Endophytic Bacteria from White Cambodia Stems (Plumeria

acuminata) Have Strong Inhibition Against Escherichia coli

Debie Rizqoh

1,* a

, Alvi Jalilul Hakim

, Novriantika Lestari

, Sipriyadi

and Oktoviani

Department of Microbiology, Faculty of Medicine and Health Sciences, University of Bengkulu,

WR. Supratman Street, Bengkulu City, Indonesia

Undergraduate Student, Faculty of Medicine and Health Sciences, University of Bengkulu,

WR. Supratman Street, Bengkulu City, Indonesia

Department of Pharmacology, Faculty of Medicine and Health Sciences, University of Bengkulu,

WR. Supratman Street, Bengkulu City, Indonesia

Department of Microbiology, Faculty of Mathematics and Natural Sciences, University of Bengkulu,

WR. Supratman Street, Bengkulu City, Indonesia

Keywords: Endophytic Bacteria, Plumeria acuminata, Escherichia coli, Antibacterial Compounds.

Abstract: The health issues caused by Escherichia coli are a primary trigger for infectious diseases in Indonesia.

Bacterial infections can be treated with antibiotics, leading to an increase in their usage. The rising use of

antibiotics has led to irrational usage, resulting in the development of antibiotic-resistant bacteria. Therefore,

there is a need for new antibiotic sources that can combat bacterial infections, especially from various

biological resources. Due to its antimicrobial compound properties, white cambodia (Plumeria acuminata) is

a biological resource with potential as an antibiotic. One way to utilize this potential is by isolating endophytic

bacteria. The stem of the white cambodia provides a suitable environment for endophytic bacteria. Thus, this

study aims to determine the antibacterial potential of endophytic bacterial isolates from the stem of white

Cambodia to inhibit the growth of E. coli. This research uses a qualitative data collection method with

laboratory experimental research. In the first stage, endophytic bacteria were isolated from the stem of P.

acuminata using the serial dilution method. Subsequently, the characteristics of the colonies were observed

based on their shape, edges, elevation, texture, pigmentation, and the result of Gram staining. In the final

stage, the antagonistic test of the endophytic bacterial isolates against E. coli was conducted using the double-

layer agar method. The isolation of endophytic bacteria from P. acuminata resulted in 3,817 colonies. Based

on the observation of colony morphology, 93 isolates have different colony morphologies due to the diverse

characteristics of the colonies and bacterial shapes. The Gram staining test showed that 83 endophytic

bacterial isolates were Gram-positive, and ten were Gram-negative. The antagonistic test revealed that seven

positive endophytic bacterial isolates could inhibit the growth of E. coli. Endophytic bacterial isolates from

P. acuminata can produce antibacterial compounds that can inhibit the growth of E. coli.

1 INTRODUCTION

Infectious diseases still cause health problems in

developing countries like Indonesia. Based on

Indonesia's health profile in 2021 still has infectious

diseases are the reason death is most frequently post-

neonatal. In 2021, pneumonia and diarrhea still

https://orcid.org/0000-0002-0327-5881

https://orcid.org/0000-0003-1042-2576

https://orcid.org/0000-0002-9195-0321

became the reason for death most during the post-

neonatal period 14.4 % of deaths were due to

pneumonia, and 14% were because of diarrhea. The

leading causes of death in the group of child toddlers

(12-59 months) are diarrhea by 10.3% and pneumonia

by 9.4% (Ministry of Health RI, 2021). Most of these

infectious diseases are caused by bacteria.

Rizqoh, D., Hakim, A. J., Lestari, N., Sipriyadi, and Oktoviani,

Endophytic Bacteria from White Cambodia Stems (Plumeria Acuminata) Have Strong Inhibition Against Escherichia Coli.

