Molecular Identification of Fusarium spp. Associated with Eaglewood Based on Its Internal Transcribed Spacer Sequence
MOLECULAR IDENTIFICATION OF Fusarium spp.
ASSOCIATED WITH EAGLEWOOD BASED ON ITS
INTERNAL TRANSCRIBED SPACER SEQUENCE
RURI PRIHATINI ARIMBI
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT OF MINI-THESIS AND SOURCE OF
INFORMATION AND DEVOLVING COPYRIGHT *
I hereby declare that mini-thesis entitled Molecular Identification of
Fusarium spp. Associated with Eaglewood Based on Its Internal Transcribed
Spacer Sequence is a true work of me with the direction of the supervisor and has
not been submitted in the form of anything to any college. A source of
information derived or quoted of works issued or published or not published from
other authors mentioned in the text and listed in the bibliography at the end of this
dissertation.
I hereby assign copyright of my paper to the Bogor Agricultural University.
Bogor, January 2014
Ruri Prihatini Arimbi
NIM G34060327
2
ABSTRACT
RURI PRIHATINI ARIMBI. Molecular Identification of Fusarium spp.
Associated with Eaglewood Based on Its Internal Transcribed Spacer Sequence.
Under supervision GAYUH RAHAYU and UTUT WIDYASTUTI.
Eaglewood was thought to be formed through fungal infection. Acremonium
and Fusarium were able to induce symptom of eaglewood formation in 2 year-old
eaglewood trees (Aquilaria crassna, A. malaccensis, and A. microcarpa). The
objective of this research is to identify nine fungal isolates from eaglewood that
morphological character tentatively determined belonging to Acremonium and
Fusarium from eaglewood by molecular approach using its Internal Transcribed
Spacer region. The primers ITS 1 and ITS 4 had successfully amplified the ITS
region (500-700 bp) of all investigated strains. Fungal isolates from eaglewood
comprises of 1 isolate Fusarium acaciae mearnsii, 1 isolate Fusarium oxysporum,
6 isolate Fusarium solani complex and 1 isolate Fusarium solani fsp. Batatas.
The molecular identification was not supported the morphological identification.
Based on molecular analysis, the Acremonium-like morphological genus was
apparently Fusarium sp. in the Section Martiella sensu Booth 1971.
Keywords: eaglewood clump, Fusarium, ITS primer.
ABSTRAK
RURI PRIHATINI ARIMBI. Molecular Identification of Fusarium spp.
Associated with Eaglewood Based on Its Internal Transcribed Spacer Sequence.
Dibimbing oleh GAYUH RAHAYU dan UTUT WIDYASTUTI.
Gaharu dapat terbentuk melalui infeksi cendawan. Acremonium dan
Fusarium mampu menyebabkan gejala pembentukan kayu gaharu di pohon-pohon
gaharu berusia 2 tahun (Aquilaria crassna, A. malaccensis, dan A. microcarpa).
Tujuan dari penelitian ini ialah mengidentifikasi 9 isolat cendawan asal pohon
gaharu yang secara morfologi termasuk Acremonium dan Fusarium melalui
pendekatan molekuler berdasarkan daerah Internal Transcribe Spacer-nya.
Daerah ITS (500-700 bp) semua isolat telah berhasil diamplifikasi oleh primer
ITS 1 dan ITS 4. Cendawan yang berasal dari gaharu terdiri dari 1 isolat Fusarium
acaciae mearnsii, 1 isolat Fusarium oxysporum, 6 isolat Fusarium solani complex
dan 1 isolat Fusarium solani fsp. Batatas. Identifikasi molekuler tidak
mendukung identifikasi sementara cendawan secara morfologi. Berdasarkan
analisis molekuler cendawan yang secara morfologi mirip Acremonium adalah
Fusarium dalam seksi Martiella sensu Booth 1971.
Kata kunci: gaharu, Fusarium, primer ITS.
3
MOLECULAR IDENTIFICATION OF Fusarium spp.
ASSOCIATED WITH EAGLEWOOD BASED ON ITS
INTERNAL TRANSCRIBED SPACER SEQUENCE
RURI PRIHATINI ARIMBI
An Undergraduate Thesis
Intended to Acquire Bachelor Degree
In
Departement of Biologi
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
4
5
Title
Name
NIM
: Molecular Identification of Fusarium spp. Associated with
Eaglewood Based on Its Internal Transcribed Spacer Sequence
: Ruri Prihatini Arimbi
: G34060327
Approved by
Dr Ir Gayuh Rahayu
Supervisor I
Dr Ir Utut Widyastuti, MSi
Supervisor II
Endorsed by
Dr Ir Iman Rusmana, M. Si
Head of Department
Graduation Date:
6
7
PREFACE
This mini-thesis is made through a research entitled Molecular Identification
of Fusarium spp. Associated with Eaglewood Based on Its Internal Transcribed
Spacer Sequence. It is intended to confirm the identification that was made by
morphological approach. Hopefully, it might be useful information for readers and
science development especially in eaglewood project.
I would firstly thank to Dr. Gayuh Rahayu and Dr. Ir. Utut Widyastuti, MSi.
as my supervisors. They gave me a lot of trust and flexibility on the project so I
could dealt with such a challenging project, I could collect data in a short time and
above all complete this project. I am thanking them for the great support.
I would also like to thank to all staffs and lecturers in RCBio and IPBCC for
all knowledge and time they shared to me. To all Biology 43, thank you for all the
best time we spent together. At last, I would like to sent acknowledgement to Bu
Sri Listyowati and to Uncle Jo for helping me personally in facing all the
problems while making this mini-thesis.
