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
Copyright
c
2021 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
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
g
ula
r
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
L.
)
a
g
ainst
B
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
y
Da
y
5Da
y
6Da
y
7
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.
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