DOI: 10.5220/0013671700003873

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Medical Science and Health (ICOMESH 2023), pages 303-310

ISBN: 978-989-758-740-5

303

One of the most common bacteria that causes

infection in the ducts digestion is Escherichia coli,

which has been diagnosed by personnel health as one

reason diarrhea is the highest in Indonesia, reaching

10% (Ministry of Health RI, 2018). E. coli is a bacil

Gram-negative bacteria with a size range between 1.0-

1.5 μm x 2.0-6.0 μm. E. coli are many opportunistic

bacteria found in the large intestine of humans as a

microbiota. Most E. coli strains are commensal

bacteria that help, but some strains are pathogenic and

can cause disease. The disease that E. coli can cause is

diarrhea, which is caused by consuming contaminated

food or water. It also causes stomach cramps, malaise,

and fever (Joeginjantoro R, 2019).

Antibiotics are the primary choice for treating

bacterial infections due to their significant benefits in

reducing pain and death consequences of infectious

diseases. Its practical usage of antibiotics has rapidly

improved in overcoming and preventing infectious

diseases. Unfortunately, its high demand for

antibiotics has caused its use to be inappropriate and

excessive. The cause is the availability of easy

antibiotics obtained by society without instructions or

recipes from medical personnel, especially doctors.

Improper use of antibiotics can result in the

development of resistant bacteria to antibiotics, which

later becomes a severe issue in treating bacterial

infections (Andiarna et al., 2020).

Resistance to antibiotics is a problem that society

needs to handle seriously. When bacteria become

resistant to antibiotics, medicine has lost its

effectiveness in treating human infections and

diseases. Resistance to antibiotics can happen due to

mutation or transfer of resistance genes through a

horizontal process. Resistance genes can inherited or

acquired from an element genetically mobile, like a

plasmid that can transferred between bacteria.

Mutations known as single-step mutations cause

appearance resistance levels quickly and in a short

time (Nurjanah et al., 2020). Incident resistance in

Indonesia is sporadic, selective, and impossible to

overcome fully (Lia Yunita et al., 2021). Based on the

problem above, it is vital to research new antibiotics

from various sources that are effective in treating

bacterial infections.

One example of source life that can utilized is the

plant white cambodia (Plumeria acuminata), which is

frequently used as a source of traditional drugs. P.

acuminata is a plant originating from Central America

that belongs to the Apocynaceae family. P. acuminata

are often found in Indonesia. Apart from being an

ornamental plant, P. acuminata also has compounds

that have antimicrobial properties. P. acuminata is one

of the plants with potential as an alternative antibiotic

to treat infections caused by bacteria (Zulkifli et al.,

2022).

Endophytic bacteria reside and live inside network

plants and then form colonies without harming the

host plants (Tangapo et al., 2018). The connection

between endophytic bacteria and host plants own

mutual relationship, beneficial or symbiotic

mutualism. In this connection, plants supply nutrients

for bacteria, while bacteria protect plants from seed

disease, help produce phytohormones, and stimulate

the absorption of minerals, especially nitrogen. P.

acuminata stems provide a suitable environment for

endophytic bacteria that can do nitrogen-fixing. This

ability is beneficial in a biological way because it

helps plants obtain nitrogen (N). The N elements

comprise essential proteins in photosynthesis,

increasing plant resistance (Koomnok et al., 2007).

The presence of endophytic bacteria in plants is

significant because these bacteria can produce

bioactive compounds with characteristics similar to

those produced by host plants. This is because there is

an evolutionary genetic exchange between host and

microbe endophyte (Hasan Basri et al., 2021). This

compound provides profit for the plant and has

potency benefits, especially in matter health.

One of the uses of endophytic bacteria is research

conducted by Zulkifli (2022), showing that

endophytic bacteria in the bark of P. acuminata plants

have the potential to be a source of antibacterial

substances against the growth of Staphylococcus

aureus, Bacillus cereus, Pseudomonas aeruginosa,

and Klebsiella pneumoniae. Further research was

carried out by Hidayati (2019), where the results of

phytochemical tests showed that endophytic bacteria

in white Cambodia stems had secondary metabolites

in the form of alkaloids.