Bogor, November 2013
Ruri Prihatini Arimbi
8
CONTENT
Page
INTRODUCTION
Background ................................................................................................ 9
Objective .................................................................................................... 10
Research hypotheses .................................................................................. 10
METHODS
Materials ..................................................................................................... 10
Equipment .................................................................................................. 11
Data Analysis Procedures .......................................................................... 11
Maintenance of working culture ................................................................ 11
DNA extraction and PCR amplification .................................................... 11
Electrophoresis ........................................................................................... 12
DNA sequencing and Analysis ................................................................... 12
Time and Place ........................................................................................... 12
RESULT AND DISCUSSION ............................................................................. 12
CONCLUSION ................................................................................................... 16
REFERENCES .................................................................................................... 16
CURRICULUM VITAE ..................................................................................... 18
LIST OF FIGURE
Page
1 Microscopic features of conidial heads of Acremonium IPBCC 07.563 .......... 13
2 The position of Internal Transcribed Spacer gene segment .............................. 13
3 Electrophoresis DNA fragment of nine fungal isolates from eaglewood .......... 13
3 Molecular phylogenetic tree .............................................................................. 15
LIST OF TABLE
Page
1 Strain isolates from eaglewood and the references were used in this study ...... 10
2 Fungal isolates from eaglewood identification result based on ITS region ...... 14
9
INTRODUCTION
Background
Eaglewood is a non-timber forest product with high economy value
especially from its fragrant resin deposit. This resinuous substance is accumulated
in wood tissue as a response toward wounding or pathogens infection. According
to Nobuchi and Siripatanadilok (1991), eaglewood clump was thought to be
formed through fungal infection. Various species of Fusarium such as Fusarium
oxysporum, Fusarium bulbigenium, and Fusarium lateritium have been isolated
by Santoso (1996). Rahayu et al. (1999) stated that several isolates of
Acremonium sp. From eaglewood clump of Gyrinops versteegii and Aquilaria
malaccensis were able to induce symptom of clumps formation in 2 year-old
eaglewood trees (Aquilaria crassna, Aquilaria malaccensis, and Aquilaria
microcarpa). IPB Culture Collection (IPBCC) has a number of collection of
mitosporic fungi from eaglewood that have been identified tentatively on
morphological bases to either Acremonium or Fusarium (Gayuh Rahayu, personal
communication 14 February 2013).
Acremonium and Fusarium are closely related genera which are
differentiated on the bases of the presence of macroconidia. Acremonium lacks
macroconidia and Fusarium have macroconidia. The genus Acremonium
(Summerbell et al. 2011) includes some of the most simply structured of all
filamentous anamorphic fungi. The characteristic morphology consists of septate
hyphae giving rise to thin, tapered, mostly lateral phialides produced singly or in
small groups conidia. Conidia tend to be unicellular, produced in mucoid heads or
unconnected chains. They can be hyaline or melanised, but the hyphae are usually
hyaline. Colonies can produce conidia in less than a week in a warm, moist
environment, making Acremonium a very fast growing fungus. The colony
typically has a slightly powdery texture, and a color which can vary from gray to
pink.
Fusarium colonies are also usually fast growing, pale or brightly colored
(depending on the species) and may or may not have a cottony aerial mycelium.
The color of the mycelia varies from whitish to yellow, brownish, pink, reddish or
lilac shades. These fungi is a large genus and widely distributed in soil and in
association with plants. Most species are harmless saprobes and relatively
abundant members of the soil microbial community, but some are phytopatogenic
and human pathogens (Summerbell and Schroers 2002).
Using morphotypic characters are apparently difficult to identify the isolates
from eaglewood to specific level. Choi et al. (1999) stated that cultural
characteristics may be useful and have traditionally been fundamental. However,
currently morphological identification often mislead Therefore, morphological
identification of Acremonium and Fusarium up to specific level are usually
supported by molecular identification using its ITS (Internal Transcribe Spacer)
sequence. The ITS regions of fungal ribosomal DNA are highly variable
sequences of great importance in distinguishing fungal species by PCR analysis.
Summerbell and Schroers (2002) found that Acremonium falciforme is
10
phylogenetically considered as a variant of Fusarium solani complex based on
their 28S rDNA.
Accurate identification of these fungi is, therefore, essential prior to broad
application of these fungi for artificial inoculation and provide IPBCC with
information for building up a molecular reference for fungal identification.
Objective
The objective of this research is to identify to species level the tentatively
determined fungal culture belonging to Acremonium and Fusarium from
eaglewood by molecular approach.
Research Hypotheses
Acremonium and Fusarium are not identifiable to specific level by
conventional methods or morphological character. Accurate identification using
molecular approach of these fungi is essential to determine appropriate phylogenic
and discover its genetic information.
METHODS
Materials
Materials used in this research were 9 fungal isolates from eaglewood
(Table 1) and 1 isolate of Fusarium oxysporum from Cucumis sativus IPBCC
88.012 (CBS 254.52), PDA (Potato Dextrose Agar) media, PDB (Potato Dextrose
Broth) media, alcohol (70%), antibiotics cloramfenicol, chemical for DNA
extraction, primers ITS 1 (reverse primer) and ITS 4 (forward primers).
Table 1. Strain isolates from eaglewood and the references were used in this study
ITS
No
.
1
2
3
4
5
6
7
8
9
Accession
Number
IPBCC 07.525
IPBCC 07.526
IPBCC 08.563
IPBCC 08.565
IPBCC 08.566
IPBCC 08.561
IPBCC 08.568
IPBCC 08.569
IPBCC 08.570
Name of Isolates
Fusarium sp.
Fusarium sp.
Acremonium-like
Acremonium-like
Acremonium-like
Acremonium-like
Fusarium sp.
Fusarium sp.
Fusarium sp.
Origin
Lombok
West Papua
Central Bangka
Central Bangka
Central Bangka
Sukabumi
Central Bangka
Central Bangka
Genebank
Accession
Number
-
11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
IPBCC
Isolate By125
NRRL 42499
NRRL 13459
NRRL 26419
NRRL34207
NRRL 52789
ISPaVe 2010
NRRL 34033
NRRL 28505
NRRL 34014
CBS 116522
CBS 116521
CBS 108944
CBS 110138
CBS 318.34
FMR 8038
NRRL 22400
CBS 109028
CBS 117481
FRC S-2438
CNUN180
P266_D1_10
Isolate 1125
Fusarium oxysporum
F.oxysporum
F. oxysporum
F. concolor
F.equiseti
Fusarium poae
F. acaciae mearnsii
F. acuminatum
F. lateritium
F. brachygibbosum
F. nelsonii
Fusarium sp.
