Studi Perbandingan Struktur Komunitas Bakteri Endofit Pada Empat Kultivar Padi Asal Indonesia Berdasarkan Sekuen 16s Rrna

COMPARATIVE STUDY OF ENDOPHYTIC BACTERIAL
COMMUNITY STRUCTURES IN FOUR INDONESIAN
RICE CULTIVARS BASED ON 16S rRNA SEQUENCE

YENI KHAIRINA

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

THE THESIS STATEMENT AND SOURCES OF INFORMATION
ALONG COPYRIGHT DEVOLUTION
I hereby declare that the thesis entitled “Comparative study of
endophytic bacterial community structures in four Indonesian rice cultivars
based on 16S rRNA sequence” is true of my research under the guidance of the
supervisor committee and has not been submitted in any form to any college.
Sources of information derived or citated from published and unpublished works
from other writers have been mentioned in the text and listed in the references in
the end of this thesis.
I hereby assign the copyright of my thesis to Bogor Agricultural

University.

Bogor,

March 2016

Yeni Khairina
NIM G351124121

RINGKASAN
YENI KHAIRINA. Studi Perbandingan Struktur Komunitas Bakteri Endofit pada
Empat Kultivar Padi asal Indonesia berdasarkan Sekuen 16S rRNA. Dibimbing
oleh YULIN LESTARI dan ANJA MERYANDINI.
Mikrob endofit telah banyak dilaporkan memiliki pengaruh yang positif
bagi tanaman inangnya, misalnya dengan mendukung pertumbuhan tanaman,
memperkuat pertahanan melawan bakteri patogen, dan meningkatkan asupan
nutrisi. Namun, studi tentang diversitas mikrob endofit khususnya pada kultivar
padi asal Indonesia dengan agro-ekosistem yang berbeda masih terbatas
disebabkan banyaknya penggunaan metode kultur biasa. Denaturing Gradient Gel
Electrophoresis (DGGE) merupakan salah satu pendekatan metagenomik

berdasarkan pada pemisahan fragmen-fragmen DNA dengan ukuran yang sama
namun memiliki sekuen basa yang berbeda. Metode ini dapat mengungkapkan
diversitas mikrob yang sulit untuk dikulturkan dalam media buatan. Berdasarkan
latar belakang di atas, penelitian ini bertujuan untuk menganalisis dan
membandingkan struktur komunitas bakteri endofit dan spesifik takson pada
bakteri yaitu aktinomiset, menggunakan gen 16S rRNA pada empat kultivar padi
asal Indonesia dengan metode PCR-DGGE.
Bagian tanaman (akar, batang, dan daun) yang sehat diambil dari 4
kultivar padi asal Indonesia yaitu Ciherang, IR64, Inpara 2, dan Situ Patenggang,
DNA total diekstraksi menggunakan Genomic DNA Mini Kit Plant. Amplifikasi
PCR untuk komunitas bakteri dilakukan pada sampel akar, batang, dan daun
tanaman padi yang menghasilkan panjang fragmen sekitar ±600 bp. Sementara
itu, amplifikasi PCR untuk komunitas aktinomiset dilakukan pada sampel batang
dan daun menggunakan strategi PCR 2 tahap yang menghasilkan fragmen dengan
ukuran sekitar ±1087 bp (PCR pertama) dan ±195 bp (PCR kedua). Analisis
diversitas untuk komunitas bakteri dan aktinomiset endofit dilakukan dengan
metode DGGE pada gel poliakrilamid. Pita target dipotong dan kemudian
diamplifikasi kembali dengan menggunakan primer tanpa GC clamp. Sekuensing
pada produk PCR dari pita DGGE dilakukan sesuai dengan standar protocol
menggunakan ABI PRISM sequencer. Analisis hubungan antar DNA yang

disekuensing dilakukan berdasarkan pohon filogeni menggunakan metode
neighbor-joining pada perangkat lunak MEGA 5.0.
Analisis Shannon-Wiener dan profil DGGE menunjukkan diversitas
bakteri endofit pada padi kultivar Ciherang dan IR64 lebih tinggi dibandingkan
dengan Inpara 2 dan Situ Patenggang. Analisis dice similarity coefficient
menunjukkan bahwa struktur komunitas pada masing-masing sampel relative
cukup mirip, sedangkan analisis kluster menunjukkan tingginya kesamaan
struktur antara Ciherang dan IR64 dan antara Inpara 2 dan Situ Patenggang.
Distribusi anggota-anggota bakteri pada takson filogeni bervariasi antara kultivar
padi dimana sekuen yang didapatkan dekat kekerabatannya dengan
Gammaproteobacteria, Bacilli, Flavobacteria, β-Proteobacteria, dan αProteobacteria. Kelas Gammaproteobacteria memiliki beberapa afiliasi spesies
seperti Cellvibrio japonicus strain UEDA 107, Pseudomonas putida strain
ZJUTBX04, Escherichia fergusonii strain NBRC, Pseudomonas poae RE*1-1-14
strain RE*1-1-14, Cellvibrio mixtus strain J3-8, Cellvibrio mixtus strain ACM

2601, and Pseudomonas brassicacearum subsp. brassicacearum NFM421 strain
NFM421 dengan identitas kesamaan berkisar antara 90-97%. Kelas βProteobacteria memiliki afiliasi spesies seperti Burkholderia cepacia strain 106
dengan identitas kesamaan 93%. Kelas α-Proteobacteria memiliki afiliasi spesies
yaitu Brevundimonas olei strain DUCC3718 dengan identitas maksimum 92%.
Kelas Bacilli memiliki afiliasi spesies seperti Sporosarcina koreensis strain F73