Based on the background above, research on the

isolation of endophytic bacteria in stems of P.

acuminata is critical because there is still limited

scientific information about the existence of P.

acuminata endophytic bacteria and their benefits as

agents producing potential antibacterial compounds to

inhibit the activity of E. coli.

2 METHODS

The type of research used by researchers is

experimental research. The data collection method

used by researchers was qualitative to determine the

antibacterial activity of the endophytic bacteria of P.

acuminata stems against E. coli. This research was

conducted at the Microbiology Laboratory, Faculty of

Medicine and Health Sciences, Bengkulu University.

ICOMESH 2023 - INTERNATIONAL CONFERENCE ON MEDICAL SCIENCE AND HEALTH

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The P. acuminata plant used in this research was

taken from Bengkulu City. The samples needed are

three stems from three P. acuminata plants.

2.1 Isolation of Endophytic Bacteria

Isolation of endophytic bacteria was carried out using

dilution methods. The samples that must be prepared

are three P. acuminata stems from three plants. The

P. acuminata stems were cut with a size of 2-5 cm,

then sterilized surface with running water, and peeled

the skin stem. The piece sample was sterilized by

soaking in alcohol 70% for 1 minute, then moving it

into 5.25% Sodium Hypochlorite (NaOCl) for 5

minutes, then moving it back inside 70% alcohol

three times with an interval of 30 seconds (Hidayati,

2019). After that, the sample is crushed or crushed

using a sterile mortar and pistil by adding 3-5 ml of

sterile distilled water. Prepare 5 test tubes filled with

9 ml of sterile distilled water, then take 1 ml of

solution from the first tube containing the sample that

has been ground with a micropipette and put it in the

second tube, then homogenized using a vortex mixer.

Please do the same thing; take another 1 ml sample in

the second tube filled with 9 ml of sterile distilled

water and put it in the third tube, then homogenize.

The same step is done until the fifth tube. After

dilution, take 0.1 ml of the suspension from each

dilution and distribute it in King's B medium

aseptically, then spread it evenly using a spreader,

then incubate the isolate for 24 hours in an incubator

(Rizqoh et al., 2021).

2.2 Colony Characteristics and

Morphology of Endophytic

Bacteria

The characterization of bacterial morphology is done

in macroscopic and microscopic ways. Macroscopic

observation involves elevation, edges, shape, and

bacterial colony formation. Meanwhile, microscopic

observations were done using Gram staining (Oktavia

& Pujiyanto, 2018).

2.3 Antagonist Test

Escherichia coli are cultured into Nutrient Broth

media. The media was incubated at room temperature

for 24 hours. Then, spectrophotometry will measure

turbidity (OD = 0.3 concentration 10 6 – 10 7 cells

/mL).

Antagonist tests of endophyte isolates against

target microorganisms are carried out using the

double-layer technique. This procedure involves the

use of semi-solid nutrient media and solid nutrient

media. E. coli put into NB medium. Furthermore, E.

coli culture is mixed into semi-solid NA media, then

placed on previously solid NA media as a layer first

on the plate. Isolate endophytic bacteria dotted atop

the layer, then incubated for 24 hours at room

temperature. Bacterial isolates are said to be positive

and produce compound antibacterial if an inhibition

zone is formed in the test. The inhibition zone then

measured the diameter of the inhibition zone based on

Morales category (2003) (Table 1).

Table 1. Inhibitory Power Categories (Morales, 2003)

Inhibition Zone Diamete

Cate

≥ 20

–

30 m

Ver

stron

10-20 m

Strong

5-10m

Moderate

≤ 5m

Wea

3 RESULTS

3.1 Isolation of Endophytic Bacteria

The results of calculating the number of endophytic

bacterial colonies that grow in a total of 3,810

bacterial colonies (Table 2) and after viewing based

on characteristics colony 93 isolates were found to

have characteristics different colonies.

