Endophytic fungi of wild zingiberaceae from Mount Halimun Salak National Park

 
 
 

i 

ENDOPHYTIC FUNGI
OF WILD ZINGIBERACEAE FROM
MOUNT HALIMUN SALAK NATIONAL PARK

NICHO NURDEBYANDARU

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013

 

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iii 

STATEMENT

Hereby I declare this thesis entitled Endophytic Fungi of Wild
Zingiberaceae from Mount Halimun Salak National Park is my research
report that was carried out and written under guidance of my supervisory boards.
This thesis has not been submitted in any form to any higher education institution
except to Bogor Agricultural University (IPB). All information either originated
or cited from published or unpublished works of other author have been
acknowledged in the text of this thesis and listed in the reference. I also, hereby,
give my copyright to Bogor Agricultural University.

Ibaraki, September 2013

Nicho Nurdebyandaru
NIM G35111021

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SUMMARY

NICHO NURDEBYANDARU. Endophytic Fungi of Wild Zingiberaceae from Mount
Halimun Salak National Park. Under supervision of GAYUH RAHAYU, IMAN
HIDAYAT and KAZUHIKO NARISAWA.
The genus Colletotrichum, Diaporthe and Guignardia can live as plant
pathogens on a wide variety of tropical woody, herbaceous and graminicoulos plants
as well as fungal endophytes of many healthy plants. These two life styles are
apparently caused by their biotrophic life strategies as such that they live as
symptomless endophytes of living plant tissues. The species in Colletotrichum,
Diaporthe and Guignardia have been reported to cause serious diseases in various
economic plants in Indonesia. The exploration of these fungal endophytes was mainly
done on economic plants. Those from non-cultivated plants in natural habitats are
much less studied, with most studies being of endophytic strains. No reports are found

on fungal endophyte from wild Zingiberaceae plant, therefore it is necessary to
conduct research on the endophytic fungi on Zingiberaceae in order to provide
information related fungal endophyte of wild Zingiberaceae in Indonesia.
All fungal species recorded from Indonesia were mostly identified based on
morphological characters. In this research, isolates of the fungal endophyte was
collected by conventional isolation method. The isolates were then identified
molecularly based on their ITS1F-ITS4 sequence. These sequences were aligned with
MAFFT online. Phylogenetic analyses with all reference strains were performed using
MEGA5 to produce the phylogram. The position and bootstrap value for branch
support became the criteria of naming the isolates.
Eleven isolates of fungal endophytes were obtained from different part of four
species of Zingiberaceae at Mount Halimun Salak National Park (MHSNP) namely
Alpinia malaccensis, Ammomum gracile, Etlingera punicea and Zingiber odoriferum.
These consisted of nine isolates of Colletotrichum spp., one each isolate of Diaporthe
infecunda and Guignardia mangiferae. Three of Colletotrichum isolates were belong
to gloeosporioides clade, and six others were separate and made its own clade. The
Colletotrichum species distributed widely on those four plant species at different plant
part, one isolate of Colletotrichum kahawae subsp. kahawae from root of Etlingera
punicea, one isolate of Colletotrichum aff. siamense from root of Alpinia
malaccensis, and seven isolates of unidentified Colletotrichum species either from

root of Alpinia malaccensis, pseudostem and leaf of Ammomum gracile, and from
root of Zingiber odoriferum. One isolate of Diaporthe infecunda was from
pseudostem of Alpinia malaccensis and one isolate Guignardia mangiferae was from
pseudostem of Zingiber odoriferum. All of this finding is new records on fungal
endophytes from wild species of Zingiberaceae and may contribute to the
management of domestication of these plants.
Keywords: endophyte, ITS, Zingiberaceae

 
 

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RINGKASAN

NICHO NURDEBYANDARU. Fungi Endofit dari Zingiberaceae liar asal Taman
Nasional Gunung Halimun Salak. Dibimbing oleh GAYUH RAHAYU, IMAN
HIDAYAT, dan KAZUHIKO NARISAWA.
Genus Colletotrichum, Diaporthe dan Guignardia dapat hidup sebagai patogen
pada berbagai jenis tumbuhan tropis berkayu, herba, rerumputan atau sebagai endofit

pada tanaman yang sehat. Colletotrichum, Diaporthe dan Guignardia telah dilaporkan
menyebabkan penyakit pada berbagai tanaman bernilai ekonomi tinggi di Indonesia.
Eksplorasi fungi endofit ini lebih banyak dilakukan pada tanaman budidaya yang
memiliki nilai ekonomi tinggi, sedangkan studi pada tanaman liar sangat jarang
dilakukan padahal fungi endofit dilaporkan banyak diisolasi tanaman liar pada habitat
aslinya. Beberapa spesies liar Zingiberaceae telah ditemukan pada daerah sekitar
Taman Nasional Gunung Halimun Salak (MHSNP). Sebagian dari tanaman tersebut
didomestikasi oleh penduduk sekitar karena diduga memiliki kandungan yang
bermanfaat. Studi mengenai fungi endofit dari Zingiberaceae liar tersebut belum
pernah dilaporkan, oleh karena itu diperlukan penelitian tentang endofit dari
Zingiberaceae liar untuk memberikan informasi mengenai keragaman fungi endofit di
Indonesia.
Jenis endofit yang telah dilaporkan dari Indonesia umumnya diidentifikasi
hanya berdasarkan karakter morfologinya. Pada penelitian ini, fungi diidentifikasi
dengan sekuen ITS. Sampel diambil dari bagian tanaman Zingiberaceae yang sehat
tanpa penyakit untuk disterilisasi permukaan. Sampel kemudian ditanam di media
MEA dan hifa yang tumbuh dari sampel kemudian diisolasi dan dimurnikan pada
media yang sama. Isolat murni kemudian diidentifikasi bagian ITS rDNA secara
molekular dengan pasangan primer ITS1F dan ITS4. Sekuen ITS disejajarkan secara
online dengan program MAFFT. Analisis filogenetik dilakukan dengan MEGA5

dengan membandingkan posisi sekuen isolat dengan sekuen referensi pada filogram.
Sebelas isolat fungi endofit diisolasi dari bagian berbeda dari 4 jenis
Zingiberaceae di MHSNP, yaitu Alpinia malaccensis, Ammomum gracile, Etlingera
punicea dan Zingiber odoriferum. Sembilan diantaranya yaitu Colletotrichum spp,
dan masing-masing satu fungi Diaporthe infecunda dan Guignardia mangiferae. Tiga
isolat Colletotrichum tergolong ke dalam clade gloeosporioides, dan sisanya
membentuk clade tersendiri. Spesies Colletotrichum tersebut terdistribusi pada empat
spesies tanaman pada bagian tanaman yang berbeda. Isolat-isolat tersebut yaitu
Colletotrichum kahawae subsp. kahawae dari akar Etlingera punicea, Colletotrichum
aff. siamense dari akar Alpinia malaccensis, dan tujuh isolat Colletotrichum lainnya
belum teridentifikasi yaitu dari akar Alpinia malaccensis, dari batang dan daun
Ammomum gracile, dan dari akar Zingiber odoriferum. Isolat Diaporthe infecunda
diisolasi dari batang Alpinia malaccensis dan isolat Guignardia mangiferae diisolasi
dari batang Zingiber odoriferum Hasil tersebut diatas merupakan informasi baru
mengenai keberadaan fungi endofit dari Zingiberaceae liar di Indonesia yang dapat
memberikan kontribusi terhadap manajemen domestikasi tanaman Zingiberaceae
tersebut.
Kata kunci: Endofit, ITS, Zingiberaceae

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© Hak Cipta Milik IPB, Tahun 2013
Hak Cipta Dilindungi Undang-Undang
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Further reproduction prohibited without IPB permission.