F. delphinoides
F. dimerum
F. dimerum
F. biseptatum
F. penzigii
F. solani
F. solani f. batatas
Fusarium cf. solani
Fusarium cf. solani
Fusarium cf. solani
T. harzianum
Acremonium furcatum
A. atrogriseum
Cucumis sativus
-
GQ365156.1
DQ790539.1
GQ505763.1
GQ505688.1
JQ912669.1
DQ459854.1
JF740933.1
FN547465.1
GQ505450.1
GQ505436.1
GQ505441.1
EU926242.1
EU926259.1
JQ434586.1
EU926251.1
EU926258.1
AM412618.1
AF178407.1
JX435216.1
JX435205.1
JN235324.1
HM769735.1
JF311914.1
JX847767.1
Equipment
Equipments used in this research i.e. a compound microscope, bio-safety
cabinet, autoclave, micropipettes, electrophoresis and PCR machine.
Data Analysis Procedures
Maintenance of Working Culture
Culture stock of Acremonium sp. and Fusarium sp. were subcultured onto
PDA and incubated at room temperature for five to seven days. These cultures
were then used to obtain single spore cultures. A small piece of mycelium
originating from a single spore was then cut off and transferred to new PDA plates
to obtain working cultures. From each working cultures, three pieces of mycelia
plug were transferred on to PDB and incubated for 3 days in shaker incubator.
Actively growing mycelia were used as a source of DNA genom.
DNA Extraction and PCR Amplification
DNA genome was extracted from mycelia using CTAB method of
Sambrook et al. (1989) with modification in PVP (Polivinil Pirolidon) 1%.
Genomic DNA were amplified using MJ Research PCR thermal cycle machine
with ITS1 (5’-CTTGGTCATTTAG AGGAGTAA-3’) and ITS4 (5’-CAGGAG
ACTTGTACACGGTCCAG-3’) primers. The PCR mixture composed of dNTP
12
mix 2mM 1 µL, TE buffer 1 µL, MgCl 0.2 µL, and Taq DNA Polymerase enzyme
0.1 µL, 0.25 µL primers and 0.5 µL DNA template. Amplification PCR was
performed for 35 cycles with an initial denaturation period of 3 minutes at 94oC,
denaturation period of 30 seconds at 94oC, annealing for 30 seconds, at 55oC,
elongation for 1 minute, at 72oC, and final extension period for 5 minutes at
72oC.
Electrophoresis
The DNA product from PCR was electrophoresed on 1% agarose gel. In
addition, 10 µL PCR product were mixed in 2 L loading dye which contains
charged molecules which simulates DNA fragments of a certain size and length. It
was placed in the well of an electrophoresis gel. Electrophoresis was run at 70 volt
for 45 minutes. In order to confirm insert size based on DNA marker, the PCR
product was run side by side on the same gel. Gel was stained in ethidium
bromide to visualize the DNA using UV transluminator and it was documented
using GelDoc.
DNA Sequencing and Analysis
PCR product was sent to PT. Science Genetika for sequencing. Sequences
for ITS were edited and aligned in MEGA V (Molecular Evolutionary Genetic
Analysis) V. Phylogenetic construction were made included the reference strain of
Fusarium sp. from the genebank (Table 1) using Maximum likelihood in MEGA
V. Acremonium atrogriseum, Acremonium furcatum and Trichoderma harzianum
were used as an outgroup taxa. The robustness of the phylogenetic tree was
estimate bootstrap analysis with 1.000 replications. The tree was drawn in Adobe
Reader.
Time and Place
This research was conducted from May 2010 until July 2011 in Research
Center for Bioresources and Biotechnology (RCBio), Bogor Agricultural
University.
RESULT AND DISCUSSION
Isolate IPBCC 08.568 and IPBCC 08.570 were typically has Fusarium
characteristics in which the macroconidia and microconidia are present, with a
few macroconidia. Whilst the microscopic characters of the other isolates are
typically Acremonium like (Figure 1). They have no macroconidia. The
microconidia of these isolates are produced in a slimy head of a simple phialide.
This trait closed to Fusarium solani in the Section Martiella of Booth (1971).
Such confusion was also discussed by Summerbell and Schroers (2002) on
Acremonium falciformae .
13
Conidial head
4 µm
Fig 1. Microscopic features of conidial heads of Acremonium IPBCC 08.563
The molecular identification was not supported the morphological
identification. The primers had successfully amplified the ITS region about 500700 bp (Figure 2) of all investigated strains and the DNA fragment was visualized
using UV transluminator (Figure 3). All specimens either with or without
macroconidia have been identified as Fusarium. A similar finding of Summerbell
and Schroers (2002) was Acremonium falciforme is phylogenetically considered
as a variant of Fusarium solani complex based on their ITS sequences.
Fig 2. The position of Internal Transcribed Spacer gene segment (Lilley and
Chinabut 2000)
9
8
7
6
5
4
3
(600bp)
2
1
Marker (3530 bp)
Fig 3. Electrophoresis DNA fragment of nine fungal isolates from eaglewood
Tree topology (Figure 4) indicated that both Trichoderma and Acremonium
are good outgroup, since they formed separate clade with Fusarium. Within
Fusarium there are two clades i.e. mixture species of Fusarium as clade one with
60% bootstrap value and Fusarium solani complex as a clade two with 61%.
14
Clade one can be divided into several subclades. Amongst those is Fusarium
oxysporum complex.
In the clade one, Fusarium solani FMR 8C38 seemed to be misplaced, but
actually may not. One strain (IPBCC 07.526) that is Fusarium acaciae mearnsii
with 100% bootsrap value. These situation indicated that ITS is not a good region
to differentiate species of Fusarium. For this purpose, systematicians use Tef-1α
for molecular classification of Fusarium such as FusariumID (Geiser et al. 2004,
Hsua et al. 2011).