Table 2: Calculation Results of the Number of Endophytic

Bacterial Colonies

Plant Code Dilution Number of Colonies

E 10

-1

Plate 1 209

Plate 2 181

E 10

-2

Plate 1 122

Plate 2 253

E 10

-3

Plate 1 267

Plate 2 273

E 10

-4

Plate 1 269

Plate 2 217

E 10

-1

Plate 1 137

Plate 2 15

E 10

-2

Plate 1 26

Plate 2 165

E 10

-3

Plate 1 33

Plate 2 56

-4

Plate 1 265

Plate 2 271

E 10

-1

Plate 1 TMTC

Plate 2 226

E 10

-2

Plate 1 TMTC

Plate 2 159

Endophytic Bacteria from White Cambodia Stems (Plumeria Acuminata) Have Strong Inhibition Against Escherichia Coli

305

E 10

-3

Plate 1 115

Plate 2 178

E 10

-4

Plate 1 259

Plate 2 114

Total Number of Colonies 3,810

*TMTC: too many to count

3.2 Characterization Colonies and

Morphology of Endophytic

Bacteria

From growing colonies of endophytic bacteria

screening between colonies, characteristics between

different colonies were observed by looking at the

colony's shape, edges, elevation, texture, and pigment

(Table 3). Based on the results of observations of the

characteristics of the colony, 93 isolates of

endophytic bacteria were obtained, which were

grouped into 14 different colony groups.

Gram staining is done to determine the

morphology of the bacteria by determining the cell

morphology and the Gram type (Table 4). The

observation results of microscopic Gram stain show

that there were 80 isolates of endophytic bacteria in

the coccus Gram-positive bacteria, four isolates of

endophytic bacteria were coccus Gram-negative

bacteria and nine isolates of bacillus Gram-negative.

Table 3 Characteristics Colony of P. acuminata Endophytic Bacterial Isolates

Characteristics colony

Isolate code

Number of

isolates

Shape Margin Elevation Texture Pigment

1. Circular Entire Convex Moist Shiny

white

KE 1, KE 5, KE 6, KE 9,

KE 10, KE 11, KE 21, KE

29, KE 30, KE 31, KE 32,

KE 33, KE 34, KE 35, KE

36, KE 37, KE 38, KE 49,

KE 50, KE 39

2. Circular Entire Convex Moist Yellow KE 2, KE 8, KE 17, KE

20, KE 78, KE 48, KE 51,

KE 53, KE 54, KE 81, KE

3. Circular Entire Convex Moist Shiny

white

KE 3, KE 18, KE 19, KE

22, KE 25, KE 62, KE 63,

KE 64, KE 65, KE 70, KE

71, KE 72, KE 60, KE 82,

KE 84, KE 85

4. Circular Entire Convex Moist Shiny

white

KE 4, KE 23, KE 74, KE

75, KE 52

5. Irre

ular Undulate Convex Moist White KE 7 1

6. Circular Entire Convex Moist Shiny

white

KE 12, KE 13, KE 14, KE

25, KE 26, KE 27, KE 41,

KE 42, KE 43, KE 57, KE

58, KE 61

7. Irregular Undulate Flat Moist White KE 15, KE 28, KE 66, KE

83, KE 89, KE 90

8. Circular Entire Flat Moist White KE 16, KE 45, KE 46, KE

55, KE 59, KE 67, KE 68,

KE 80

9. Irregular Undulate Flat Moist Shiny

white

KE 40 1

10. Irregular Undulate Flat Moist Yellow KE 44, KE 47, KE 76 3

11. S

indle Entire Convex Moist White KE 55 1

12 Circula

Undulate Flat Moist Yellow KE 73 1

13. Irre

ular Undulate Convex Moist Yellow KE 77, KE 79 2

14. Irregular Undulate Convex Moist Shiny

white

KE 86, KE 87, KE 88 3

Information : KE 1= Endophytic bacterial isolate 1

Cambodia and so on

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Table 4: Grouping Isolate Based on Gram Stain