 

 
 

ENDOPHYTIC FUNGI
OF WILD ZINGIBERACEAE FROM
MOUNT HALIMUN SALAK NATIONAL PARK

NICHO NURDEBYANDARU

This thesis is submitted
as a partial fulfillment of the requirement for the
Degree of Master of Science
in Microbiology

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2013

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EXTERNAL EXAMINER : Dr. Kartini Kramadibrata

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Research Title

: Endophytic Fungi of Wild Zingiberaceae from
Mount Halimun Salak National Park

Name

: Nicho Nurdebyandaru

Student Identification Number

: 0351110211

Study Program

: Microbiology

Advisory Board,

Dr. lr. Oayuh Rahayu
Supervisor

Prof. Kazuhiko Narisawa
Co-Supervisor

Acknowledged by,
Coordinator of Microbiology
Study Program

Prof. Dr. Ir. Anja Meryandini

Date of examination:
Date of submission:

Dr Ir Dahrul Syah, MScAgr

 
 

ix 

: Endophytic Fungi of Wild Zingiberaceae from

Research Title

Mount Halimun Salak National Park
Name

: Nicho Nurdebyandaru

Student Identification Number

: G351110211

Study Program

: Microbiology

Advisory Board,

Dr. Ir. Gayuh Rahayu
Supervisor

Dr. Iman Hidayat
Co-Supervisor 

Prof. Kazuhiko Narisawa
Co-Supervisor 

Acknowledged by,
Coordinator of Microbiology
Study Program

Dean of Graduate School

Prof. Dr. Ir. Anja Meryandini

Dr Ir Dahrul Syah, MScAgr

Date of examination:
Date of submission:

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PREFACE

I praise Allah for all mercy and gifts so I can complete this research entitled
Endophytic Fungi of Wild Zingiberaceae from Mount Halimun Salak National Park.
Tributes are addressed to those who contribute to crystallize this work until this thesis
was finally accomplished. This research will not success without many parties and
individuals helped, therefore I would like to acknowledge Dr. Ir. Gayuh Rahayu, Dr.
Iman Hidayat and Prof. Kazuhiko Narisawa as my supervisors for their guidance,
time, energy, motivation, advice and valuable support also boundless patience during
my study in Indonesia and Japan. My sincere thanks to Dr. Kartini Kramadibrata as
external examiner commission and Prof. Dr. Ir. Anja Meryandini as coordinator of
Microbiology study program for the advice, input, critic and suggestion until this
thesis become better.
I would like to thank to Sepriyadi Rihi S.Si., Israwati Harahap M.Si, and all
staff of Mycology Laboratory LIPI-Cibinong who helped so much at this research.
Thanks to Mycology IPB lab member: Eris Septiana, Ivan Permana Putra, Tatik
Hartanti, Kemala S. Nagur, for togetherness, discussion and motivation during first
year at Indonesia, also Oktan Dwi Nurhayat S.Si., Erwin S.Pd., for their time and
support at Mycology laboratory of IPB. Rola Sameer Mahmoud, Yuki Komatsuzaki,
Erika Usui, Saki Shingaki and Surono for their support, help and discussion during
this research in Japan. I also thanks to Prof. Nobuo Sakagami for all the
administrative assistance.
I want to thank my father, Dr. Ir. Rubiyo M.Si., my mother Ir. Endang
Mufrihati, my brother Briliandaru MP S.T., Dhimas Upadyandaru, Dhaksa Buwana,
my sister Azzalia and Adelia, and my fiancé Angelia Rezty for their patience, love
and pray.
I hope this paper benefiting others.

Ibaraki, September 2013

Nicho Nurdebyandaru

 
 

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CONTENTS
SUMMARY

iv

PREFACE

x

CONTENT

xi

LIST OF TABLES

xii

LIST OF FIGURES

xii

LIST OF APPENDIXES

xii

1 INTRODUCTION

1

Background

2

Objectives

2

Hypothesis

2

Uses

2

2 MATERIAL AND METHOD
Time and Place

3

Plant Material

3

Isolation of Endophytic Fungi

3

DNA Extraction, Amplification and Sequencing of Fungal ITS

4

Phylogenetic Analysis

4

Morphology Observation

5

3 RESULT AND DISCUSSION
Endophytic Species of Colletotrichum

5

Endophytic Species of Diaporthe

14

Endophytic Species of Guignardia

19

4 CONCLUSION

20

REFERENCES

21

APPENDIX

23

BIOGRAPHY

26

 
 

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LIST OF TABLES
1
2
3
4

List of reference Colletotrichum strains and out group used in this study
The shape and size of Colletotrichum appressoria (Weir et al. 2012)
List of reference Diaporthe strain used in this study
List of reference Guignardia strain used in this study

6
14
15
19

LIST OF FIGURES
1 Habitus of Alpinia malaccensis (A), Ammomum gracile (B), Etlingera punicea
(C) and Zingiber odoriferum (D)
2 Phylogramme of Colletotrichum derived from an ML analyses of ITS1F-ITS4
(+500bp) sequence using Kimura-2 model, with gamma distribution (G) and
evolutionary invariable (I), bootstrap 1000x
3 Subtrees of Colletotrichum gloeosporioides complex with C. musae subclade
(A) and C. kahawae subclade (B)
4 Hyphal appressoria (red arrow) of the isolate A) 133A1, B) 511A2, C)
611A4A1 and D) 6Fr2C1
5 Phylogramme of Diaporthe derived from an ML analyses of ITS1F-ITS4
(+500bp) sequence using Tamura-Nei model, with gamma distribution (G) and
evolutionary invariable (I), bootstrap 1000x
6 Phylogramme of Guignardia derived from ML analyses of ITS1F-ITS4
(+500bp) sequence using kimura-2 model, with gamma distribution (G) and
bootstrap 1000x

3

11
13
13

17

20

LIST OF APPENDIXES
1 Maximum Likelihood (ML) of 24 different nucleotide substitution models for
Colletotrichum species
2 Maximum Likelihood (ML) of 24 different nucleotide substitution models for
Diaporthe species
3 Maximum Likelihood (ML) of 24 different nucleotide substitution models for
Guignardia species

23
24
25

 
 