dan Brevibacillus brevis DZBY12 dengan identitas kesamaan masing-masing 97%
dan 99%. Kelas Flavobacteria memiliki afiliasi spesies seperti Myroides odoratus
dan Flavobacterium ceti strain 454 dengan identitas kesamaan 91-99%.
Sementara itu, satu pita teridentifikasi sebagai archaea.
Hasil analisis Shannon-wiener dan profil DGGE pada spesifik takson
bakteri, aktinomiset, menunjukkan nilai diversitas dari komunitas ini tidak terlalu
berbeda antar sampel yang dibandingkan. Analisis dice similarity coefficient dan
analisis kluster menunjukkan adanya kesamaan struktur komunitas yang tinggi
antara padi kultivar Ciherang dan IR64. Analisis filogeni menunjukkan kluster
aktinomiset dengan 4 famili besar yaitu Microbacteriaceae, Streptomycetaceae,
Cellulomonadaceae, dan Micrococcaceae. Famili Microbacteriaceae memiliki
afiliasi spesies seperti Microbacterium insulae strain DS-66 dan Microbacterium
luteolum strain IFO 15 074 dengan identitas maksimum 99%. Famili
Streptomycetaceae memiliki afiliasi spesies seperti Streptomyces acidiscabies
strain ATCC 49003, Streptomyces chartreusis strain ISP 5085, dan Streptomyces
scopiformis strain NBRC 100 244 dengan identitas maksimum 98-99%. Famili
Cellulomonadacee memiliki afiliasi spesies yaitu Cellulomonas flavigena strain
DSM 20109 dengan identitas maksimum 100%. Famili Micrococaceae memiliki
afiliasi spesies seperti Kocuria polaris strain CMS 76or, Kocuria rosea strain
DSM 20447, Kocuria aegyptia strain YIM 70003, Arthrobacter aurescence TCI

strain TCI, Arthrobacter ramosus strain CCM 1646, Arthrobacter arilaitensis
strain Re117, Citricoccus nitrophenolicus strain PNP1, dan Micrococcus luteus
NCTC strain 2665 dengan identitas maksmum 99-100%.
Kata kunci: 16S rRNA, aktinomiset, bakteri, DGGE, komunitas endofit, tanaman
padi

SUMMARY
YENI KHAIRINA. Comparative Study of Endophytic Bacterial Community
Structures in Four Indonesian Rice Cultivars based on 16S rRNA Sequence.
Supervised by YULIN LESTARI and ANJA MERYANDINI.
Endophytic microbes have been reported to give beneficial effects to their
host by promoting the plant growth, strengthening the protection against
pathogen, and increasing nutritional supply. However, studies on the diversity of
microbial endophytes especially in Indonesian rice cultivars with different agroecosystems are still limited due to the mainly use of culture-dependent method.
DGGE (Denaturing Gradient Gel Electrophoresis) is one of metagenomic
approach based on the separation of the DNA fragments that have the same length
but with different sequences. It can be used to reveal the diversity of
microorganisms that are difficult to be cultured in the artificial media. Thus, this
study aimed to compare the community structure of bacterial endophytes and
specific bacterial taxon, actinomycetes, based on 16S rRNA gene in four

Indonesian rice cultivars using PCR-DGGE method.
The part of plant samples (root, stem, and leaf) were collected from four
healthy rice cultivars IR 64, Inpara 2, Situ Patenggang, and Ciherang. Total DNA
was extracted using Genomic DNA Mini Kit Plant. PCR amplification of bacteria
domain was done from root, stem, and leaf samples resulted in fragment size ±600
bp. Meanwhile, PCR amplification of actinomycetes was done from leaf and stem
using two-stage PCR strategy resulted fragment size ±1087 bp (first PCR) and
±195 bp (second PCR). Diversity analysis of endophytic bacteria and
actinomycetes community was conducted using DGGE on polyacrylamide gel.
Bands of interest were excised and re-amplified using the primer without GCclamp. Sequencing of re-amplification product of DGGE bands was done
according to standard protocols using DNA sequencer ABI PRISM. The
sequencing results were compared to the GenBank nucleotide sequence database
of NCBI BLAST.N. Relationship analysis among the sequenced DNA was
performed based on phylogenetic tree using the neighbor-joining method and
software MEGA 5.0
Shannon-Wiener analysis and DGGE profiles showed the diversity of
endophytic bacteria in Ciherang and IR64 rice cultivars were higher compared
with that of Inpara 2 and Situ Patenggang. The dice similarity coefficient showed
that all of the samples have a quite similar community structures, however cluster
analysis of endophytic bacteria demonstrated high similarity of the community

structure between Ciherang and IR64 rice cultivars and community structure
between Inpara 2 and Situ Patenggang. Distributions of bacterial members to
phylogenetic taxon were varied among the rice cultivars in which the majority of
the sequences obtained were closely related to Gammaproteobacteria, Bacilli,
Flavobacteria, β-Proteobacteria, and α-Proteobacteria. Gammaproteobacteria class
was affiliated to Cellvibrio japonicus strain UEDA 107, Pseudomonas putida
strain ZJUTBX04, Escherichia fergusonii strain NBRC, Pseudomonas poae
RE*1-1-14 strain RE*1-1-14, Cellvibrio mixtus strain J3-8, Cellvibrio mixtus
strain ACM 2601, and Pseudomonas brassicacearum subsp. brassicacearum

NFM421 strain NFM421 with 90-97% of maximum identity. β-Proteobacteria
class was affiliated to Burkholderia cepacia strain 106 with 93% of maximum
identity. α-Proteobacteria was affiliated to Brevundimonas olei strain DUCC3718
with 92% of maximum identity. Bacilli class was affiliated to Sporosarcina
koreensis strain F73 and Brevibacillus brevis DZBY12 with 97% and 99% of
maximum identity, respectively. Flavobacteria class was affiliated to Myroides
odoratus and Flavobacterium ceti strain 454 with 91-99% of maximum identity.
Meanwhile, one band was identified into archaea.
Focusing on specific bacterial taxon, actinomycetes, Shannon-Wiener
analysis and DGGE profiles showed the diversity of this community were not

different among the samples. The dice similarity coefficient and cluster analysis
of endophytic actinomycetes demonstrated high similarity of the community
structure between Ciherang and IR64 rice cultivars. Phylogenetic analysis showed
actinomycetes cluster with 4 large families, they were Microbacteriaceae,
Streptomycetaceae, Cellulomonadaceae, and Micrococcaceae. Microbacteriaceae
was affiliated to Microbacterium insulae strain DS-66 and Microbacterium
luteolum strain IFO 15 074 with maximum identity 99%. Streptomycetaceae was
affiliated with Streptomyces acidiscabies strain ATCC 49003, Streptomyces
chartreusis strain ISP 5085 and Streptomyces scopiformis strain NBRC 100 244
by maximum identity 98-99%. Cellulomonadaceae was affiliated with
Cellulomonas flavigena strain DSM 20109 with maximum identity 100%.
Micrococaceae was affiliated to Kocuria polaris strain CMS 76or, Kocuria rosea
strain DSM 20447, Kocuria aegyptia strain YIM 70003, Arthrobacter aurescence
TCI strain TCI, Arthrobacter ramosus strain CCM 1646, Arthrobacter arilaitensis
strain Re117, Citricoccus nitrophenolicus strain PNP1, and Micrococcus luteus
NCTC strain 2665 with maximum identity 99-100%.
Key words: 16S rRNA gene, actinomycetes, bacteria, endophytic community,
DGGE, rice plant