Form Isolate Gram Amount

Cocci KE 1, KE 2, KE 3,

KE 4, KE 5, KE 6,

KE 7, KE 8, KE 9,

KE 10, KE 11, KE

12, KE 13, KE 14,

KE 15, KE 16, KE

18, KE 20, KE 23,

KE 24, KE 25, KE

26, KE 27, KE 28,

KE 29, KE 30, KE

31, KE 32, KE 33,

KE 34, KE 35, KE

36, KE 37, KE 38,

KE 39, KE 40, KE

41, KE 42, KE 43,

KE 44, KE 45, KE

46, KE 47, KE 48,

KE 49, KE 50, KE

51, KE 52, KE 53,

KE 54, KE 55, KE

56, KE 57, KE 58,

KE 59, KE 60, KE

61, KE 62, KE 63,

KE 64, KE 65, KE

66, KE 67, KE 69,

KE 70, KE 71, KE

72, KE 73, KE 74,

KE 75, KE 76, KE

80, KE 86, KE 87,

KE 88, KE 89, KE

90, KE 91, KE 92,

KE 93

Positive 80

Cocci KE 22, KE 17, KE

79, KE 78

Negative 4

Bacil KE 21, KE 19, KE

68, KE 77, KE 84,

KE 81, KE 82, KE

83, KE 85

Negative 9

3.3 Antagonist Test Endophytic

Bacterial Isolate to Escherichia coli

Of the 93 isolates obtained, an antagonist test was

carried out. The antagonist test was carried out using

a 2-layer agar method consisting of solid and semi-

solid nutrient media. Antagonist test results can be

seen in Table 5.

Antagonist test gets that result endophytic

bacterial isolates tested on E. coli. Seven isolates can

inhibit the growth of E. coli. Inhibiting activity

growth of E. coli bacteria can be seen from the clear

zone formed around the tested endophytic bacterial

isolate (Figure 1).

Figure 1. Clear zone formed around endophytic bacterial

isolates tested (A) KE 19, (B) KE 44, (C) KE 76, (D) KE

77, (E) KE 78, (F) KE 79, (G) KE 81

Calculating the zone of inhibition and endophytic

bacteria that grow using a compass tool shove. The

calculation method is by calculating the diameter of

the isolate and the zone of inhibition, subtracting the

diameter of the isolate, then obtaining a mark of the

diameter of the inhibition zone. The value of the

diameter of the inhibition zone formed is categorized

based on classification Morales (2003). Table 6

shows that isolates with codes KE 19 and KE 44 have

power activity hampered by categories strong against

E. coli bacteria. Isolate others with codes KE 76, KE

77, KE 78, KE 79, and KE 81 have power activity in

the moderate category of inhibition against E. coli

bacteria.

4 DISCUSSIONS

Endophytes are microorganisms in the form of

bacteria that live well in the tissues of the plant host.

In the network, plant endophytes do not give rise to

damage to plants. During co-evolution, endophytic

bacteria estimated origin from outside the

environment plants, then enter the network plant

passed various pathways, such as stomata (pores

small on the surface leaves), lenticels (holes in the

skin wood), lenticels (holes in the skin stem), wound

plant, area of emergence of shoots, roots side (root

shoots) and sprouts (Siregar et al., 2020). The number

of endophytic bacteria generally ranges between 10 3

-10 5 cfu /g network plants (Tangapo, 2018). In this

research, the results of the isolation of endophytic

bacteria from stem White Cambodia on King'B agar

that has been incubated for 24 hours, 3,810 colonies

of endophytic bacteria that grew, are presented.

There are differences in endophytic bacteria

colony numbers from the stem. It is in line

according to Afzal et al., which exists influencing

factors diversity of endophytic bacteria something

plants, besides competent with bacteria to colonize

plants as endophytic bacteria, plants host and factors

Endophytic Bacteria from White Cambodia Stems (Plumeria Acuminata) Have Strong Inhibition Against Escherichia Coli

307

the environment also has an influence on the diversity

of endophytic bacteria from something plant (Afzal et

al., 2019). Afzal et al. also mentioned that the total

population of endophytic bacteria in plants can vary

depending on the type of growth medium used for

isolation and the level of dilution when doing

isolation. In this study, endophytic bacteria were

isolated using King's B media. King's B media

consists of glycerol, peptone, dyspotassium

phosphate, magnesium sulfate, and agar, which

supports the growth of bacteria. King's B media was

selected because it has content similar to the situation

inside plants and is also non-selective, so it is possible

for endophytic bacteria to live and grow (Rizqoh et

al., 2021).