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1 INTRODUCTION

Background
Endophytic fungi have been defined as fungi that inhabit host plant tissue in
their life cycle without causing visible symptoms of disease (Schulz & Boyle 2005).
This group of fungi can be found in every part of the plant and also in various kinds of
plants (Rodriguez et al. 2009). Every plant examined to date harbors at least one
species of endophytic fungus. Many plants, especially woody plants, may contain
hundreds or thousands of fungal species. Arnold et al. (2000) for example, isolated
more than a thousand fungal strains that consist of 418 endophytic morphospecies
from leaves of Heisteria concinna (Olaceae) and Ouratea lucens (Ochnaceae) at
tropical rain forest of Panama. Huang et al. (2008) reported that they were able to get
1,160 fungal isolates representing 31 taxonomic groups, including 73 morphospecies
from 29 Chinese traditional medicinal plants. Taxa from Alternaria, Colletotrichum,
Phoma, Phomopsis and Xylariales are the dominant fungal endophytes. In Indonesia,
Ilyas et al. (2009) isolated 53 isolates of endophytic fungi from Uncaria gambier
Roxb. (Rubiaceae), a spice plant, in West Sumatra. These endophytic fungi belong to
either Coelomycetes such as Phomopsis, Pestalotiopsis, Phoma or Hypomycetes, such
as Aspergillus, Cladosporium, Fusarium and Penicillium.
Zingiberaceae is known as one of medicinal and spices plants that expectedly to
harbor high number of endophytic fungi. This family is distributed mainly in tropical
Asia (South and Southeast), America (Wu & Larsen 2000) and also Africa (Dhetchuvi
et al. 2011) with its greatest diversity in Southeast Asia. However, information
regarding endophytic fungal diversity isolated from zingiberaceous plants is scarce.
Several reports have been recorded from Southeast Asia, for example, 36 species of
fungal endophytes has been reported to associate with wild ginger, Ammomum
siamese (Zingiberaceae) from Thailand (Bussaban et al. 2001). Of those endophytes,
some coelomyctes such as Colletotrichum gloeosporoides and Phomopsis spp. were
regarded as the dominant fungi. They found two new Ascomycetes species,
Gaeumannomyces amomi and Leiosphaerella amomi, and three new species of
Pyricularia and found other fungi such as Aspergillus, Cladosporium, Diaporthe,
Fusarium and Penicillium. They also noted that Fusarium spp. occured from different
plant parts such as pseudostem and rhizome of Ammomum siamense (Bussaban et al.
2001).
Photita et al. (2005) obtained C. gloeosporoides from Alpinia malaccensis also
from Thailand. These C. gleosporoides divided into three subgroups indicated the
complexity of this species. Some pathogenic fungi have been reported from
Zingiberacae and none of them are endophytes. For example, Palarpawar and Gurdhe
(1988) reported the occurrence of C. curcuma causing turmeric (Curcuma longa) leaf
spot disease in India. Colletotrichum musae causing anthracnose on C. alismatifolia
has been reported from Thailand (Mahadtanapuk et al. 2007).

 
 

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Although Indonesia is rich of Zingiberaceae biodiversity, thorough information
on its pathogenic and endophytic diversity and fungal community profile is still
lacking. Noverita et al. (2009) was the only report of fungal endophytes on cultivated
Zingiberaceae from Indonesia. They only studied on Zingiber ottensii (Bangle ghost),
and were able to isolate 10 endophytic fungi from leaf and rhizome without further
identification. Above all, Indonesia has about 650 species, of which ten species of
Zingiberaceae are found in Mount Halimun Salak National Park (MHSNP) (Priyadi et
al. 2010). Some of those species are considered as wild plants and under
domestication process. The information on endophyte of these wild zingiberaceous
plant is lacking.
The information on diversity of endophytic fungi on Zingiberacae is important
as bases for the Zingiberaceae domestication strategy. There are possibilities of
changes from biotrophic to necrotrophic life style in Colletotrichum graminicola
(Thon et al. 2002). Elucidation of fungal endophytes assemblages on various host
plants using conventional method through isolation (Guo et al. 1998) has been
reported successful. Therefore, this method will be used to explore the endophyte
associated with wild Zingiberaceae from MHSNP.

Research Objective
This study is aimed to study endophytic fungi diversity some Zingiberaceae
from MHSNP.

Hypothesis
1. Wild Zingiberaceae hosted many endophytic fungi.
2. Some of endophytic fungi belong to coelomycetes.

Uses
1. First report of endophytic fungi from wild species of Zingiberaceae in Indonesia.
2. Contribute to the knowledge of fungal biodiversity in Indonesia
3. Increase the fungal collections in Indonesia, then can be explored for any bio
prospective purposes.

 
 

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2 METHODS

Time and Place
The research had been conducted from March 2012 - August 2013 at the
laboratory of Mycology, Division of Mycology, Department of Biology, Bogor
Agricultural University and for sequencing at Gene Research Center (GRC), Ibaraki
University.

Plant material
Four species of healthy wild Zingiberaceae were collected from Gunung
Halimun Reserve Area (1086-1134 m asl). These plants namely Alpinia malaccensis
were found at S: 06° 44' 27.7", E: 106° 31' 46.8", Ammomum gracile (S: 06° 44'
27.5", E: 106° 31' 46.1"), Etlingera punicea (S: 06° 44' 24.4", E: 106° 31' 49.3") and
Zingiber odoriferum (S: 06° 44' 27.7", E: 106° 31' 46.8") (Figure 1). All plants were
identified morphologically.

A 

B 

C 

D 

Figure 1 Habitus of Alpinia malaccensis (A), Ammomum gracile (B), Etlingera
punicea (C) and Zingiber odoriferum (D).

Isolation of Endophytic Fungi
A total of 10 samples of plant parts (leaf, stem, flower, fruit and root) from each
species were first washed with running tap water. The plant parts were cut into 5 x 5
cm pieces to be surface sterilized with 0.1% clorox for 2 minutes followed by 70%
alcohol for 1 minute before rinsing with distilled water for 3 times. These pieces were
then dried up by placing over the sterile tissue paper in sterile Petri dishes for at least
6 hours. Five pieces of one plant part were placed onto half strength MEA agar and
kept for a month in room temperature. Whitish to dark colonies were selected to be
purified. Pure cultures were then maintained on Potato Dextrose Agar (Oxoid) slants.