Most of the strains i.e. IPBCC 07.525, IPBCC 08.561. IPBCC 07.563,
IPBCC 07.566, IPBCC 07.568, IPBCC 08.569 and IPBCC 08.570 belonged to
Fusarium solani complex. and another strain, IPBCC 07.563 in same species
complex belongs to Fusarium solani fsp. batatas. Two strains (IPBCC 07.565,
IPBCC 88.012) belonged to Fusarium oxysporum complex with 98% bootstrap
value. This identification might be true until Tef-1α is made. This analysis
indicated that the Fusarium from eaglewood comprises of Fusarium acaciae
mearnsii, Fusarium oxysporum, Fusarium solani complex and Fusarium solani
fsp. batatas.
Table 2. Fungal isolates from eaglewood identification result based on ITS region
No
1
2
3
4
5
6
7
8
9
Accession
Number
IPBCC 07.525
IPBCC 07.526
IPBCC 08.563
IPBCC 08.565
IPBCC 08.566
IPBCC 08.561
IPBCC 08.568
IPBCC 08.569
IPBCC 08.570
Origin
Lombok
West papua
Central Bangka
Central Bangka
Central bangka
Sukabumi
Central Bangka
Central Bangka
Belonging to
Fusarium solani complex
Fusarium acasiae mearnsii
Fusarium solani f. batatas
Fusarium oxysporum
Fusarium solani complex
Fusarium solani complex
Fusarium solani complex
Fusarium solani complex
Fusarium solani complex
Sequence analysis of ITS gene showed two isolates namely IPBCC 07.565
and IPBCC 88.012 has the closest similarity of Fusarium oxysporum complex.
Fusarium oxysporum comprises a group of soil inhabitants that can exist as
saprophytes in the soil debris but also as pervasive plant endophytes colonizing
the plant roots. It stands in the ground as clamidiospora that can be found on the
roots of those who are ill Many strains of these species are pathogenic to plant
crops. The Fusarium oxysporum species complex causes catastrophic crop losses
in over 150 plant species and is considered one of the world’s most economically
significant soil borne plant pathogens. This species is, however, primarily nonpathogenic and forms endophytic, saprophytic, and latent pathogenic relationships
with its plant hosts (The Royal Botanical Garden 2013).
IPBCC 07.526 had similarity with Fusarium acaciae mearnsii NRRL 34207
with 100% bootstrap value. Bootstrap values measure how consistently the data
support given taxon bipartitions (Hedges 1992). High bootstrap values (close to
100%) nearly all of the characters informative for this group agree that it is a
group for a certain clade.
15
Figure 4. Molecular phylogenetic tree constructed on the bases of ITS1-ITS4
sequence. Bootstrap value >50% are shown at branches
Isolate IPBCC 07.563 has similarity with Fusarium solani f batatas NRRL
22400 with bootstrap value reached for 99%. It means both of these fungi can be
recognized as the same species. However, the other isolates can be recognized as
Fusarium solani complex though the bootstrap value showed low level (61%64%). It meant isolate IPBCC 08.561. IPBCC 07.563. IPBCC 07.566, IPBCC
07.568, IPBCC 08.569 and IPBCC 08.570 nearly close to Fusarium solani species
than any other Fusarium species. Fusarium solani is one of the most frequently
isolated fungi from soil and plant debris ubiquitous in soil and also decaying plant
material, where they act as decomposers. However, they are also host-specific
pathogens of a number of agriculturally important plants, including pea, cucurbits,
and sweet potato (Zhang et al, 2006).
Acremonium were used as an outgroup for these isolates. According to
Thomas et al. (2003), the similarities between Fusarium and Acremonium may
produce single-celled conidia of similar shapes on erect phialides, which were a
characteristic of the case isolate. While Fusarium also produces sickle-shaped
multicellular macroconidia in sporodochia, these are not always observed in the
laboratory. Colonies of Fusarium sp. often produce various shades of red, blue, or
purple, but these can be absent or subtle. Furthermore, Acremonium sp. may
produce similar pigments (Thomas et al. 2003).
Trichoderma were also used as an outgroup is a genus of asexually
reproducing fungi that are often the most frequently isolated soil and free-living
16
fungi that are highly interactive in root, soil, and foliar environment (Gary et al.
2004). They show a high level of genetic diversity, and can be used to produce a
wide range of products of commercial and ecological interest. They are prolific
producers of extracellular proteins, and are best known for their ability to produce
enzymes that degrade cellulose and chitin, although they also produce other useful
enzymes (Gary et al. 2004).
CONCLUSION
Molecular identification on the base of Internal Transcribed Spacer
indicated that nine fungal isolates from eaglewood comprises of 1 isolate
Fusarium acaciae mearnsii, 1 isolate Fusarium oxysporum, 6 isolates Fusarium
solani complex and 1 isolate Fusarium solani fsp. Batatas.
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Hsuan HM, Salleh B, and Zakaria L. 2011. molecular identification of Fusarium
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Gary et al. 2004. Trichoderma species — opportunistic, avirulent plant symbionts.
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TJ, Zhang N, Kuldau GA, and O’Donnell K. 2004. FUSARIUM-ID v. 1.0: A
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18
CURRICULUM VITAE
The author was born in Padang on June 17th 1988 as the first child of
Rumzi Jani and Tri Koryani. In 2006, the author was graduated from SMA Negeri
5 Kota Jambi and entered Department of Biology, Faculty of Mathematics and
Natural Science, Bogor Agricultural University. In 2008, she conducted Field
Study with the title ―Eksplorasi Rhizobakteria Asal Perakaran Legum dari
Kawasan Taman Wisata Alam Situ Gunung‖. In 200λ, she did field work with the
title ―Pembiakan Apantheles sp. sebagai Biokontrol Penggerek Batang pada
Tanaman Tebu‖ in PT. PG. Rajawali II at Subang city.
As an academia, author assisted practical class of Basic Biology in 2009
until 2010, Molecular Genetics in 2010, and Mycology in 2010. She became
finalist in PKM and in 2010 she also won the competition of student debate in
Faculty of Mathematic and Natural Science.