Table 5. Antagonist Test Results Endophytic Bacterial

Isolate to Escherichia coli

Results Isolate Code Number of

isolates

Positive (+) KE 19, KE 44, KE 76, KE

81, 78, KE 79, KE 77

Negative (-) KE 1, KE 2, KE 3, KE 4,

KE 5, KE 6, KE 7, KE 8,

KE 9, KE 10, KE 11, KE

12, KE 13, KE 14, KE 15,

KE 16, KE 17, KE 18, KE

20, KE 21, KE 22, KE 23,

KE 24, KE 25, KE 26, KE

27, KE 28, KE 29, KE 30,

KE 31, KE 32, KE 33, KE

34, KE 35, KE 36, KE 37,

KE 38, KE 39, KE 40, KE

41, KE 42, KE 43, KE 45,

KE 46, KE 47, KE 48, KE

49, KE 50, KE 51, KE 52,

KE 53, KE 54, KE 55, KE

56, KE 57, KE 58, KE 59,

KE 60, KE 61, KE 62, KE

63, KE 64, KE 65, KE 66,

KE 67, KE 68, KE 69, KE

70, KE 71, KE 72, KE 73,

KE 74, KE 75, KE 76, KE

80, KE 82, KE 83, KE 84,

KE 85, KE 86, KE 87, KE

88, KE 89, KE 90, KE 91,

KE 92, KE 93

Note: Positive (+) = potentially produce compound

antibiotics by forming an inhibition zone, Negative (-) = no

potentially produce compound antibiotics

Endophyte isolates of this research have various

types of colonies, including shape, edges, elevation,

texture, pigment, and cells. Diversity colonization

endophytes are formed and influenced by several

factors related to the environment, plants, and

bacteria. In addition to the ability of bacteria to

colonize plants as endophytes, the plant's host and the

environment in which bacteria grow can influence the

growth and size of endophytic bacterial cells in

certain plants. The age of the plant host, genotype,

location, geographic location, and even analyzed

network can determine the type of endophytic

bacteria it contains. Apart from that, the growth

stages of the host can also determine the diversity of

endophytes. Nutrient availability tends to experience

enhanced bacterial diversity where the plant stage is

enriched. Not only that, but the climate can also

influence the colonization of endophytes in plants

(Afzal et al., 2019). The isolates obtained were then

observed characteristics of the colony. Observation

morphology Bacterial colonies are needed to

facilitate the identification process of the type of

bacteria (Wardhani et al., 2020).

Table 6. Inhibitory Power Category Endophytic Bacterial

Isolate

No. Isolate

code

Inhibition

zone diameter

(

)

Category

1. KE 19 13 ± 1.65 Stron

2. KE 44 10, 55 ± 0.05 Stron

3. KE 76 7.05 ± 0.4 Moderate

4. KE 77 7.3 ± 0.6 Moderate

5. KE 78 7.5 ± 0.6 Moderate

6. KE 79 7.35 ± 0.6 Moderate

7. KE 81 6.15 ± 0.7 Moderate

This result of the Gram stain also showed various

types of cell morphology. The type of endophytic

bacteria found in one plant host is not limited to only

types of endophytic bacteria, but consists of various

genera and types. Based on the results of research

conducted by Zulkifli (2022), skin stem P. acuminata

produces endophytic bacteria from the genus

Bacillus, Pseudomonas, and Alcaligenes. This is in

line with the statement Tangapo (2018) stated that the

genera Pseudomonas, Bacillus, Agrobacterium, and

Enterobacter are the most abundant genera found

(Tangapo, 2018).