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DNA Extraction, Amplification and Sequencing of Fungal ITS
Five days old colony grown on Potato Dextrose Broth was used as a DNA
source. Isolates were selected for molecular identification. Fungal genomic DNA was
prepared with the NucleonTMPhytoPureTM (GE Healthcare, UK) extraction kit
following the manufacturer’s protocol. Amplification of rDNA ITS region on samples
was carried out by PCR machine using universal primer pair ITS1F
(CTTGGTCATTTAGAGG) (Gardes & Bruns 1993) and ITS4 (CCTCCGCTTATTG)
(White et al. 1990). For each sample, amplification was performed in a 50 µl reaction
volume, which contained 5µl PCR buffer, 4 µl dNTPS, 2.5 µl each primer, 0.15 µl
Taq DNA polimerase (Takara), 0.5 µl DNA template and were added with miliQ
water until 50 µl volume. Thermal cycle consisted of 5 minutes initial denaturation at
94˚C, followed by 35 cycles of 35 seconds denaturation at 94˚C, 55 seconds primer
annealing at 55˚C, 2 minutes extension at 72˚C, and a final 10 min extension at 72˚C.
PCR products were kept at 4˚C. Five µl of PCR products from each PCR reaction
were examined by electrophoresis in a 1% agarose gel in 1×TAE buffer at 100 V for
20 minutes and visualized under UV light after being stained with GelRed (Wako
Pure Chemical, Japan).
PCR products were purified by adding 43.5 µl of PCR purification solution (12
µl 3M CH3COONa pH 4.8, 30 µl 40% PEG, and 1.5 µl 200 mM MgCl2) into each
PCR product tube and these solutions were vortexed and incubated at 4˚C overnight.
After incubation time solution were centrifuged at 15000 rpm at 4˚C for 15 minutes,
and supernatant were removed. About 180 µl 80% ethanol were added and solution
were centrifuged at 15000 rpm at 4˚C for 15 minutes also supernatant were removed.
Absolute ethanol were then added and centrifuged with the same condition as
mentioned above. Sequencing PCR was done by adding 0.5 µl DNA template onto 9.5
µl solution system (1.5 µl Buffer, 0.5 µl BigDye, 0.32 µl primer ITS1F, and miliQ
water until volume reached 9.5 µl) in 0.2 ml PCR tube. Sequencing PCR cycle were
set as 2 minutes for initial denaturation at 96˚C, followed by 25 cycles of 30 second
denaturation at 96˚C, 15 second primer annealing at 50˚C, and 3 minute extension at
60˚C.
Purification of sequencing product were done by adding purification solution
(12 µl 3M CH3COONa pH 4.8, 30 µl 40% PEG, and 1.5 µl 200 mM MgCl2) three
times of volume of sequencing product. The solutions were centrifuged at 15000 rpm
at 4˚C for 20 minutes and supernatant were removed. A hundred fifty µl of ethanol
70% were added and were centrifuged as the same as above. Pellet were dried and
covered by tissue paper at room temperature. About 20 µl HIDI were added into each
pellet and were vortexed at 2000 rpm for 2 minutes, then solution were pipetted into
the 96-cell-plate and were analyzed into sequencing machine ABI 3130 Genetic
Analyzer (Applied Biosystem).
Phylogenetic Analysis
ITS data sequence was aligned online with MAFFT ver. 7 (http://mafft.cbrc.jp/).
Phylogenetic analysis was conducted using Maximum Likelihood (ML) approach, by
finding the best model and treatment in MEGA 5 software. Statistical support for the

 
 

5 

internal branches was estimated by bootstrap analysis based on 1,000 replications.
Monilichaetes infuscans CBS 869.96, Diaporthella corylina CBS 121124 and
Neofusicoccum luteum STE-U 4594 were used as an out-group, respectively for
Colletotrichum, Diaporthe and Guignardia.
Morphology Observation
A single colony from culture stock was grown at MEA and PDA in a 9 mm
Petridishes and incubated at room temperature until the sporulation was observed. All
reproductive structures if present were noted.

3 RESULT AND DISCUSSION

Totally eleven white to dark isolates were collected from Zingiberaceae from
MHSNP. These isolates showed different morphological features. However, it is not
possible to determine further to species level since all this isolates were unsporulated.
In the beginning of the isolation process some of these isolates showed orange
pustules that indicate the acervuli of the Colletotrichum. Some others showed
distinctive fruit body without spores in it. When the ITS sequence from these isolates
were aligned to the Genbank data, most of the isolates showed highly similarity with
Colletotrichum gleosporioides, one isolates each with either Colletotrichum
boninense or Colletotrichum crassipes. The two other isolates, one is similar to a
different species of Diaporthe (D. longicola and D. helianthi (100%) or D.
chimonanti and D. infecunda (99%) and another isolate with Guignardia vaccini
(100%). These identification results were further optimized and checked for gaining
more reliable result. Analyses the identities of the isolates to species level were made.
Endophytic Species of Colletotrichum/Glomerella (Teleomorph/Anamorph)
Of those isolates with white to dark colonies, nine isolates are apparently
Colletotricum. These composed of four isolates from root, pseudostem and fruit of
Alpinia malaccensis, two isolates from pseudostem and leaf Ammomum gracile, one
isolate from root of Etlingera punicea, and two isolates from root of Zingiber
odoriferum. These isolates showed orange pustules during first step of isolation.
However, the capability of forming conidia and acervuli apparently lost during
purification.
Identification using morphology of the colony is not possible. These isolates
were then analyzed molecularly. The amplicon of PCR ITS1F-ITS4 yielded about 420
bp lengths, similarly as Manter and Vivanco (2007) research that showed amplicons
length around 420-825 bp. They were sequenced and the nucleotide blast to the NCBI
indicated that all isolates showed highly similarity (>95%) with C. gloeosporioides
Genbank data. However, according to Nilsson et al. (2006) about 20% of the entries

 
 

6 

in the Genbank might be incorrectly identified to species level. In particular, Hyde et
al. (2010) stated that sequence lodged in Genbank, as C. gloeosporioides were
doubtful and belonged to many different species. Further, those ITS sequences of C.
gloeosporioides and Glomerella cingulata (teleomorph of C. gloeosporioides) were
found throughout the C. gloeosporioides species complex and outside the C.
gloeosporioides complex. Those outside C. gloeosporioides applied to more than 100
of the about 750 ITS sequences of C. gloeosporioides in GenBank. Therefore,
identification of these isolates used phylogenetic analyses.
The alignment in the phylogenetic analyses of the isolates collected, included
sequences from all species of Colletototrichum were reported by Cannon et al. (2012)
of which all are either holotypes or epitypes (Table 1). The data was analyzed by
maximum likelihood (ML), which performed the clades that meet with that of Cannon
et al. (2012). All nine clades in the Colletotrichum complex were shown in the
phylogram eventhough they are supported by low bootstrap value, except for C.
truncatum and C. orbiculare clade (Figure 2). The branches of these clades were
supported by 87 and 99% bootstrap values, respectively. However, Cannon et al.
(2012) stated that C. gloeosporioides complex is one amongst nine well-established
major clades species in the genus Colletotrichum. In this analysis, C. gloeosporioides
complex separated from the other species complex or clades similar with Cannon et
al. (2012) result.
Table 1 List of reference Colletotrichum strains and out group used in this study
Culture accession number

ITS Genbank accession
number

Colletotrichum acerbum

CBS 128530

JQ948459

C. acutatum*
C. aenigma**
C. aeschynomenes**
C. agaves
C. alatae**
C. annellatum**
C. alienum**
C. anthrisci**
C. aotearoa**
C. asianum**
C. australe**
C. axonopodi**
C. beeveri**
C. boninense**
C. brasiliense**
C. brassicicola**
C. brisbaniense**
C. carthami

