Molecular Characterization of Begomovirus Infecting Yard Long Bean and Construction of Its Specific Primers
MOLECULAR CHARACTERIZATION OF BEGOMOVIRUS INFECTING
YARD LONG BEAN (Vigna unguiculata subsp. sesquipedalis L.) AND
CONSTRUCTION OF ITS SPECIFIC PRIMERS
SARI NURULITA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
AUTHOR’S STATEMENT ON THESIS AND ITS SOURCE OF
INFORMATION AND DELEGATION OF COPYRIGHT
I declare that this thesis entitled Molecular Characterization of Begomovirus
Infecting Yard Long Bean (Vigna unguiculata subsp. sesquipedalis L.) and
Construction of Its Specific Primers is my own and authentic work under
supervision of a thesis commitee and it is not yet submitted to any universities for
any degree fulfillment. Source of information, both published or unpublished by it
authors, used for quotations in this thesis is already cited appropriately and
present in thesis‟s Literature Cited chapter.
I hereby delegate my thesis copyright to the Bogor Agricultural University.
Bogor, August 2014
Sari Nurulita
Student ID number: A352110081
Copyright delegation of scientific paper from research collaboration with other
organization must be referred on its collaboration agreement.
SUMMARY
SARI NURULITA. Molecular Characterization of Begomovirus Infecting Yard
Long Bean (Vigna unguiculata subsp. sesquipedalis L.) and Construction of Its
Specific Primers. Supervised by SRI HENDRASTUTI HIDAYAT and KIKIN
HAMZAH MUTAQIN.
Begomovirus is an important plant viral pathogen group causing serious
diseases in several tropical and sub-tropical countries. Infection of Begomovirus
was reported to cause economic losses up to million dollars. Infection of
Begomovirus on horticultural crops in Indonesia was first reported on chilli pepper
in 1999 with disease incidence reached 30 to 100%. Infection of Begomovirus on
chili pepper is obviously recognized by its typical symptoms involving leaf
curling and yellowing.
Yellow mosaic disease outbreaks have also been reported on yard long bean
(Vigna unguiculata subsp. sesquipedalis L.) growing area in Java since 2008. At
the same time, similar yellow mosaic disease was first reported in Pakistan and
Nepal on Leguminosae. The causal agent of yellow mosaic disease in South Asia
was identified as a member of Begomovirus. Yellow mosaic disease on yard long
bean in Java was considered to be caused by Begomovirus.
Begomovirus is the largest genus of the Family Geminiviridae. Molecular
characteristics of Begomovirus genome is very unique, comprising twin circular
particles, each particle is consisting of single stranded DNA (2.6 – 2.7 kb),
bipartite or monopartite. DNA-A and DNA-B of bipartite Begomovirus have
different genome organization and function except for their common region. On
the other hand, monopartite Begomovirus has all combination function of DNA-A
and DNA-B in one genome. There are three unique characteristics of common
region which is observed on every Begomovirus species, i.e. TATA box, repeated
motives (iterons), and hairpin-loop structure.
Begomovirus is only transmitted in nature by insect vector, B. tabaci
(Hemiptera: Aleyrodidae). Studies on virus – vector relationship indicates that
Begomovirus is transmitted in circulative persistent manner. Begomovirus can not
be transmitted by mechanical nor seed manner. The nature of Begomovirus of
being insect transmissable and having a wide host range may contribute to its
potential to cause high disease incidence and being difficult to control.
Common method for detection of Begomovirus is based on polymerase
chain reaction (PCR). PAL1v1978/PAR1c715 and AV494/AC1048 primer pairs
have been reported to work well for different species of Begomovirus. The former
pair amplifies common region (CR), whereas the latter amplifies coat protein gene
of Begomovirus. PAL1v1978/PAR1c715 was successfully used to detect several
geminivirus from Solanaceae, Leguminosae, Euphorbiaceae, and Malvaceae
plants.
The objective of this research is to determine the importance of
Begomovirus infection on yard long bean, to identify species of Begomovirus on
yard long bean and to analyse its molecular character, and to design specific
primers for detection of Begomovirus species infecting yard long bean.
Research was conducted in four activities: (i) field survey and sample
collection in yard long bean fields located in Central Java (Tegal, Magelang, and
Klaten), Jogyakarta (Sleman and Klaten), and West Java (Bogor and Subang)
provinces; (ii) virus detection using I-ELISA, PCR, cloning, and sequencing; (iii)
molecular characterization of Begomovirus using software BioEdit v.7.0.5, CLC
Sequence Viewer, and MEGA 6.06; (iv) construction and use of specific primer;
and (v) Koch‟s Postulate trial using whitefly transmission.
Yellow mosaic disease was found in almost all fields surveyed. Infection of
Potyvirus and Begomovirus was detected using I-ELISA and PCR, respectively.