This antagonist test stage tests between

endophytic bacterial isolates that have been obtained

in the isolation process with the target bacteria, E.

coli. The purpose of this antagonist test is to see the

ability of isolates of these endophytic bacteria to

inhibit pathogenic bacteria, namely E. coli. Results

from the antagonist test of endophytic bacterial

isolates to E. coli showed that seven positive isolates

of endophytic bacteria inhibit the growth of E. coli.

Seven isolates of this bacteria are KE 19, KE 44, KE

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308

76, KE 77, KE 78, KE 79 and KE 81. These isolates

have different cell types; KE 44, KE 76, KE 78, and

KE 79 have cocci form, and KE 19, KE 77, and KE

81 have basil form. Every bacterias has a different

genetic composition, metabolism pathway, and

biochemical ability, so each has produced different

compounds and metabolites (Afzal et al., 2019).

Endophytic bacterial isolates that showed positive

results can be observed by forming a clear zone

around the colony of endophytic bacterial isolates. A

clear zone is formed because target microorganisms

cannot grow around the isolate. This can be

interpreted that the isolate from P. acuminata can kill

and inhibit the growth of pathogenic bacteria.

Endophytic bacteria benefit indirectly; bacteria as

biocontrol can role against or as controller microbe

pathogen through the production of antipathogen

compounds, one of them being antibiotics. Endophyte

microbes generally can produce compounds with

structures similar to those produced by plant hosts

with the help of enzyme activity. Some endophytes

also can produce compound antibiotics that can

oppose pathogen microbes. Compound antibiotics in

the form of metabolites Secondary bacteria, created

by endophytic bacteria, act as active substances,

antibiotics, or products that help protect plants from

attacking insects or pathogen microbes. Hence,

endophytic bacteria have the potential to be utilized

as biological agents or biocontrol agents to protect

plants from pathogens (Tangapo, 2018).

The formation of an inhibition zone around the

endophytic bacterial isolate that was inoculated on the

test medium indicates that endophytic bacteria from

P. acuminata have antibacterial activity. This finding

is in accordance with the results of research by Yuli

(2019), which states that fraction test ethanol from

flower P. acuminata has an antibacterial effect on E.

coli. Based on research conducted by Hidayati

(2019), endophytic bacteria from P. acuminata stems

produce secondary metabolites in alkaloids.

Additionally, P. acuminata stems have content in the

form of tannins, flavonoids, alkaloids, and

triterpenoids. An alkaloids compound in endophytic

bacteria present in P. acuminata stem have an

antibacterial effect. The alkaloid compound works

with mechanisms inhibition that interferes with

components shaper peptidoglycan inside bacterial

cells, resulting in the layer from bacterial cell wall no

longer forming correctly, resulting in cell death.

Apart from that, Alkaloids can also prevent protein

synthesis, which can influence bacterial metabolism.

This alkaloid compound can also prevent the

development of Gram-negative bacteria (Anggraini et

al., 2019).

5 CONCLUSIONS

Based on the results of the research, the following

conclusion is derived. Results of isolation of P.

acuminata endophytic bacteria obtained as many as

3,810 colony endophytic bacterial isolates from stem

of P. acuminata. Result of observation of

characteristics colonies and morphology of

endophytic bacteria of 93 samples of P. acuminata

endophytic bacterial isolates get various type shapes,

edges, elevations, textured, bacterial pigments and

also shape bacterial cells. The result of the Gram stain

shows that there were 80 isolates in the Gram-positive

group and 13 isolates in the Gram-negative group.

Antagonist test results of the 93 endophytic bacterial

isolates tested against E. coli bacteria showed seven

endophytic bacterial isolates in P. acuminata have the

potency to inhibit the growth of E. coli.

ACKNOWLEDGEMENTS

Thanks for the support from the Faculty of Medicine,

Universitas Bengkulu, laboratory staffs, and all

parties who have helped this research process. A grant

from the Non-tax Revenue of the Faculty of

Medicine, Universitas Bengkulu, supports this

research.

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