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COMPARATIVE STUDY OF ENDOPHYTIC BACTERIAL
COMMUNITY STRUCTURES IN FOUR INDONESIAN
RICE CULTIVARS BASED ON 16S rRNA SEQUENCE

YENI KHAIRINA

Thesis
as one of the requirements to obtain the degree
Master of Science
on
Microbiology Major

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2016

Examiner of Beyond Commission on Thesis Examination: Dr. Ir. I Made Artika, M.App.Sc.

Thesis Title

Name
NIM

: Comparative Study of Endophytic Bacterial Community
Structures in Four Indonesian Rice Cultivars based on 16S
rRNA Sequence
: Yeni Khairina
: G351124121

Approved by
Supervisor Commission

Dr Yulin Lestari
Head

Prof Anja Meryandini, MS
Member

Discovered by

Head of Microbiology
Major

Dean of Graduate School

Prof Dr Anja Meryandini, MS

Dr Ir Dahrul Syah, MScAgr

Date of Examination: 6 January 2016

Date of Graduation:

FOREWORD
Praise and gratitude to God for all His gifts so this thesis has been
completed successfully. The theme chosen in this research was endophytic
microbial diversity that has been conducted from July 2014 until March 2015. The
title of this thesis research is “Comparative Study of Endophytic Bacterial
Community Structures in Four Indonesian Rice Cultivars Based on 16S rRNA
Sequence”.
The author thanks to Dr.Yulin Lestari and Prof. Dr. Anja Meryandini
MS.as supervisor.Thanks is also delivered to Prof. Yasuyuki Hashidoko that has
supervised the author during Population Activities Resources and Environment
(PARE) exchange program. The author also thanks to Dr. Ir. I Made Artika,
M.App.Sc. as an external examiner has given advices during thesis examination.
The author also thanks to the staff of Laboratory of Microbiology and Intergrated
Laboratory, Department of Biology IPB, and Laboratory of Ecological Chemistry,
Hokaido University.
During the study and research, the author thanks to the author’s family.
The author also gives special thanks to Mahyarudin, Randi, Sari, Mei, Reika,
Sharon, Nie, Ciko, Sipri, Gegek, Chessa, Septi, Sasha, Rara, Wahyu, Vita, Asril,
all of friends in Microbiology Major Batch 2012-2013, Graduate School of IPB,
all of friends in Laboratory of Ecological Chemistry, and all of PARE members
who have helped the author during this research. The author wished this research
can give contribution for the knowledge development specifically in agricultural
sector.

Bogor, March 2016

Yeni Khairina

TABLE OF CONTENTS
LIST OF TABLES

xiv

LIST OF APPENDIX

xv

INTRODUCTION

1

Background

1

Problem Identification

2

Objective of Study

2

Significant of Study

2

Research Scope

3

LITERATURE REVIEW

3

Endophytic Bacteria

3

Rice Plant

7

PCR-DGGE

8

METHODS
Research Framework

9
9

Time and Place

10

Sample Collection and Sterilization

10

DNA Isolation

11

Amplification of 16S rRNA Gene-specific Bacteria and Actinomycetes

12

Analysis and Cloning of DGGE Bands

13

Sequencing of the 16S rRNA Gene and Construction of Phylogenetic Tree

14

Sequence Variation Analysis

15

RESULT AND DISCUSSION

15

Result

15

Discussion

31

CONCLUSION AND SUGGESTION

34

Conclusion

34

Suggestion

34

REFERENCES

36

APPENDIX

43

BIOGRAPHY

58

LIST OF FIGURES
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Mechanism of colonization of Burkholderia kururiensis in rice
screened by SEM at 7 dpi
Research framework
PCR amplification of 16S rRNA gene specific bacteria from four
Indonesian rice plant cultivars
Bacterial community in four Indonesian rice cultivars
Shannon diversity index of 16S rRNA DGGE profiles of four
Indonesian rice cultivars for endophytic bacterial community
Hierarchical cluster analysis results of bacteria DGGE profiles
The closest sequence homology of bacterial 16S rRNA gene-targeted
DGGE band
Position of DGGE band region amplified by 799F-mod3/1389R
primer set
PCR amplification of 16S rRNA gene specific actinomycetes from
four Indonesian rice plant cultivars
Actinomycetes community in four Indonesian rice cultivars
Shannon diversity index of 16S rRNA DGGE profiles of four
Indonesian rice cultivars for endophytic actinomycetes community
Hierarchical cluster analysis result of actinomycetes DGGE profiles
The closest sequence homology of actinobacterial 16S rRNA genetargeted DGGE band
Position of the actinomycetes community amplified by 338F/518R
primer set

5
10
16
17
18
19
21
22
24
25
26
26
28
29

LIST OF TABLES
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Endophyte classification based on colonization in the plant tissue
Endophyte classification based on the lifestyle
Rice plant profile in Indonesia
Some endophytic bacteria isolated from rice plant
Characteristics (I) of agricultural soil from sampling site
Characteristics (II) of agricultural soil from sampling site
List of the primers used in this study
The quantity and quality of DNA extracted from rice plant tissue
Dice similarity coefficient analysis of endophytic bacterial community
Phylogenetic Affiliation of Endophytic Bacterial Community
Variation of specific sequence pattern in DGGE bands originated from
Indonesian rice plant
Dice similarity coefficient analysis of endophytic actinomycetes
community
Phylogenetic Affiliation of Endophytic Actinomycetes Community
Variation pattern of DGGE bands for actinomycetes community

4
4
7
8
11
11
12
16
18
20
23
26
27
30

LIST OF APPENDIX
1
2
3
4
5

Sequence of DGGE bands for bacterial community based on 16S
rRNA gene
Sequence of DGGE bands of actinomycetes community based on 16S
rRNA
Variation pattern of DGGE bands for bacterial community
Data of DGGE band volume from Phoretix 1D software for bacterial
community
Data of DGGE bands volume from Phoretix 1D software for
actinomycetes community