In non academic life, author joined Himabio as a grand visioner in period
2008-2009 and became a chief and coordinator of Save Our Surrounding (SOS)
event program in Himabio.
19
20
ASSOCIATED WITH EAGLEWOOD BASED ON ITS
INTERNAL TRANSCRIBED SPACER SEQUENCE
RURI PRIHATINI ARIMBI
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCE
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
STATEMENT OF MINI-THESIS AND SOURCE OF
INFORMATION AND DEVOLVING COPYRIGHT *
I hereby declare that mini-thesis entitled Molecular Identification of
Fusarium spp. Associated with Eaglewood Based on Its Internal Transcribed
Spacer Sequence is a true work of me with the direction of the supervisor and has
not been submitted in the form of anything to any college. A source of
information derived or quoted of works issued or published or not published from
other authors mentioned in the text and listed in the bibliography at the end of this
dissertation.
I hereby assign copyright of my paper to the Bogor Agricultural University.
Bogor, January 2014
Ruri Prihatini Arimbi
NIM G34060327
2
ABSTRACT
RURI PRIHATINI ARIMBI. Molecular Identification of Fusarium spp.
Associated with Eaglewood Based on Its Internal Transcribed Spacer Sequence.
Under supervision GAYUH RAHAYU and UTUT WIDYASTUTI.
Eaglewood was thought to be formed through fungal infection. Acremonium
and Fusarium were able to induce symptom of eaglewood formation in 2 year-old
eaglewood trees (Aquilaria crassna, A. malaccensis, and A. microcarpa). The
objective of this research is to identify nine fungal isolates from eaglewood that
morphological character tentatively determined belonging to Acremonium and
Fusarium from eaglewood by molecular approach using its Internal Transcribed
Spacer region. The primers ITS 1 and ITS 4 had successfully amplified the ITS
region (500-700 bp) of all investigated strains. Fungal isolates from eaglewood
comprises of 1 isolate Fusarium acaciae mearnsii, 1 isolate Fusarium oxysporum,
6 isolate Fusarium solani complex and 1 isolate Fusarium solani fsp. Batatas.
The molecular identification was not supported the morphological identification.
Based on molecular analysis, the Acremonium-like morphological genus was
apparently Fusarium sp. in the Section Martiella sensu Booth 1971.
Keywords: eaglewood clump, Fusarium, ITS primer.
ABSTRAK
RURI PRIHATINI ARIMBI. Molecular Identification of Fusarium spp.
Associated with Eaglewood Based on Its Internal Transcribed Spacer Sequence.
Dibimbing oleh GAYUH RAHAYU dan UTUT WIDYASTUTI.
Gaharu dapat terbentuk melalui infeksi cendawan. Acremonium dan
Fusarium mampu menyebabkan gejala pembentukan kayu gaharu di pohon-pohon
gaharu berusia 2 tahun (Aquilaria crassna, A. malaccensis, dan A. microcarpa).
Tujuan dari penelitian ini ialah mengidentifikasi 9 isolat cendawan asal pohon
gaharu yang secara morfologi termasuk Acremonium dan Fusarium melalui
pendekatan molekuler berdasarkan daerah Internal Transcribe Spacer-nya.
Daerah ITS (500-700 bp) semua isolat telah berhasil diamplifikasi oleh primer
ITS 1 dan ITS 4. Cendawan yang berasal dari gaharu terdiri dari 1 isolat Fusarium
acaciae mearnsii, 1 isolat Fusarium oxysporum, 6 isolat Fusarium solani complex
dan 1 isolat Fusarium solani fsp. Batatas. Identifikasi molekuler tidak
mendukung identifikasi sementara cendawan secara morfologi. Berdasarkan
analisis molekuler cendawan yang secara morfologi mirip Acremonium adalah
Fusarium dalam seksi Martiella sensu Booth 1971.
Kata kunci: gaharu, Fusarium, primer ITS.
3
MOLECULAR IDENTIFICATION OF Fusarium spp.
ASSOCIATED WITH EAGLEWOOD BASED ON ITS
INTERNAL TRANSCRIBED SPACER SEQUENCE
RURI PRIHATINI ARIMBI
An Undergraduate Thesis
Intended to Acquire Bachelor Degree
In
Departement of Biologi
DEPARTMENT OF BIOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
4
5
Title
Name
NIM
: Molecular Identification of Fusarium spp. Associated with
Eaglewood Based on Its Internal Transcribed Spacer Sequence
: Ruri Prihatini Arimbi
: G34060327
Approved by
Dr Ir Gayuh Rahayu
Supervisor I
Dr Ir Utut Widyastuti, MSi
Supervisor II
Endorsed by
Dr Ir Iman Rusmana, M. Si
Head of Department
Graduation Date:
6
7
PREFACE
This mini-thesis is made through a research entitled Molecular Identification
of Fusarium spp. Associated with Eaglewood Based on Its Internal Transcribed
Spacer Sequence. It is intended to confirm the identification that was made by
morphological approach. Hopefully, it might be useful information for readers and
science development especially in eaglewood project.
I would firstly thank to Dr. Gayuh Rahayu and Dr. Ir. Utut Widyastuti, MSi.
as my supervisors. They gave me a lot of trust and flexibility on the project so I
could dealt with such a challenging project, I could collect data in a short time and
above all complete this project. I am thanking them for the great support.
I would also like to thank to all staffs and lecturers in RCBio and IPBCC for
all knowledge and time they shared to me. To all Biology 43, thank you for all the
best time we spent together. At last, I would like to sent acknowledgement to Bu
Sri Listyowati and to Uncle Jo for helping me personally in facing all the
problems while making this mini-thesis.