Both viruses were detected as either single or mixed infection. Begomovirus
infections were detected more often than Potyvirus, which indicated that
Begomovirus is predominant in all locations. Begomovirus infecting yard long
bean in all samples has a bipartite genome based on detection using
PAL1v1978/PAR1c715 universal primer pair and PBL1v2040/PCR1c, i.e.
possessing DNA-A and DNA-B.
Common region sequence of DNA-A has identity >85% to that ofMungbean
yellow mosaic India virus (MYMIV). Further phylogenetic analysis to study their
relationship showed that all Begomoviruses infecting mungbean (MYMIV and
MYMV) are belong to similar cluster and they are separated from Begomoviruses
which have been reported previously. All MYMIV isolates from Java have three
unique characteristics, i.e. three repetitive sequences ATCGGTGT and one invert
sequence ACACCGAT, TATA box sequence, and hairpin loop structure sequence
which
consist
of
32
nucleotides
GGGCACTCAGCTATA
ATATTACCTGAGTGCCC. All CR components of all MYMIV isolates from
Java has similarity with MYMIV from India, Pakistan, and Nepal with >85%
homology; whereas homology among Java isolates reached >90 – 100%. Spesific
primer pairs MYF/MYR and MY1/MY2 were successfully amplified specific
DNA target of 1000 bp and 238 bp, respectively and can be used to distinguish
MYMIV from other Begomoviruses.
Transmission study to fulfill Koch‟s Postulateusing three yard long bean
varieties resulted in similar yellow mosaic symptom with those in the field.
„Parade‟ is the most susceptible variety with 100% (5/5) disease incidence,
followed by „New Jaliteng‟ and „Wulung‟ with both caused 40% (2/5) disease
incidence. Incubation period of Begomovirus in all varieties was 14 days in
average.
Diagnosis based on molecular detection and transmission study confirmed
that Mungbean yellow mosaic India begomovirus is the main causal agent of
yellow mosaic disease of yard long bean. Disease control strategy to suppress
incidence, severity and dissemination of the disease should be undertaken
promptly.
Keywords: common region, DNA sequencing, ELISA, MYMIV, PCR
RINGKASAN
SARI NURULITA. Karakterisasi Molekuler Begomovirus yang Menginfeksi
Kacang Panjang (Vigna unguiculata subsp. sesquipedalis L.) dan Pembuatan
Primer Spesifik. Dibimbing oleh SRI HENDRASTUTI HIDAYAT dan KIKIN
HAMZAH MUTAQIN.
Begomovirus merupakan salah satu patogen penting yang menginfeksi
beberapa komoditas hortikultura utama di negara tropis dan sub-tropis. Kerugian
ekonomi akibat infeksi Begomovirus di beberapa negara dilaporkan mencapai
jutaan dolar. Infeksi Begomovirus di Indonesia pada tanaman hortikultura pertama
kali dilaporkan tahun 1999 pada tanaman cabai dengan kejadian penyakit 30 –
100%. Tanaman cabai yang terinfeksi Begomovirus mempunyai gejala yang khas,
yaitu daunnya mengeriting dan menguning.
Gejala mosaik kuning juga mulai ditemukan di sejumlah pertanaman kacang
panjang pada tahun 2008 dan kejadian penyakit semakin meningkat terutama di
beberapa daerah di Jawa. Gejala yang sama juga dilaporkan pada tanaman
Leguminosae di India, Bangladesh, Pakistan, dan Nepal. Penyakit mosaik kuning
di kawasan Asia Selatan ini disebabkan oleh infeksi Begomovirus. Penyakit
mosaik kuning di Jawa diduga juga disebabkan oleh Begomovirus.
Begomovirus merupakan salah satu genus terbesar dari famili
Geminiviridae. Begomovirus mempunyai karakter molekuler yang unik dengan
partikel kembar (bipartit) atau tunggal (monopartit). Partikel bipartit Begomovirus
terdiri dari DNA-A dan DNA-B, masing-masing berukuran 2.6 – 2.7 kb dengan
fungsi yang berbeda. Partikel monopartit mempunyai fungsi gabungan dari DNAA dan DNA-B. Identitas setiap spesies Begomovirus ditemukan pada bagian
common region (CR). Setiap CR Begomovirus mempunyai tiga komponen unik,
yaitu sekuen berulang (iteron), sekuen TATA box, dan struktur hair pin loop.
Penularan Begomovirus di lapangan sebagian besar melalui vektornya
Bemisia tabaci Gen. (Hemiptera: Aleyrodidae) secara persisten sirkulatif.
Begomovirus tidak dapat ditularkan baik secara mekanis maupun benih. Kisaran
inang yang luas dan penyebaran melalui vektor membuat kejadian penyakit
Begomovirus tinggi dan sulit dikendalikan.