43
48
50
56
57

INTRODUCTION

Background
Indonesia is the third-largest rice producer in the world (USDA 2015).
However, the forecast data showed the increase of rice imports around 2% in 2015
compared than previous year (Statista 2015). It indicated that the high rate of rice
production cannot achieve self-sufficiency of the greater demand of rice. High
demand of rice can be attributed due to the population increase. In order to
achieve food stability and security in Indonesia, various efforts should be
conducted, one of them is by increasing rice productivity. The improvement of
cultivation techniques by utilizing endophytic microbes is one of good alternatives
to achieve both food and environment sustainability.
In recent years, much attention has been paid to the study of endophytic
microbes due to their great potential as less exploited resources. Some of the
endophytic microbes were reported to give beneficial effects to their host by
promoting the plant growth, strengthening the protection against pathogen, and
increasing nutritional supply (Singh et al. 2006; Nagendran et al. 2014; Sari et al.
2014). A wide range of bioactive compounds has also been produced by
endophytic microbes, which were commonly derived from bacterial group
especially a member of phylum Actinomycetes (Berdy 2005). These microbes
have huge potential to synthesis numerous novel compounds that can be applied
in pharmaceutical, agricultural and industries (Golinska et al. 2015). Nowadays,
the exploration of microbial diversity of endophytes and their potential was still
limited in the certain types of plants. Thus, the novel potential of endophytes and
their distribution throughout the diverse plant species in various ecosystems is still
being a subject of interest.
Practice in agriculture is one of the factors known to affect the diversity of
microbial community in the soil (Lopes et al. 2011), but very little knowledge
about the effects of these practices on the existence of endophytic microbial
communities, especially in rice plant. Some of superior rice cultivars in Indonesia
are very interesting to be studied, for example IR 64 and Ciherang that currently
hold about 31% and 22% of total national rice area, respectively (USDA 2012),
and the other varieties like Inpara 2 and Situ Patenggang that survive very well in
unfavorable environmental condition. Those rice cultivars have been adapted to
different kind of agro-ecosystems and cultivation techniques. IR 64 and Ciherang
are commonly cultivated in irrigated rice field, Inpara 2 is planted on tidal
swampland, while other rice variety such as Situ Patenggang is well adapted in
dry land (Suprihatno et al. 2009).
Previous researches have explored the diversity and potential role of
culturable endophytic actinomycetes in Indonesian rice plants. A number of
endophytic actinomycetes have been successfully isolated from rice cultivars,
IR64, Ciherang, Inpago 4, Inpari 9, Elo, and Inpara 2, by using cultivationdependent method (Jelita 2012). Some isolates of endophytic actinomycetes have
potential role to produce indole-3-acetic acid, protect the host from pathogen, and
fix dinitrogen gas (Sari et al. 2014; Lestari et al. 2014; Hastuti et al. 2012). It ia

2

known that only small portion of microbes (0.1-10%) from the total population
can be culturad. Meanwhile, more than 99% of microbes are still difficult to
culture in artificial media (Sekiguchi 2006). A solution to overcome the
difficulties and limitations associated with the cultivation technique is
metagenomic approaches.
DGGE (Denaturing Gradient Gel Electrophoresis) is one of a
metagenomic approach based on the separation of the DNA fragments that have
the same length but with different sequences (Fischer and Lerman 1994). The use
of PCR-DGGE technique in the study of biodiversity has benefits to track and
describe the dominant population within the samples. In its application, DGGE is
applicable for overviewing the succession and diversity of microbial community
structure because it can proceed many different samples simultaneously based on
environmental change (Piterina et al. 2012). Using PCR-DGGE, Mahyarudin et
al. (2015) has successfully revealed the community structure of actinomycetes in
soil and root of some Indonesian rice plants, including four cultivars as described
above. To obtain more comprehensive data, further DGGE analysis of
actinomycetes community in stem and leaf as well as bacterial community
structure in root, stem, and leaf of these Indonesian rice varieties were done. This
study aimed to compare the community structure of bacterial endophytes and a
specific bacterial taxon, Actinomycetes, in the Indonesian rice cultivars using 16S
rRNA gene-targeted PCR-DGGE analysis.
Problem Identification
Endophytic bacteria have been reported to have an important role for
supporting the growth of rice plants. However, data of the overall diversity of
endophytic bacteria in Indonesian rice plants with difference agro-ecosystems are
still not available yet. It is because only 1% of microbes that can be cultured,
whereas approximately 99% of microorganisms cannot be cultured in artificial
media.

Objective of Study
This study aimed to compare the community structure of bacterial
endophytes and specific bacterial taxon, actinomycetes, based on 16S rRNA gene
in four Indonesian rice cultivars using PCR-DGGE analysis.

Significant of Study
Genetic diversity analysis of endophytic bacteria and specific bacterial
taxon, actinomycetes, in rice plant are expected to answer the whole diversity of
endophytic bacteria community which colonize in these plants. The diversity data
of endophytic bacteria in rice plant can be used as a basic information for
developing further study in order to find the method to increase the production of
rice plants.

3

Research Scope
The research involves sample collection and sterilization, the isolation of
genomic DNA in rice plants tissues, the amplification of 16S-rRNA gene-specific
bacteria and actinomycetes, diversity analysis using PCR-DGGE, and
phylogenetic tree construction.