CBS 1129996
ICMP 18608
ICMP 17673
CBS 118190
ICMP 17919
CBS 129826
ICMP 12071
CBS 125334
ICMP 18537
ICMP 18580
CBS 116478
IMI 279189
CBS 128527
CBS 123755
CBS 128501
CBS 101059
CBS 292.67
SAPA100011

JQ005776
JX010244
JX010176
DQ286221
JX01019
JQ005222
JX010251
GU227845
JX010205
FJ972612
JQ948455
EU554086
JQ005171
JX010292
JQ005235
JQ005172
JQ948291
AB696998

Species

 
 

C. cereale
C. chlorophyti**
C. chrysanthemi
C. circinans
C. clidemiae**
C. cliviae
C. coccodes
C. colombiense**
C. constrictum
C. cosmi
C. costaricense**
C. curcumae
C. cuscutae**
C. cymbidiicola**
C. cordylinicola**
C. dacrycarpi**
C. dematium*
C. destructivum
C. dracaenophilum**
C. echinochloae
C. eleusines
C. eremochloae**
C. falcatum***
C. fioriniae
C. fructi
C. fructicola
C. fuscum
C. gloeosporioides*
C. godetiae**
C. graminicola*
C. guajavae**
C. hanaui**
C. hemerocallidis**
C. higginsianum
C. hipperastri
C. horii***
C. indonesiense**
C. jacksonii
C. jasminigenum**
C. johnstonii
C. karstii
C. kahawae subsp. kahawae**
C. kahawae subsp. ciggaro**

7 

CBS 129663
IMI 103806
SAPA 100010
CBS 221.81
ICMP18658
CBS 125375
CBS 369.75
CBS 129818
CBS 128504
CBS 853.73
CBS 330.75
IMI 288937
IMI 304802
IMI 347923
ICMP18579
CBS 130241
CBS 125.25
CBS 149.34
CBS 118199
MAFF 511473
MAFF 511155
CBS 129661
CBS 147945
CBS 128517
CBS 346.37
ICMP 172921
CBS 130.57
ICMP 17821
CBS 133.44
CBS 130836
IMI 350839
MAFF 305404
CDLG5
IMI 349063
CBS 241.78
ICMP 10492
CBS 127551
MAFF 305460
MFLUCC 10 0273
CBS 128532
CBS 132134
ICMP 17816
ICMP 18539

DQ126177
GU227894
AB696999
GU227855
JX010265
GQ485607
HM171679
JQ005174
JQ005238
JQ948274
JQ948180
GU227893
JQ948195
JQ005166
JX010226
JQ005236
GU227819
AJ301942
DQ286209
AB439811
EU554131
JQ478447
JQ005772
JQ948292
FJ972603
JX010165
JQ005762
JX010152
JQ948402
DQ003110
JQ948270
JX519217
JQ400005
JQ005760
JQ005231
GQ329690
JQ948288
EU554108
HM1311513
JQ948444
HM585409
JX010231
JX010231

8 

 
 

C. kinghornii**
C. laticiphilum**
C. lilii
C. limetticola*
C. lindemuthianum
C. lineola*
C. linicola
C. liriopes**
C. lupine***
C. melonis**
C. miscanthi**
C. musae*
C. navitas**
C. nicholsonii**
C. novae-zelandiae**
C. nupharicola**
C. nymphaeae*
C. oncidii**
C. orchidophilum**
C. orbiculare
C. parsonsiae**
C. paspali**
C. paxtonii**
C. petchii*
C. phaseolorum#
C. phormii*
C. phyllanthi**
C. pseudoacutatum**
C. psidii#
C. pyricola**
C. quenslandicum*
C. rhombiforme**
C. rusci**
C. salicis*
C. salsolae**
C. sansevieriae**
C. scovillei**
C. siamense**
C. simmondsii**
C. sloanei**
C. spaethianum*
C. spinaciae
C. sublineola*

CBS 198.35
CBS 112989
CBS 109214
CBS 114.14
CBS 144.31
CBS 125337
CBS 172.51
CBS 119444
CBS 109225
CBS 159.84
MAFF 510857
ICMP 19119
CBS 125086
MAFF 511115
CBS 128505
ICMP 18187
CBS 515.78
CBS 129828
CBS 632.80
CBS 514.97
CBS 128525
MAFF 305403
IMI 165753
CBS 378.94
CBS 157.36
CBS 118194
CBS 175.67
CBS 436.77
ICMP 19120
CBS 128531
ICMP 1778
CBS 129953
CBS 119206
CBS 607.94
ICMP 19051
MAFF 239721
CBS 126529
ICMP 18578
CBS 122122
IMI 364297
CBS 167.49
CBS 128.57
CBS 131301

JQ948454
JQ948289
GU227810
JQ948193
JQ005779
GU227829
JQ005765
GU227804
DQ286119
JQ948194
EU554121
JX010146
GQ919067
EU554126
JQ005228
JX010187
JQ948197
JQ005169
JQ948151
JQ005778
JQ005233
EU554100
JQ948285
JQ005223
GU227896
JQ948446
JQ005221
JQ948480
JX010219
JQ948445
JX010276
JQ948457
GU227818
JQ948460
JX010242
AB212991
JQ948267
JX010171
JQ948276
JQ948287
GU227807
GU227847
DQ003114

 
 

C. tabacum
CBS 161.53
JQ005763
C. theobromicola***
ICMP 18649
JX010294
C. ti**
ICMP 4832
JX010269
C. tofieldiae
CBS 495.85
GU227801
C. torulosum**
CBS 128544
JQ005164
C. trichellum
CBS 217.64
GU227812
C. tropicale**
ICMP 18653
JX010264
C. truncatum*
CBS 151.35
GU227862
C. verruculosum**
IMI 45525
GU227806
C. walleri**
CBS 125472
JQ948275
C. xanthorrhoeae
BRIP 45094
GU048667
C. yunnanense**
CBS 132135
EF369490
Monilochaetes infuscans
CBS 869.96
GU180626.1
* : epitype, ** : ex-holotype, *** : ex-neotype, # : authentic culture
 

The phylogram of the ITS region showed that the nine isolates Colletotrichum
distributed into 2 different clades i.e. C. gloeosporioides and a presumably new clades
(Figure 2). Identification further within clades is problematic. Weir et al. (2012) also
stated that ITS can only be used to separate clades.
The isolates in C. gloeosporioides clades were belong to isolate 611A4, 133A1
and 6Fr2C1. Two verification approaches were taken to confirm the position of these
isolates in the C. gloeosporioides complex. The first verification used unique string of
C. gloeosporioides i.e 5’-GGGCGGGT-3’. According to Weir et al. (2012) about
139-142 bases after the ITS1F primer binding site, all of the isolates accepted in the
C. gloeosporioides complex share the string 5’-GGGCGGGT-3’. They also found this
string appeared to be specific to isolates of the C. gloeosporioides complex on the
bases of their comparison with GenBank data. In this study only two isolates (133A1
and 611A4) have such kind of string proofing that the isolates are members of
gloeosporioides complex. In contrast, isolates 6Fr2C1 did not have such a string.
The isolates that are in C. gloeosporioides clades belong to two subclades, i.e.
C. musae (611A4) and C. kahawae (133A1 and 6Fr2C1) subclades (Figure 3). The
position of isolate in the C. musae clades cannot be determined yet since the branch
has low supported bootstrap value (39%). Therefore isolates 133A1 is tentatively
identified as Colletotrichum aff. siamense. A low bootstrap value (37%) also
supported the branch of kahawae subclade. Therefore, those in kahawae should be
determined by comparison using the available morphological data.
The second confirmation was done by using morphological observation. All of
these isolates are unsporulated in all media tested, thus made the confirmation limited.
All isolates showed the hyphal appressoria (Figure 4), a character of the
Colletotrichum. Confirmation using appressoria did not solve the problem;
eventhough the presence of hyphal appressoria in Colletotrichum has been stated by
several mycologists (Photita et al. 2005, Weir et al. 2012), amongst other characters
such as acervuli, setae, conidia and ascospores. Of the species in the gloeosporioides