Bogor, November 2013
Ruri Prihatini Arimbi
8
CONTENT
Page
INTRODUCTION
Background ................................................................................................ 9
Objective .................................................................................................... 10
Research hypotheses .................................................................................. 10
METHODS
Materials ..................................................................................................... 10
Equipment .................................................................................................. 11
Data Analysis Procedures .......................................................................... 11
Maintenance of working culture ................................................................ 11
DNA extraction and PCR amplification .................................................... 11
Electrophoresis ........................................................................................... 12
DNA sequencing and Analysis ................................................................... 12
Time and Place ........................................................................................... 12
RESULT AND DISCUSSION ............................................................................. 12
CONCLUSION ................................................................................................... 16
REFERENCES .................................................................................................... 16
CURRICULUM VITAE ..................................................................................... 18
LIST OF FIGURE
Page
1 Microscopic features of conidial heads of Acremonium IPBCC 07.563 .......... 13
2 The position of Internal Transcribed Spacer gene segment .............................. 13
3 Electrophoresis DNA fragment of nine fungal isolates from eaglewood .......... 13
3 Molecular phylogenetic tree .............................................................................. 15
LIST OF TABLE
Page
1 Strain isolates from eaglewood and the references were used in this study ...... 10
2 Fungal isolates from eaglewood identification result based on ITS region ...... 14
9
INTRODUCTION
Background
Eaglewood is a non-timber forest product with high economy value
especially from its fragrant resin deposit. This resinuous substance is accumulated
in wood tissue as a response toward wounding or pathogens infection. According
to Nobuchi and Siripatanadilok (1991), eaglewood clump was thought to be
formed through fungal infection. Various species of Fusarium such as Fusarium
oxysporum, Fusarium bulbigenium, and Fusarium lateritium have been isolated
by Santoso (1996). Rahayu et al. (1999) stated that several isolates of
Acremonium sp. From eaglewood clump of Gyrinops versteegii and Aquilaria
malaccensis were able to induce symptom of clumps formation in 2 year-old
eaglewood trees (Aquilaria crassna, Aquilaria malaccensis, and Aquilaria
microcarpa). IPB Culture Collection (IPBCC) has a number of collection of
mitosporic fungi from eaglewood that have been identified tentatively on
morphological bases to either Acremonium or Fusarium (Gayuh Rahayu, personal
communication 14 February 2013).
Acremonium and Fusarium are closely related genera which are
differentiated on the bases of the presence of macroconidia. Acremonium lacks
macroconidia and Fusarium have macroconidia. The genus Acremonium
(Summerbell et al. 2011) includes some of the most simply structured of all
filamentous anamorphic fungi. The characteristic morphology consists of septate
hyphae giving rise to thin, tapered, mostly lateral phialides produced singly or in
small groups conidia. Conidia tend to be unicellular, produced in mucoid heads or
unconnected chains. They can be hyaline or melanised, but the hyphae are usually
hyaline. Colonies can produce conidia in less than a week in a warm, moist
environment, making Acremonium a very fast growing fungus. The colony
typically has a slightly powdery texture, and a color which can vary from gray to
pink.
Fusarium colonies are also usually fast growing, pale or brightly colored
(depending on the species) and may or may not have a cottony aerial mycelium.
The color of the mycelia varies from whitish to yellow, brownish, pink, reddish or
lilac shades. These fungi is a large genus and widely distributed in soil and in
association with plants. Most species are harmless saprobes and relatively
abundant members of the soil microbial community, but some are phytopatogenic
and human pathogens (Summerbell and Schroers 2002).
Using morphotypic characters are apparently difficult to identify the isolates
from eaglewood to specific level. Choi et al. (1999) stated that cultural
characteristics may be useful and have traditionally been fundamental. However,
currently morphological identification often mislead Therefore, morphological
identification of Acremonium and Fusarium up to specific level are usually
supported by molecular identification using its ITS (Internal Transcribe Spacer)
sequence. The ITS regions of fungal ribosomal DNA are highly variable
sequences of great importance in distinguishing fungal species by PCR analysis.
Summerbell and Schroers (2002) found that Acremonium falciforme is
10
phylogenetically considered as a variant of Fusarium solani complex based on
their 28S rDNA.
Accurate identification of these fungi is, therefore, essential prior to broad
application of these fungi for artificial inoculation and provide IPBCC with
information for building up a molecular reference for fungal identification.
Objective
The objective of this research is to identify to species level the tentatively
determined fungal culture belonging to Acremonium and Fusarium from
eaglewood by molecular approach.
Research Hypotheses
Acremonium and Fusarium are not identifiable to specific level by
conventional methods or morphological character. Accurate identification using
molecular approach of these fungi is essential to determine appropriate phylogenic
and discover its genetic information.
METHODS
Materials
Materials used in this research were 9 fungal isolates from eaglewood
(Table 1) and 1 isolate of Fusarium oxysporum from Cucumis sativus IPBCC
88.012 (CBS 254.52), PDA (Potato Dextrose Agar) media, PDB (Potato Dextrose
Broth) media, alcohol (70%), antibiotics cloramfenicol, chemical for DNA
extraction, primers ITS 1 (reverse primer) and ITS 4 (forward primers).
Table 1. Strain isolates from eaglewood and the references were used in this study
ITS
No
.
1
2
3
4
5
6
7
8
9
Accession
Number
IPBCC 07.525
IPBCC 07.526
IPBCC 08.563
IPBCC 08.565
IPBCC 08.566
IPBCC 08.561
IPBCC 08.568
IPBCC 08.569
IPBCC 08.570
Name of Isolates
Fusarium sp.
Fusarium sp.
Acremonium-like
Acremonium-like
Acremonium-like
Acremonium-like
Fusarium sp.
Fusarium sp.
Fusarium sp.
Origin
Lombok
West Papua
Central Bangka
Central Bangka
Central Bangka
Sukabumi
Central Bangka
Central Bangka
Genebank
Accession
Number
-
11
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
IPBCC
Isolate By125
NRRL 42499
NRRL 13459
NRRL 26419
NRRL34207
NRRL 52789
ISPaVe 2010
NRRL 34033
NRRL 28505
NRRL 34014
CBS 116522
CBS 116521
CBS 108944
CBS 110138
CBS 318.34
FMR 8038
NRRL 22400
CBS 109028
CBS 117481
FRC S-2438
CNUN180
P266_D1_10
Isolate 1125
Fusarium oxysporum
F.oxysporum
F. oxysporum
F. concolor
F.equiseti
Fusarium poae
F. acaciae mearnsii
F. acuminatum
F. lateritium
F. brachygibbosum
F. nelsonii
Fusarium sp.