LITERATURE REVIEW
Endophytic Bacteria
Endophyte is derived from "endon" which means "inside" and "python"
which means plant (Schulz and Boyle 2006). Endophytism is a phenomenon
where mutualistic relationship happened between plant and microbe in which the
microbes live inside the plant without causing any symptoms or diseases (Wani et
al. 2015). Endophytic bacteria was defined by Ryan et al. (2008) as a bacteria that
occupy different areas in the plant tissue and does not cause pathogenicity. While
Hallmann et al. (1997) stated that endophytic bacteria are bacteria that colonize
latently or actively and locally or systematically of plant tissue. However, all of
the definition is still very limited because it does not provide any information
about the other types of symbiosis between endophytic bacteria and plants except
beneficial relationship. Sturz et al. (2000) defined that endophytic bacteria are
nonpathogenic bacteria that form various types of relationship with its host such
as beneficial, neutral, or detrimental. From the previous definition, the endophytic
bacteria can be summed as bacteria isolated from sterile surface tissue and
colonize plant tissue both locally and systemically and form various types of with
its host such as beneficial, neutral, or detrimental.
Based on Wani et al. (2015), endophyte can be divided into two
categories: systemic and non-systemic endophytes (Table 1). Petrini (1991) has
defined systemic endophyte as organisms that occupy the parts of plant tissue at
least one part of their life cycle, perform a symbiotic relationship with its host,
and does not cause any symptoms or certain diseases. While the non-systemic
endophyte is endophyte that occupies the part of plant tissue in a relatively short
time and form a different relationship with its host plant depend on environmental
conditions (Salud et al. 2011).
Based on the lifestyle, endophytic bacteria can be divided into two
categories namely facultative and obligate endophyte (Table 2). Obligate
endophytic can survive depend on the presence of its host. Whereas facultative
endophytic have at least one cycle of its life living outside of the host plant
(Hardoim et al. 2008).

4

Table 1 Endophyte classification based on colonization in the plant tissue
(Wani et al. 2015)
No

Criteria

1

Relationship

2

Mode of
transmission
Colonization

3
4
5
6

Plant defense
response
Coevolution
Diversity

Endophyte
Systemic
Form a mutualistic relationship
with the host
Perform vertical or horizontal gene
transmission
Systemic colonization in
intercellular space
Response not actively to plant
defense
Coevolution within the plant exist
Low diversity

Non-systemic
Form a various relationship with its host
(mutualism, latent saprophyte, etc)
depend on environmental condition.
Perform horizontal gene transmission
Local colonization in intercellular space
Response actively to plant defense
Coevolution within the plant rarely exist
High diversity

Table 2 Endophyte classification based on the lifestyle (Hardoim et al. 2008)
No

Criteria

1

Habitat

2
3

Host range
Mode of
Transmission
Source of
endophytic
Mode of
Entrance

4
5

Endophyte
Facultative

Obligate

Soil, surface of plant, inside the
plant tissue, or artificial nutrient
Broad-host range
Horizontal

Inside the plant tissue

Commonly from rhizosphere or
phyllosphere
Commonly enter from wounds or
natural openings

Exist in the plant tissue in a long
period
Transmit gene to the next offspring
and occupy vegetative tissue or seed

Narrow-host range
Vertical or use vector

Endophytic bacteria can come from various sources, but rhizosphere and
phyllosphere are believed to be the main sources of endophytic bacteria (Hallm
ann et al. 1997). It is indicated by the similarities of community existing in the
plant tissue with community in the rhizosphere and phyllosphere. However, the
further study stated that the rhizosphere community has the greater opportunities
to occupy niches in the plant tissue compared by phyllosphere community. The
assumption can be justified for several reasons. According to Hallmann et al.
(1997), the rhizosphere community is a major source of endophytic bacteria
because at the beginning of plant growth, root emerges and exposes to the
rhizosphere organisms first, so it is possible that the rhizosphere microbes enter
into the root tissue and colonize systemically. Phyllosphere community has a
small possibility to occupy a niche as endophyte in the plant tissue. It is because
when the phyllosphere bacteria enter and colonize plant tissue, there will be
competition between the bacteria that already exist in the plant tissue with the
phyllosphere bacteria. Beside rhizosphere and phyllosphere, seeds and vegetative
material are also believed to be the source of endophytic bacteria (Kaga et al.
2009).
Interaction between the candidates of endophytic bacteria with the plants
had been initiated before the bacteria colonizing plant tissue. In conclusion, the
success of endophytic bacteria to enter the plant tissue depend on some important

5

stages include pre-colonization, colonization, and post-colonization. Precolonization involves the finding and recognizing the host. Host finding is carried
out by the movement of the candidate of endophytic bacteria towards plant tissue
in various ways for example by chemotaxis. After finding a suitable host,
candidate of endophytic bacteria will attach to the surface of host tissue and cause
the host plasma membrane damage. After the attachment, the next stage is the
introduction. Results from the introduction stage between candidates of
endophytic bacteria and plants can be positive or negative. The introduction with
a negative response (incompatible) between bacteria and the host will induce
defense system in plants such as the induction of resistance, hypersensitivity,
phytoalexin production, the destruction of the cell wall, formation of papillae, and
so on. Meanwhile, if the introduction is positive (compatible), the plant will
produce nutrients required for bacteria growth. The recognition process is a preselection of endophyte communities in the plant (Hallmann et al. 1997)

A

B

A

B

CC

D

D

E

E
Figure 1 Mechanism of colonization of Burkholderia kururiensis in rice plant
screened by SEM at 7 dpi. A) SEM showed no bacteria that colonize the
surface of the base or root hairs. B) SEM showed that the root surface
covered by bacteria which allows the invasion site occurs at the base of
the hair root (marked with elliptical area). C) Magnification of bacteria
attachment on the surface of the root hairs. D,E) The invasion of
bacteria into the root tissue through the root hairs. EC, epidermal cells;
h, the root hairs ; b, bacteria (Mattos et al. 2008).
Colonization stage of endophytic bacteria in plant tissue (Figure 1)
generally happened through natural opening such as lenticels and stomata, natural
wound (due to biotic or abiotic factor), the emergence of lateral roots area,