9 

10 

 
 

complex, Weir et al. (2012) only gave the illustration and description of the hyphal
appressoria of eight member of C. gloeosporioides complex (Table 2). Comparison to
the illustration of appressoria confirmed that isolate 133A1 is C. kahawae subsp.
kahawae. The hyphal appressoria of 6Fr2C1 did not meet with any illustration in
kahawae subclades, thus identification to species level cannot be made. Hyphal
appressoria of 611A4 did not meet with any illustration available for species in musae
subclade as well and thus cannot confirm the position as Colletotrichum aff. siamense.
The appressoria of isolate 133A1 are mostly globose with slightly lobed, whilst that of
511A2 and 6Fr2C1 are mostly lobed and lumped (Figure 4). The available
information on the appressoria (Tabel 2) is poor, thus it cannot be used as a key factor
to differentiate the species. Therefore, confirmation using other markers is needed.
The other six isolates (412D2, 423C1, 511A2, 511A3, 622C1 and 631C2) are
grouped forming a strong clade with 96% bootstrap value. There is a possibility that
this clade is a new clade and new species. All the isolates also showed hyphal
appressoria. However, this could only confirm the isolates until genus level.
Confirmation into species level using other markers is needed.
This study reveals new result of the presence of endophytic Colletotrichum from
Zingiberaceae from MHSNP. Colletotrichum kahawae subsp. kahawae is a new
record in Indonesia. In Africa, C. kahawae subsp. kahawae is an important pathogen
of coffee berry (Weir et al. 2012) and has never been reported as an endophyte. This
evidence of endophytic Colletotrichum species confirmed the report of Bussaban et
al. (2001) who found the endophytic C. gloeosporioides from wild species of
Zingiberaceae (Ammomum siamense) and Photita et al. (2005) who found that species
in Alpina malaccensis in Thailand.
This study found that one individual plant might harbor more than one species
of Colletotrichum. Alpinia malaccensis hosted Colletotrichum cf. siamense on root
and two of unidentified diferrent lineage of Colletotrichum in the fruit and
pseudostem. Ammomum gracile hosted presumably the same species on their
pseudostem and leaf. Freeman and Katan (1997) reported that Colletotrichum has
been known to infect all plant part above the ground and also root.
The other eight isolates needs confirmation using other markers. Actually, Weir
et al. (2012) stated that ITS sequences can be used to distinguish Colletotrichum
alatae, C. asianum, C. cordylinicola, C. gloeosporioides, C. horii, C. kahawae, C.
musae, C. manihotis, C. psidii, C. theobromicola, C. xanthorrhoeae from all other
taxa. In constrast, ITS sequences are insufficient to separate C.aenigma, C.
aeschynomenes C. alienum, C. aotearoa, C. clidemiae, C. fructicola, C. nupharicola,
C. queenslandicum, C. siamense and C. tropicale isolates. It is best distinguished
these taxa using TUB2, GAPDH, or GS.

 
 

11 
Colletotrichum costaricense CBS:330.75
Colletotrichum cuscutae IMI:304802
Colletotrichum limetticola CBS:114.14
Colletotrichum melonis CBS:159.84
Colletotrichum tamarilloi CBS:129814
Colletotrichum lupini CBS:109225
51

Colletotrichum walleri CBS:125472
Colletotrichum scovillei CBS:126529
Colletotrichum sloanei IMI:364297
Colletotrichum indonesiense CBS:127551

acutatum
clade

Colletotrichum cosmi CBS:853.73
Colletotrichum acutatum CBS:112996
78

Colletotrichum acerbum CBS:128530
Colletotrichum rhombiforme CBS:129953
Colletotrichum fioriniae CBS:128517

Colletotrichum australe CBS:116478
Colletotrichum phormii CBS:118194
Colletotrichum salicis CBS:607.94
Colletotrichum johnstonii CBS:128532
Colletotrichum pyricola CBS:128531
Colletotrichum kinghornii CBS:198.35
Colletotrichum godetiae CBS 133.44
Colletotrichum destructivum CBS 149.34
Colletotrichum fuscum CBS 130.57
Colletotrichum higginsianum IMI 349063
Colletotrichum tabacum CBS 161.53

destructivum
clade

Colletotrichum linicola CBS 172.51
Glomerella truncata CBS:127604

truncatum
clade

Colletotrichum curcumae IMI:288937
Colletotrichum truncatum CBS:151.35

87
98

Colletotrichum jasminigenum MFLUCC 10-0273

Colletotrichum constrictum CBS:128504

73

Colletotrichum dacrycarpi CBS:130241
Colletotrichum brasiliense CBS:128501

91
63

Colletotrichum parsonsiae CBS:128525

56

Colletotrichum hippeastri CBS:125376
Colletotrichum phyllanthi CBS:175.67
77

Colletotrichum karstii CGMCC 3.14194
Colletotrichum annellatum CBS:129826

Colletotrichum novae-zelandiae CBS:128505
Colletotrichum petchii CBS:378.94

boninense
clade

Colletotrichum beeveri CBS:128527

53
61

Colletotrichum brassicicola CBS:101059
Colletotrichum colombiense CBS:129818
Colletotrichum torulosum CBS:128544

58

Colletotrichum boninense CBS:123755
Colletotrichum cymbidiicola IMI:347923
Colletotrichum oncidii CBS:129828
Colletotrichum paspali MAFF:305403
Colletotrichum nicholsonii MAFF:511115
Colletotrichum navitas CBS 125086
Colletotrichum falcatum CBS:147945
Colletotrichum eleusines MAFF:511155

graminicola
clade

Colletotrichum miscanthi MAFF:510857
Colletotrichum hanaui MAFF:305404
Colletotrichum jacksonii MAFF:305460

92
91

Colletotrichum echinochloae MAFF 511473

Colletotrichum eremochloae CBS:129661
99

Colletotrichum sublineoa CBS 131301

Colletotrichum cereale CBS:129663
95

Colletotrichum circinans CBS:221.81
Colletotrichum spinaciae CBS 128.57
95

Colletotrichum dematium CBS:125.25
Colletotrichum lineola CBS:125337

95

dematium
clade

Colletotrichum fructi CBS:346.37
52
51

Colletotrichum anthrisci CBS:125334

Colletotrichum spaethianum CBS:167.49
Colletotrichum lilii CBS:109214

67

Colletotrichum verruculosum IMI:45525
Colletotrichum liriopes CBS:119444
Colletotrichum tofieldiae CBS 495.85

spaethianum
clade

12 

 
 