F. delphinoides
F. dimerum
F. dimerum
F. biseptatum
F. penzigii
F. solani
F. solani f. batatas
Fusarium cf. solani
Fusarium cf. solani
Fusarium cf. solani
T. harzianum
Acremonium furcatum
A. atrogriseum
Cucumis sativus
-
GQ365156.1
DQ790539.1
GQ505763.1
GQ505688.1
JQ912669.1
DQ459854.1
JF740933.1
FN547465.1
GQ505450.1
GQ505436.1
GQ505441.1
EU926242.1
EU926259.1
JQ434586.1
EU926251.1
EU926258.1
AM412618.1
AF178407.1
JX435216.1
JX435205.1
JN235324.1
HM769735.1
JF311914.1
JX847767.1
Equipment
Equipments used in this research i.e. a compound microscope, bio-safety
cabinet, autoclave, micropipettes, electrophoresis and PCR machine.
Data Analysis Procedures
Maintenance of Working Culture
Culture stock of Acremonium sp. and Fusarium sp. were subcultured onto
PDA and incubated at room temperature for five to seven days. These cultures
were then used to obtain single spore cultures. A small piece of mycelium
originating from a single spore was then cut off and transferred to new PDA plates
to obtain working cultures. From each working cultures, three pieces of mycelia
plug were transferred on to PDB and incubated for 3 days in shaker incubator.
Actively growing mycelia were used as a source of DNA genom.
DNA Extraction and PCR Amplification
DNA genome was extracted from mycelia using CTAB method of
Sambrook et al. (1989) with modification in PVP (Polivinil Pirolidon) 1%.
Genomic DNA were amplified using MJ Research PCR thermal cycle machine
with ITS1 (5’-CTTGGTCATTTAG AGGAGTAA-3’) and ITS4 (5’-CAGGAG
ACTTGTACACGGTCCAG-3’) primers. The PCR mixture composed of dNTP
12
mix 2mM 1 µL, TE buffer 1 µL, MgCl 0.2 µL, and Taq DNA Polymerase enzyme
0.1 µL, 0.25 µL primers and 0.5 µL DNA template. Amplification PCR was
performed for 35 cycles with an initial denaturation period of 3 minutes at 94oC,
denaturation period of 30 seconds at 94oC, annealing for 30 seconds, at 55oC,
elongation for 1 minute, at 72oC, and final extension period for 5 minutes at
72oC.
Electrophoresis
The DNA product from PCR was electrophoresed on 1% agarose gel. In
addition, 10 µL PCR product were mixed in 2 L loading dye which contains
charged molecules which simulates DNA fragments of a certain size and length. It
was placed in the well of an electrophoresis gel. Electrophoresis was run at 70 volt
for 45 minutes. In order to confirm insert size based on DNA marker, the PCR
product was run side by side on the same gel. Gel was stained in ethidium
bromide to visualize the DNA using UV transluminator and it was documented
using GelDoc.
DNA Sequencing and Analysis
PCR product was sent to PT. Science Genetika for sequencing. Sequences
for ITS were edited and aligned in MEGA V (Molecular Evolutionary Genetic
Analysis) V. Phylogenetic construction were made included the reference strain of
Fusarium sp. from the genebank (Table 1) using Maximum likelihood in MEGA
V. Acremonium atrogriseum, Acremonium furcatum and Trichoderma harzianum
were used as an outgroup taxa. The robustness of the phylogenetic tree was
estimate bootstrap analysis with 1.000 replications. The tree was drawn in Adobe
Reader.
Time and Place
This research was conducted from May 2010 until July 2011 in Research
Center for Bioresources and Biotechnology (RCBio), Bogor Agricultural
University.
RESULT AND DISCUSSION
Isolate IPBCC 08.568 and IPBCC 08.570 were typically has Fusarium
characteristics in which the macroconidia and microconidia are present, with a
few macroconidia. Whilst the microscopic characters of the other isolates are
typically Acremonium like (Figure 1). They have no macroconidia. The
microconidia of these isolates are produced in a slimy head of a simple phialide.
This trait closed to Fusarium solani in the Section Martiella of Booth (1971).
Such confusion was also discussed by Summerbell and Schroers (2002) on
Acremonium falciformae .
13
Conidial head
4 µm
Fig 1. Microscopic features of conidial heads of Acremonium IPBCC 08.563
The molecular identification was not supported the morphological
identification. The primers had successfully amplified the ITS region about 500700 bp (Figure 2) of all investigated strains and the DNA fragment was visualized
using UV transluminator (Figure 3). All specimens either with or without
macroconidia have been identified as Fusarium. A similar finding of Summerbell
and Schroers (2002) was Acremonium falciforme is phylogenetically considered
as a variant of Fusarium solani complex based on their ITS sequences.
Fig 2. The position of Internal Transcribed Spacer gene segment (Lilley and
Chinabut 2000)
9
8
7
6
5
4
3
(600bp)
2
1
Marker (3530 bp)
Fig 3. Electrophoresis DNA fragment of nine fungal isolates from eaglewood
Tree topology (Figure 4) indicated that both Trichoderma and Acremonium
are good outgroup, since they formed separate clade with Fusarium. Within
Fusarium there are two clades i.e. mixture species of Fusarium as clade one with
60% bootstrap value and Fusarium solani complex as a clade two with 61%.
14
Clade one can be divided into several subclades. Amongst those is Fusarium
oxysporum complex.
In the clade one, Fusarium solani FMR 8C38 seemed to be misplaced, but
actually may not. One strain (IPBCC 07.526) that is Fusarium acaciae mearnsii
with 100% bootsrap value. These situation indicated that ITS is not a good region
to differentiate species of Fusarium. For this purpose, systematicians use Tef-1α
for molecular classification of Fusarium such as FusariumID (Geiser et al. 2004,
Hsua et al. 2011).