6

epidermis conjugation, radicle germination, or also penetration using hydrolytic
enzyme such as cellulase and pectinase (Hallmann et al. 1997). The penetration
process of endophytic bacteria through natural openings happened either actively
or passively. Penetration is done passively assisted by fluid flowing from the leaf
through the stomata. While active penetration can be done by using hydrolysis
enzyme. However, there are regulations that cause the enzyme is only produced
when the penetration process, but after entering into the plant tissue, the enzyme
is no longer produced. This is acceptable because if the enzyme is continuously
produced, it will damage the plant tissue and create negative relationship between
endophyte and its host. The patterns of endophytic bacteria colonization also vary
depend on the strain and its species. Endophytic bacteria commonly colonize plant
in the intercellular tissue. But others colonize the intracellular part, as well as
vascular tissue. Interaction between endophytic bacteria and plants depends on the
type of its endophytic bacteria.
Endophytic community structure is dynamic. It is influenced by several
factors, both abiotic and biotic. Biotic factors include conditions in its host plants,
such as the availability of nutrients, specificity, the stage of development, health,
and etc. While the influences of abiotic consist of physical and chemical factors.
Physical factors include temperature, rainfall, UV radiation, and moisture
(Hallmann et al. 1997). While chemical factors include differences of the soil
profile like pH, salinity, and other chemical compounds that may indirectly affect
the composition of the bacterial communities in the rhizosphere which is
considered as the main source of endophytic bacteria (Berg and Smalla 2009).
Marschner et al. (2001) stated that the composition of the rhizosphere bacteria
affected by complex interactions between soil type, plant species, and the location
of the root zone. Ahlholm et al. (2007) also reported that the success of foliar
endophytic bacteria infect the host plant depends on the interaction between the
environment and the host genotype. It means that different environmental
conditions can influence the selection of endophytic bacteria in plant tissue to
survive in favorable environmental conditions.
Endophytic actinomycetes is a type of bacteria that recently developed
mainly in the field of agriculture. Actinomycetes are a group of microorganisms
that are most distributed in nature. In the natural habitat, Streptomyces is the most
common actinomycetes group and often found on the total population of
actinomycetes. Some genera of actinomycetes such as Actinoplanes,
Amycolatopsis, Catenuloplanes, Dactylosporangium, Kineospora, Microbispora,
Micromonospora, Nonomuraea are very difficult to be isolated and grown,
usually referred to rare actinomycetes (Monisha et al. 2011). Some endophytic
actinomycetes were reported to have antagonistic activity against various kinds of
pathogens in rice (Tian et al. 2004; Hastuti et al. 2012). Streptomyces, Nocardia
sp., and Streptosporangium sp. showed significant antagonistic activity against
root pathogens such as Pythium sp. and Phytophthora sp. (Verma et al. 2009).
Endophytic actinomycetes can also protect its host from environmental stress, for
example by accelerating the activity of cellulose and lignification process to
survive in the dry condition (Thaecowisan et al. 2005). Actinomycetes also
contributed to plant growth factor by dissolving phosphate (Gangwar et al. 2011),
fixing nitrogen (Valdes et al. 2005), and producing IAA (Lestari et al. 2014).

7

Rice Plant
Rice (Oryza sativa L.) is an annual plant that has a fibrous roots and short
stem which form leaf midrib to support the leaves. Rice is known as a source of
carbohydrates, especially the endosperm. Other parts of rice commonly known as
industrial raw materials for example the outer shell of rice (bran) as an oil, rice
husk as fuel or material for paper and fertilizer. The taxonomical classification of
the rice plant is as follows:
Kingdom
Division
Class
Ordo
Family
Genus
Species

: Plantae
: Magnoliophyta
: Monocot
: Poales
: Poaceae
: Oryza
: Oryza sativa L.

Rice used in this study consists of several varieties, namely Situ
Patenggang, IR 64, Ciherang and Inpara 2 (Table 3). Selection of those varieties
based on different types of agro-ecosystems. Situ Patenggang is commonly
planted on dry land; IR 64 and Ciherang are cultivated on irrigated land; Inpara 2
is planted on tidal swampland.
Table 3 Rice plant profile in Indonesia (Suprihatno et al. 2009)
Rice plant varieties
Characteristics
IR64
Age (days)
Height (cm)
Productive tiller (per
number of stem)
Shattering
Resilience
Amylose content
1,000-kernel weight of
milled rice (gram)
Production average
(ton/ha)
Yield potency (ton/ha)
Resistance to disease

Ciherang

Situ Patenggang

116-125
107-115
14-17

±128
±103
16

±124
±134
11

Tight
Tight
27
24.1

Intermediate
Intermediate
23
27-28

Intermediate
Intermediate
22,05
25.66

Intermediate
Intermediate
21,9
25

5

6

5,49

4,1

Stress tolerance

5
5-8.5
Intermediate
Resistance to
resistance level to bacterial leafXoo and rice
blight strain III
ragged stunt virus and IV
(RRSV)
Lowland irrigated Lowland irrigated
field
field in 5000 m
above the sea
level
-

Year of production

1986

Cultural adaptation

Inpara 2

±115
±85
25

2000

6,08
6,1
Resistance to
Resistance to some blast
bacterial leaf-blight disease
patotype III

Lowland swamps
and tidal

Dry-land

Toxicity of
Fe and Al
2008

Toxicity of AI (60 ppm)
2010

8

A wide variety of microorganisms, including fungi, actinomycetes, and
other bacteria have been found in plants and referred to as endophyte. Endophytic
bacteria that have been isolated from various parts of the rice plant for last 5 years
by both culturable and unculturable methods can be seen in Table 4.
Table 4 Some endophytic bacteria isolated from rice plant
Rice Part
Grains, root,
and leaves
Leaves, stem,
and roots
Seed

Bacteria Taxon
(Bacteria and Actinomycetes)
Sphingomonas, Pseudomonas, Burkholderia,
Enterobacter, Pantoea,
Azospirillum, and Herbaspirillum
Acinetobacter oryzae sp. nov.

Rice Species

Ref.

Oryza sativa L.

Loaces et al. 2011

Oryza alta

Chaudhary et al 2011

Oryza sativa L.

Gangwar et al. 2011

Singh et al. 2011

Root
Root

Burkholderia cepacia, Citrobacter
sp., Citrobacter sp., Citrobacter sp., Bacillus
amyloliquefaciens, B. amyloliquefaciens,
and B. thuringiensis
Burkholderia cepacia
Streptomyces sp. GMKU 3100

Leaves

Pantoea ananatis and Pseudomonas syringae

Oryza sativa L.
Oryza sativa L.
cv. KDML105
Oryza sativa L.

Root

Stenotrophomonas maltophilia

Oryza sativa L.

Zhu et al. 2012

Root

Pesudomonas sp.,Bacillus, Azotobacter,
and Enterobacter
Penibacillus, Microbacterium, Bacillus, and
Klebsiella.

Oryza sativa L.

Narayanasamy 2012

Oryza sativa L.

Ji et al. 2014

Oryza sativa L.

Jiang et al. 2013

Seed

Staphylococcus, Rhizobium,
Microbacterium and Methylobacterium.
Pantoea sp. Sd-1

Oryza sativa L.

Xiong et al. 2014

Root

Rhizobium rhizoryzae sp. nov.

Oryza sativa L.