Colletotrichum salsolae ICMP 19051
Colletotrichum gloeosporioides IM 356878
Colletotrichum aotearoa ICMP 18537
Colletotrichum horii ICMP 10492
Colletotrichum nupharicola ICMP 18187
Colletotrichum siamense ICMP 18578
Colletotrichum theobromicola ICMP 18649
Colletotrichum aeschynomenes ICMP 17673
Colletotrichum fructicola ICMP 17921
Colletotrichum alienum ICMP 12071
Colletotrichum queenslandicum ICMP 1778
Colletotrichum aenigma ICMP 18608
Colletotrichum tropicale ICMP 18653
Colletotrichum ti ICMP4832

gloeosporioides
clade

Colletotrichum clidemiae ICMP 18658
Colletotrichum musae CBS 116870
Colletotrichum asianum ICMP 18580
611A4
Colletotrichum alatae ICMP 17919
Colletotrichum xanthorrhoeae ICMP:17903
Colletotrichum kahawae ICMP:17816
Colletotrichum psidii ICMP 19120
133A1
Colletotrichum cordynicola ICMP 18579
6Fr2C1
99

Colletotrichum lindemuthianum CBS:144.31

orbiculare clade

Colletotrichum orbiculare CBS 514.97
Gloeosporium carthami SAPA100011
Colletotrichum nymphaeae CBS:515.78
Colletotrichum simmondsii CBS:122122
Colletotrichum paxtonii IMI:165753

acutatum clade

Colletotrichum guajavae IMI:350839
Colletotrichum chrysanthemi IMI:364540
Colletotrichum laticiphilum CBS:112989
Colletotrichum brisbanense CBS:292.67
412D2
423C1
511A3

96

631C2
87

other clade

511A2
622C1
Monilochaetes infuscans CBS 869.96

0.05

Figure 2 Phylogramme of Colletotrichum derived from an ML analyses of ITS1FITS4 (+500bp) sequence using Kimura-2 model, with gamma distribution
(G) and evolutionary invariable (I), bootstrap 1000x.

 
 

13 

A 

B 

Figure 3 Subtrees of Colletotrichum gloeosporioides complex with C. musae
subclade (A) and C. kahawae subclade (B).

A 

B 

C 

D 

Figure 4 Hyphal appressoria (red arrow) of the isolate A) 133A1, B) 511A2, C)
611A4A1 and D) 6Fr2C1.

14 

 
 

Table 2 The shape and size of Colletotrichum appressoria (Weir et al. 2012)
Species
Colletotrichum
aenigma

Shape

Size

Subglobose or with a few broad lobes

6-10 µm

C. crassipes

Mostly elliptic to subfusoid, deeply
lobed.
Mostly simple, elliptic to fusoid in shape,
sometime developing broad, irregular
lobes
Mostly simple, globose to shortcylindric, a few with broad, irregular
lobes
Variable in shape, simple to broadly
lobed, sometimes in groups, sometimes
intercalary,
Variable in shape, some simple,
subglobose, but often with a small
number of broad, irregular lobes
Appressoria deeply lobed.

C. kahawae subsp.
ciggaro

Typically cylindric to fusoid in shape,
deeply lobed

C. kahawae subsp.
kahawae

*

C. queenslandicum

Globose to short-cylindric, rarely lobed

C. aeschynomenes
C. alatae

C. alienum

C. atoteroa

C. clidemiae

7–13.5 × 5–10.5
µm

7–17 × 4–9.5 µm

6–12 µm in φ

*only illustration available
Endophytic Species of Diaporthe
In this study the identification of Diaporthe is only used ITS region, eventhough
according to Gomes et al. (2013), the locus could distinguish only 75 of the 95
species (79 % success). They suggested that identification should be based on
multigene, a combination of ITS, TEF1, TUB, HIS and CAL region. All Diaporthe
species recognized by Gomes et al. (2013) were used as reference strains (Table 3)
with Diaporthella corylina as outgroup. Phylogenetic study of the isolate originated
from pseudostem of Alpinia malaccensis indicated that the isolate (6Fr2C3) belongs
to Diaporthe infecunda with 86% bootstrap value (Figure 5). Gomes et al. (2013) also
stated that D. infecunda has ITS positions 108 (T), 279 (C), 292 (G), 359 (C) and 360
(G). Yet, confirmation using this character has not been done. The isolate being
studied has no reproductive structure and this evidence is similar to that of Gomes et
al. (2013), which was also sterile.
The presence of D. infecunda on Zingiberaceae in Indonesia is the first report.
The specimens of D. infecunda studied by Gomes et al. (2013) were also endophyte
from non-Zingiberaceus medicinal plants growing in Brazil. Those isolates were from

 
 

15 

petiole of Maytenus ilicifolia (Celastraceae), and from leaves of Schinus
terebinthifolius (Anacardiaceae). Endophytic Diaporthe sp. has also found on
Cinchona ledgeriana in Indonesia (Maehara et al. 2012). Other than endophytic
species, pathogenic species of Diaporthe, Diaporthe phaseolorum (Gomes et al.
2013) and Diaporthe citri had been reported to occur on Glycine max and unknown
plant, respectively in Indonesia.
Table 3 List of reference Diaporthe strain used in this study
Species
Diaporthe acaciigena*
D. acerina
D. alleghaniensis*
D. alnea
D. ambigua**
D. ampelina
D. amygdali
D. angelicae**
D. arctii
D. arecae***
D. arengae*
D. aspalathi*
D. australafricana*
D. batatas
D. beckhausii
D. brasiliensis*
D. carpini
D. caulivora****
D. celastrina
D. chamaeropis
D. cinerascens
D. citri
D. conorum
D. convolvuli
D. crataegi
D. crotalariae*
D. cuppatea*
D. cynaroidis*
D. decedens
D. detrusa
D. elaeagni
D. endophytica*
D. eres
D. eugeniae
D. fibrosa
D. foeniculacea
D. ganjae*
D. gardeniae

Culture accession
number
CBS 129521
CBS 137.27
CBS 495.72
CBS 146.46
CBS 114015
STE-U 2673
CBS 111811
CBS 111592
CBS 136.25
CBS 161.64
CBS 114979
CBS 117169
CBS 111886
CBS 122.21
CBS 138.27
CBS 133183
CBS 114437
CBS 127268
CBS 139.27
CBS 454.81
CBS 719.96
CBS 230.52
CBS 186.37
CBS 124654
CBS 114435
CBS 162.33
CBS 117499
CBS 122676
CBS 109772
CBS 109770
CBS 504.72
CBS 133811
CBS 101742
CBS 444.82
CBS 109751
CBS 111553
CBS 180.91
CBS 288.56