Most of the strains i.e. IPBCC 07.525, IPBCC 08.561. IPBCC 07.563,
IPBCC 07.566, IPBCC 07.568, IPBCC 08.569 and IPBCC 08.570 belonged to
Fusarium solani complex. and another strain, IPBCC 07.563 in same species
complex belongs to Fusarium solani fsp. batatas. Two strains (IPBCC 07.565,
IPBCC 88.012) belonged to Fusarium oxysporum complex with 98% bootstrap
value. This identification might be true until Tef-1α is made. This analysis
indicated that the Fusarium from eaglewood comprises of Fusarium acaciae
mearnsii, Fusarium oxysporum, Fusarium solani complex and Fusarium solani
fsp. batatas.
Table 2. Fungal isolates from eaglewood identification result based on ITS region
No
1
2
3
4
5
6
7
8
9
Accession
Number
IPBCC 07.525
IPBCC 07.526
IPBCC 08.563
IPBCC 08.565
IPBCC 08.566
IPBCC 08.561
IPBCC 08.568
IPBCC 08.569
IPBCC 08.570
Origin
Lombok
West papua
Central Bangka
Central Bangka
Central bangka
Sukabumi
Central Bangka
Central Bangka
Belonging to
Fusarium solani complex
Fusarium acasiae mearnsii
Fusarium solani f. batatas
Fusarium oxysporum
Fusarium solani complex
Fusarium solani complex
Fusarium solani complex
Fusarium solani complex
Fusarium solani complex
Sequence analysis of ITS gene showed two isolates namely IPBCC 07.565
and IPBCC 88.012 has the closest similarity of Fusarium oxysporum complex.
Fusarium oxysporum comprises a group of soil inhabitants that can exist as
saprophytes in the soil debris but also as pervasive plant endophytes colonizing
the plant roots. It stands in the ground as clamidiospora that can be found on the
roots of those who are ill Many strains of these species are pathogenic to plant
crops. The Fusarium oxysporum species complex causes catastrophic crop losses
in over 150 plant species and is considered one of the world’s most economically
significant soil borne plant pathogens. This species is, however, primarily nonpathogenic and forms endophytic, saprophytic, and latent pathogenic relationships
with its plant hosts (The Royal Botanical Garden 2013).
IPBCC 07.526 had similarity with Fusarium acaciae mearnsii NRRL 34207
with 100% bootstrap value. Bootstrap values measure how consistently the data
support given taxon bipartitions (Hedges 1992). High bootstrap values (close to
100%) nearly all of the characters informative for this group agree that it is a
group for a certain clade.
15
Figure 4. Molecular phylogenetic tree constructed on the bases of ITS1-ITS4
sequence. Bootstrap value >50% are shown at branches
Isolate IPBCC 07.563 has similarity with Fusarium solani f batatas NRRL
22400 with bootstrap value reached for 99%. It means both of these fungi can be
recognized as the same species. However, the other isolates can be recognized as
Fusarium solani complex though the bootstrap value showed low level (61%64%). It meant isolate IPBCC 08.561. IPBCC 07.563. IPBCC 07.566, IPBCC
07.568, IPBCC 08.569 and IPBCC 08.570 nearly close to Fusarium solani species
than any other Fusarium species. Fusarium solani is one of the most frequently
isolated fungi from soil and plant debris ubiquitous in soil and also decaying plant
material, where they act as decomposers. However, they are also host-specific
pathogens of a number of agriculturally important plants, including pea, cucurbits,
and sweet potato (Zhang et al, 2006).
Acremonium were used as an outgroup for these isolates. According to
Thomas et al. (2003), the similarities between Fusarium and Acremonium may
produce single-celled conidia of similar shapes on erect phialides, which were a
characteristic of the case isolate. While Fusarium also produces sickle-shaped
multicellular macroconidia in sporodochia, these are not always observed in the
laboratory. Colonies of Fusarium sp. often produce various shades of red, blue, or
purple, but these can be absent or subtle. Furthermore, Acremonium sp. may
produce similar pigments (Thomas et al. 2003).
Trichoderma were also used as an outgroup is a genus of asexually
reproducing fungi that are often the most frequently isolated soil and free-living
16
fungi that are highly interactive in root, soil, and foliar environment (Gary et al.
2004). They show a high level of genetic diversity, and can be used to produce a
wide range of products of commercial and ecological interest. They are prolific
producers of extracellular proteins, and are best known for their ability to produce
enzymes that degrade cellulose and chitin, although they also produce other useful
enzymes (Gary et al. 2004).
CONCLUSION
Molecular identification on the base of Internal Transcribed Spacer
indicated that nine fungal isolates from eaglewood comprises of 1 isolate
Fusarium acaciae mearnsii, 1 isolate Fusarium oxysporum, 6 isolates Fusarium
solani complex and 1 isolate Fusarium solani fsp. Batatas.
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Gary et al. 2004. Trichoderma species — opportunistic, avirulent plant symbionts.
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TJ, Zhang N, Kuldau GA, and O’Donnell K. 2004. FUSARIUM-ID v. 1.0: A
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18
CURRICULUM VITAE
The author was born in Padang on June 17th 1988 as the first child of
Rumzi Jani and Tri Koryani. In 2006, the author was graduated from SMA Negeri
5 Kota Jambi and entered Department of Biology, Faculty of Mathematics and
Natural Science, Bogor Agricultural University. In 2008, she conducted Field
Study with the title ―Eksplorasi Rhizobakteria Asal Perakaran Legum dari
Kawasan Taman Wisata Alam Situ Gunung‖. In 200λ, she did field work with the
title ―Pembiakan Apantheles sp. sebagai Biokontrol Penggerek Batang pada
Tanaman Tebu‖ in PT. PG. Rajawali II at Subang city.
As an academia, author assisted practical class of Basic Biology in 2009
until 2010, Molecular Genetics in 2010, and Mycology in 2010. She became
finalist in PKM and in 2010 she also won the competition of student debate in
Faculty of Mathematic and Natural Science.
In non academic life, author joined Himabio as a grand visioner in period
2008-2009 and became a chief and coordinator of Save Our Surrounding (SOS)
event program in Himabio.
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