Zhang et al. 2014

Stem

Streptomyces

Oryza sativa L.

Mingma et al. 2015

Leaves,
stems, and
roots
Seed

Rungin et al. 2012
Ferrando et al. 2012

PCR-DGGE
DGGE (Denaturing Gradient Gel Electrophoresis) is a molecular
technique used to separate DNA fragments from PCR products that have the same
size of base pair but different sequence arrangement. It is separated by acrylamide
gel with a gradient denaturant from low to high (Fischer and Lerman 1983; Myers
et al. 1987; Rosenbaum and Riesner 1987; Riesner et al. 1991). Separation of
DNA fragments based on the partial melting point of double-stranded DNA in
which DNA that has a high GC-content will have a higher melting point
compared with the low GC-content.
One characteristic of DGGE is the use of GC-clamp on one end of the
primer ranged between 30-50 nucleotides (Muyzer et al. 1997). DGGE has
effectiveness to detect about 50% of the sequences in the DNA fragment until 600
bp, but using GC-clamp, the percentage of effectiveness can be increased up to
100% (Myers et al. 1985; Sheffield et al. 1989). GC-rich sequences will prevent
an open double-stranded DNA totally becomes single stranded (Sheffield et al.
1989; Sheffield et al. 1992).

9

Effective staining for DGGE gel is by using SYBR green (Muyzer et al.
1997). Benefits of using SYBR green is reducing background staining on the gel,
so it can facilitate band profile both dominant and less dominant.
The principle of DGGE is the using of gradient to separate DNA
fragments. Good separation of DNA fragments can be done by optimizing the
gradient and electrophoresis time. Sequence variations would lead to the
differences in melting point and the position of the stop migration in the gel.
When the lowest melting point is reached then a portion of the double-stranded
DNA will be opened and the migration process will immediately stop. Melting
behavior of DNA fragments can be observed by using perpendicular gradient gel.
Gel perpendicular has a denaturant gradient increase from left to right,
perpendicular towards the electrophoresis direction (Fischer and Lerman 1994).
The electrophoresis proceeded for 16 hours at 100V. The optimal time of
electrophoresis is determined by the electrophoresis gradient. While the parallel
gradient gel is the gel that has an increased gradient from top to bottom, parallel to
the electrophoresis direction. DGGE is also useful for proceeding many samples
at the same time. Apparatus of DGGE can be obtained by different commercial
companies such as Bio-Rad (Herculas, USA).
DGGE technique has several advantages such as simple, easy to use for
regular laboratory, and the results are also easy to interpret. In its application to
the study of ecology, DGGE can be used to study the changes of the community
because it can analyze many different samples simultaneously based on
environmental change. In addition, this method can be used to observe the
richness of bacterial isolation, for example to analyze the results of the PCR
product of pure cultures whether the product contains one or more fragments.
Another benefit is to compare the different extraction protocol by comparing the
ability of producing different 16S rRNA fragment from different DNA extraction
protocol (Heuer and Smalla 1997; Liesack et al. 1997). It is also can be used to
screen the clone library in a suitable vector (Kowalchuk et al. 1997). Determining
PCR and colony bias to know the error rate of the DNA polymerase in DNA
synthesis (Keohavong and Thilly 1989).
DGGE has a limitation in community screening because it can proceed the
sample with maximum size around 600 bp, so it does not contain a lot of
information for accurate identification. Application of GC clamp in the PCR
process is also sometimes producing primer dimers so variable of coloring gel can
be reduced.

METHODS

Research Framework
The research framework (Figure 2) generally included the sampling of rice
plant samples, DNA Isolation, PCR amplification, DGGE analysis, and
construction of phylogenetic tree.

10

Sampling

Surface Sterilization of Rice
Plant Samples

DNA Isolation

Amplification of 16S rRNA
for Bacteria

Amplification of 16S rRNA
Specific Actinomycetes

DGGE

DGGE

Statistical Analysis of DGGE Profile

Cloning and Sequencing of DGGE Band

Construction of Phylogenetic Tree

Analysis of Sequence Variation

Figure 2 Research framework

Time and Place
The research was conducted in June 2014 until March 2015 in the
Laboratory of Microbiology, Department of Biology, Faculty of Mathematics and
Natural Science, IPB, and Ecological Chemistry Laboratory, Faculty of
Agriculture, Hokkaido University.

Sample Collection and Sterilization
Sampling site was conducted in Bogor and Cianjur, West Java, Indonesia.
The part of plant samples (root, stem, and leaf) were collected from four healthy

11

rice cultivars (IR 64, Inpara 2, Situ Patenggang, and Ciherang) at vegetative state
(30 days old) (Mahyarudin 2014). The characteristic of agricultural soil of the
sampling site has been analyzed from previous study (Mahyarudin 2014) and
presented in Table 5 and 6. Surface sterilization of the samples was conducted
based on Coombs and Franco (2003), with modification. The samples were
washed by tap water to clean the surface part. About 0.5-1 g of each samples were
immersed in 70% ethanol solution for 1 minute, washed with sodium hypochlorite
(NaOCl) 1% for 5 minutes, rinsed with 70% ethanol for 1 minute, and finally
washed three times with sterile distilled water. Surface sterilization was validated
by spreading the last washing water on culture media and incubated for 1 month.
The samples that did not show any contamination were used for further analysis.
Table 5 Characteristics (I) of agricultural soil from sampling site (Mahyarudin
2014)
Texture
No
1
2
3

Sample
Irrigated
rice soil
Dry Soil
Tidal Soil

Organic matter

Sand

Ash

Clay

16

41

43

22
21

32
61

32
61

pH

HCL 25%

C (%)

N (%)

C/N

P205
(mg/100g)

K20
(mg/100g)

5.4

5.4

0.21

0.2

4.7

29

5.6
5.2

5.6
5.2

0.44
0.35

0.4
0.4

9.2
7.6

80
47

Table 6 Characteristics (II) of agricultural soil from sampling site (Mahyarudin
2014)
Ion Exchange Value
No
1
2
3

Name of
Sample
Irrigated
rice soil
Dry soil
Tidal soil

KCL/IN

Ca

Mg

K

Na

Value

CEC

BS+ (%)

A13+
(cmolc/Kg)

H+
(cmolc/Kg)