ITS Genbank
accession number
KC343005
KC343006
KC343007
KC343008
KC343010
KC343016
KC343019
KC343027
KC343031
KC343032
KC343034
KC343036
KC343038
KC343040
KC343041
KC343042
KC343044
KC343045
KC343047
KC343048
KC343050
KC343052
KC343079
KC343054
KC343055
KC343056
KC343057
KC343058
KC343059
KC343061
KC343064
KC343065
KC343074
KC343098
KC343099
KC343101
KC343112
KC343113

16 

 
 

D. helianthi*
CBS 592.81
KC343115
D. cf. heveae 1
CBS 852.97
KC343116
D. hickoriae*
CBS 145.26
KC343118
D. hongkongensis*
CBS 115448
KC343119
D. hordei
CBS 481.92
KC343604
D. impulsa
CBS 114434
KC343121
D. inconspicua*
CBS 133813
KC343123
D. infecunda*
CBS 133812
KC343126
D. juglandina
CBS 121004
KC343134
D. longispora*
CBS 194.36
KC343135
D. lusitanicae*
CBS 123212
KC343136
D. manihotia
CBS 505.76
KC343138
D. mayteni*
CBS 133185
KC343139
D. megalospora
CBS 143.27
KC343140
D. melonis***
CBS 507.78
KC343142
D. musigena*
CBS 129519
KC343143
D. neilliae
CBS 144.27
KC343144
D. neoarctii*
CBS 109490
KC343145
D. neotheicola*
CBS 123209
KC343105
D. nobilis
CBS 113470
KC343146
D. nobilis
CBS 200.39
KC343151
D. nomurai
CBS 157.29
KC343154
D. novem*
CBS 127270
KC343156
D. oncostoma
CBS 100454
KC343160
D. oxe*
CBS 133186
KC343164
D. padi var. padi
CBS 114200
KC344137
D. paranensis*
CBS 133184
KC343171
D. perjuncta**
CBS 109745
KC343172
D. perseae
CBS 151.73
KC343173
D. phaseolorum
CBS 116019
KC343175
D. pseudomangiferae*
CBS 101339
KC343181
D. pseudophoenicicola
CBS 176.77
KC343183
D. pustulata
CBS 109742
KC343427
D. raonikayaporum*
CBS 133182
KC343188
D. rhoina
CBS 146.27
KC343189
D. saccarata*
CBS 116311
KC343190
D. schini*
CBS 133181
KC343191
D. sclerotioides*
CBS 296.67
KC343193
D. scobina
CBS 251.38
KC343195
D. sojae
CBS 100.87
KC343196
D. stictica
CBS 370.54
KC343212
D. subordinaria
CBS 101711
KC343213
D. tecomae
CBS 100547
KC343215
D. terebinthifolii*
CBS 133180
KC343216
D. toxica*
CBS 534.93
KC343220
D. vaccinii*
CBS 160.32
KC343228
D.vexans
CBS 127.14
KC343229
D. viticola
CBS 100170
KC343230
Diaporthella corylina
CBS 121124
KC343004
* : ex-type, ** : ex-epitype, *** : ex-isotype, **** : ex-neotype

 
 

17 
Diaporthe viticola strain CBS 113201
97

Diaporthe viticola strain CBS 100170

91

Diaporthe cynaroidis strain CBS 122676
Diaporthe australafricana strain CBS 111886

96
97

Diaporthe australafricana strain CBS 113487
Diaporthe beckhausii strain CBS 138 27
Diaporthe toxica strain CBS 534 93
99

Diaporthe toxica strain CBS 535 93

Diaporthe padi var padi strain CBS 114200
99

Diaporthe padi var padi strain CBS 114649
Diaporthe saccarata strain CBS 116311
Diaporthe cf heveae 1 RG 2013 strain CBS 852 97
Diaporthe caulivora strain CBS 127268

97

Diaporthe caulivora strain CBS 178 55
Diaporthe sp 5 RG 2013 strain CBS 125575
Diaporthe brasiliensis strain CBS 133183

99
99

Diaporthe brasiliensis strain LGMF926

Diaporthe decedens strain CBS 109772

99

Diaporthe decedens strain CBS 114281
Diaporthe impulsa strain CBS 114434
Diaporthe carpini strain CBS 114437
Diaporthe fibrosa strain CBS 109751
99

Diaporthe fibrosa strain CBS 113830
Diaporthe scobina strain CBS 251 38
Diaporthe ampelina strain CBS 111888

99

Diaporthe ampelina strain STE U2660
Diaporthe impulsa strain CBS 141 27
Diaporthe crataegi strain CBS 114435
Diaporthe nomurai strain CBS 157 29
Diaporthe woolworthii strain CBS 148 27
Diaporthe detrusa strain CBS 109770
99

Diaporthe detrusa strain CBS 114652
Diaporthe acerina strain CBS 137 27
Diaporthe crotalariae strain CBS 162 33
Diaporthe aspalathi strain CBS 117168

97

Diaporthe aspalathi strain CBS 117169
Diaporthe woodii strain CBS 558 93
Diaporthe amygdali strain CBS 126679

99

Diaporthe amygdali strain CBS 126680
Diaporthe pustulata strain CBS 109742
Diaporthe acaciigena strain CBS 129521
Diaporthe oncostoma strain CBS 100454

99

Diaporthe oncostoma strain CBS 109741
Diaporthe perjuncta strain CBS 109745
Diaporthe hickoriae strain CBS 145 26
Diaporthe cinerascens strain CBS 719 96
Diaporthe anacardii strain CBS 720 97
Diaporthe inconspicua strain LGMF922
Diaporthe inconspicua strain CBS 133813
Diaporthe elaeagni strain CBS 504 72
Diaporthe stictica strain CBS 370 54
Phomopsis sp strain RJGD D16
Diaporthe foeniculacea strain CBS 116957

98
82

Diaporthe foeniculacea strain CBS 123208

70

Diaporthe chamaeropis strain CBS 454 81

88

Diaporthe chamaeropis strain CBS 753 70
Diaporthe celastrina strain CBS 139 27
Diaporthe juglandina strain CBS 121004
Diaporthe alleghaniensis strain CBS 495 72
Diaporthe eres strain CBS 439 82

96
59

Diaporthe eres strain CBS 445 62

Diaporthe vaccinii strain CBS 122116

59

Diaporthe vaccinii strain CBS 160 32
Phomopsis sp strain RJGD D9
87

Diaporthe gardeniae strain CBS 288 56

Diaporthe cf nobilis RG 2013 strain CBS 113470
Diaporthe cf nobilis RG 2013 strain CBS 116953

56

Diaporthe alnea strain CBS 146 46

53
96

Diaporthe neilliae strain CBS 144 27
Diaporthe rhoina strain CBS 146 27
Diaporthe pseudophoenicicola strain CBS 176 77

91

Diaporthe pseudophoenicicola strain CBS 462 69
56

Diaporthe sp 7 RG 2013 strain CBS 458 78

54

Diaporthe sp 8 RG 2013 strain LGMF925
Diaporthe arecae strain CBS 161 64
Diaporthe cf heveae 2 RG 2013 st