Penyakit Hawar Daun Bakteri pada

Bawang Merah. Jurnal HPT. Tropika. 13(2): 167-178.

Rosita A. 2012. Isolasi dan Karakterisasi Bakteri Endofit

dari Umbi Tanaman Kentang (Solanum tuberosum L.)

menggunakan Primer PCR-RAPD. Skripsi. Fakultas

Sains dan Teknologi Universitas Islam Negeri,

Malang. p. 62-68.

Sambrook J, Russell DW. 2001. Molecular Cloning A

Laboratory Manual. Cold Spring Harbor Laboratory

Press. New York. p. 5-6.

Tan RX, Zou WX. 2001. Endophytes: A Rich Source of

Functional Metabolites. Natural Product Reports. 18:

448-459.

Screening of Antibacterial Potency and Molecular Identiﬁcation of Endophytic Bacteria from Soursop Leaf (Annona muricata L.)

175