10.5

2.86

0.49

0.45

13.85

14.62

95

0.92

0.26

37.7
24.74

3.63
3.78

0.98
0.43

0.12
0.45

42.43
29.4

37.27
36.51

>100
81

0
0.43

0.02
0.22

DNA Isolation
DNA isolation of the roots, stems, and leaves of rice plants tissues was
done in accordance to the Genomic DNA Mini Kit (Plant) protocol (Geneaid,
Sintai, Taiwan). Total of 0.1-0.5 g sample of the roots, stems, or leaves that have
been sterilized, refined with the addition of liquid nitrogen and crushed into
powder. Samples that have been refined were transferred into 2 mL microtube and
added by 700 mL GP1 buffer solution and 5 mL RNase then homogenized for 1-2
minutes. The mixture was incubated at 60 °C for 15 minutes along with the
elution buffer. During incubation, microtubes were inverted every 5 minutes.
After incubation process, GP2 buffer was added to the mixture and homogenized.
After that, the mixture was incubated for 5 minutes in the ice. A mixture that has
been incubated was transferred to the filter column that has been put on the
collection tube and centrifuged at 1000 rpm for 1 minute. In the collection tube,
supernatant was transferred into a 1.5 mL microtube and added by GP3 buffer

12

isopropanol 1.5x volume of supernatant. After that, the mixture was homogenized
for 30 seconds. The total of 700 mL of the mixture was transferred to the GD
column that has been put on the collection tube, then centrifuged at 13000 rpm for
2 minutes. Supernatant in the collection tube was discarded. The process was
repeated until the mixture in microtube dried. The total of 400 mL W1 buffer was
added to the GD column and centrifuged at 13000 rpm for 30 seconds.
Supernatant in the collection tube was discarded. After that, 600 mL washing
buffer was added to the GD column and centrifuged at 13000 rpm for 30 seconds.
GD column was centrifuged again at 13000 rpm for 3 minutes to dry the matrix
volume. GD column that has been dried was put on the 1.5 mL microtube. A total
of 30 mL of elution buffer was added to the GD column and allowed up to 20
minutes and then centrifuged for 30 seconds at 13000 rpm for obtain total DNA.
The results of DNA isolation was electrophoresed on 1% gel agarose and
visualized.

Amplification of 16S rRNA Gene-specific Bacteria and Actinomycetes
PCR amplification of bacteria domain from root, stem, and leaf was done
using 799F-mod3/1389R primer set to amplify fragment size ±600 bp. The
reaction mixture was as follows: 10 µL Ampli-Taq Gold 360 (Applied
Biosystems, Carlsbad, CA), 0.4 µL of each primer (10 µmol), 1 µL of DNA
template (30 ng), and 8.2 µL sterile MiliQ water. The amplification was
performed in TAKARA Thermal Cycler (Takara, Dalian, China) using initial
denaturing of 1 minute at 95 oC, followed by 30 cycles of 30 seconds denaturing
at 95 oC, 1 minute annealing at 55 oC, and 1 minute extension at 72 oC, completed
by 7 minutes final extension at 72 oC.
PCR amplification of actinomycetes from leaf and stem was done by twostage PCR strategy. Firstly, primer set 27F/16Sact1114R was used to amplify
fragment size ±1087 bp. Secondly, 1 µL product from the first PCR was used as a
template to amplify fragment size ±195 bp with primer set 338F-gc/518R. The
mixture for both PCR reaction was as follows: 10 µL Ampli-Taq Gold 360
(Applied Biosystems, Carlsbad, CA), 0.4 µL of each primer (10 µmol), 1 µL of
Table 7 List of the primers used in this study
No
1

Primer name
16Sact1114R

Sequence (5'->3')
GAGTTGACCCCGGCRGT

Ref.
Martina et al. 2008

2

System
Select for
actinomycetes
Universal

27F

AGAGTTTGATCCTGGCTCAG

Martina et al. 2008

3

Universal

P338F

ACTCCTACGGGAGGCAGCAG

Overeas et al. 1997

4

Universal

P518R

ATTACCGCGGCTGCTGG

Overeas et al. 1997

5

DGGE

P338F-gc

Overeas et al. 1997

6

Select for Bacteria

799F-mod3

CGCCCGCCGCGCGCGGCGGG
CGGG
GCGGGGGCACGGGGGGACTC
CTAC
GGGAGGCAGCAG
CMGGATTAGATACCCKGG

7

Universal

1389R

ACGGGCGGTGTGTACAAG

Hongoh et al. 2003

Hanshew et al. 2013

13

DNA template (30 ng), and 8.2 µL sterile MiliQ water. The amplification was
carried out using initial denaturing of 5 minutes at 94 oC, followed by 30 cycles of
1 minute denaturing at 94 oC, 30 seconds annealing at 55 oC, and 30 seconds
extension at 72 oC, completed by 3 minutes final extension at 72 oC.

Analysis and Cloning of DGGE Bands
DGGE was performed using Bio-Rad DCode system (Bio-Rad, Hercules,
CA, USA) in 6% and 8% (w/v) polyacrylamide gel (acrylamide-bisacrylamide,
37.5:1) with 30% to 70% denaturing concentrations for bacteria and
actinomycetes DGGE analysis, respectively (100% denaturant corresponding to
7M urea and 40% deionized formamide). Electrophoresis was performed at 100 V
and 60 °C for 16 hours in 1 × Tris-acetate-EDTA (TAE). The gel was stained by
15 µL of Sybr Gold dye (Molecular Probes, Invitrogen, Cergy Pontoise, France)
in 1 × TAE buffer (150 mL) for 30 minutes with dark conditions. Gel was
screened by using Typhoon imaging system (Amersham, Piscataway, NJ, USA).
Band profile image was analyzed using Phoretix 1D software (Nonlinear
Dynamics, Newcastle, UK) to estimate the total bands that appeared on
polyacrylamide gel.
Statistical analysis of DGGE profile was conducted by using alpha diversity
(Shannon-wiener/He) to estimate the diversity within each sample and beta
diversity (Dice similarity coefficient/SD) to estimate the similarity of band pattern
between the samples. The quantification of statistical analysis was conducted
using PAST Software (Hammer et al. 2001) based on the estimation analysis of
band intensity using Phoretix 1D software (Nonlinear Dynamics, Newcastle, UK).
The index was calculated by following equation:

Where, Na represented the number of bands detected in sample a; Nb represented
the number of bands detected in sample b; Nc represent