Deteksi Begomovirus umumnya dilakukan dengan metode Polymerase chain
reaction (PCR). Metode PCR merupakan salah satu teknik deteksi yang sensitif
dan cepat. Primer merupakan salah satu komponen utama dalam proses PCR.
PAL1v1978/ PAR1c715 dan AV494/ AC1048 merupakan primer universal yang
banyak digunakan untuk mendeteksi Begomovirus. Primer PAL1v1978/
PAR1c715 dilaporkan dapat mendeteksi Begomovirus yang menginfeksi beberapa
tanaman dari famili Solanaceae, Leguminosae, Euphorbiaceae, dan Malvaceae.
Pasangan primer PAL1v1978/ PAR1c715 mengamplifikasi bagian CR, sedangkan
primer AV494/ AC1048 mengamplifikasi pada bagian protein selubung.
Penelitian ini bertujuan untuk melaporkan pentingnya infeksi Begomovirus
pada tanaman kacang panjang, mengidentifikasi spesies Begomovirus pada
tanaman kacang panjang, menganalisis karakter molekuler dan membuat primer
spesifik untuk mendeteksi spesies Begomovirus yang menginfeksi tanaman
kacang panjang.
Penelitian ini dilakukan dengan beberapa tahapan, yaitu: (i) survei penyakit
di beberapa lokasi pertanaman kacang panjang di provinsi Jawa Tengah (Tegal,
Magelang, dan Klaten), D.I. Yogyakarta (Sleman dan Kalasan), serta Jawa Barat
(Bogor dan Subang); (ii) deteksi beberapa virus yang menginfeksi tanaman
kacang panjang menggunakan I-ELISA serta deteksi Begomovirus menggunakan
PCR, cloning, dan sekuensing; (iii) analisis karakter molekuler Begomovirus
menggunakan perangkat lunak BioEdit v.7.0.5, CLC Sequence Viewer, dan
MEGA 6.06; (iv) membuat primer spesifik menggunakan perangkat lunak
Oligonucleotide Calculator dan PrimerBLAST; serta (v) uji postulat Koch
menggunakan penularan kutukebul dan inokulum dari lapangan untuk
membuktikan agen penyebab gejala mosaik kuning.
Gejala mosaik kuning ditemukan hampir di semua lokasi survei.
Berdasarkan hasil deteksi menggunakan I-ELISA dan PCR ditemukan adanya
infeksi Potyvirus tunggal, Begomovirus tunggal, serta campuran antara
Begomovirus dan Potyvirus. Hasil deteksi seluruh sampel menunjukkan dominasi
infeksi Begomovirus di beberapa lokasi. Hasil deteksi menggunakan primer
universal PAL1v1978/ PAR1c715 dan PBL1v2040/ PCR1c membuktikan bahwa
Begomovirus yang menginfeksi tanaman kacang panjang di Indonesia memiliki
genom bipartite, yaitu memiliki DNA-A dan DNA-B.
Sekuen bagian CR DNA-A isolat Begomovirus hasil survei mempunyai nilai
kemiripan lebih dari 85% dengan spesies Mungbean yellow mosaic India virus
(MYMIV). Semua isolat MYMIV asal Jawa pada analisis filogenetik
mengelompok bersama isolat MYMIV asal India, Banglades, Nepal, dan Pakistan.
Isolat MYMIV ini terpisah dari spesies-spesies Begomovirus Indonesia yang telah
dilaporkan sebelumnya.
Semua isolat MYMIV asal Jawa memiliki tiga karakter unik CR, yaitu tiga
sekuen berulang ATCGGTGT dan satu invert sequence ACACCGAT, sekuen
TATA box, dan struktur pembentuk hairpin loop yang terdiri dari 32 sekuen
nukleotida
GGGCACTCAGCTATAATATTACCTGAGTGCCC.
Semua
komponen CR isolat MYMIV asal Jawa memiliki kesamaan dengan CR isolat
MYMIV asal India, Pakistan, dan Nepal. Sekuen CR semua isolat MYMIV asal
Jawa juga memiliki nilai homologi >85% dengan isolat MYMIV asal Banglades,
India, Nepal, dan Pakistan serta memiliki homologi >90 – 100% antara sesama
isolat asal Indonesia. Primer spesifik MYF/ MYR dan MY1/ MY2 yang didesain
dalam penelitian ini berhasil mengamplifikasi MYMIV secara spesifik berturutturut pada 1000 bp dan 238 bp.
Hasil penularan (uji postulat Koch) menggunakan kutukebul B. tabaci pada
tiga varietas kacang panjang menghasilkan gejala mosaik kuning sama dengan
yang ada di lapangan. Varietas „Parade‟ merupakan varietas paling rentan dengan
kejadian penyakit 100% (5/5) diikuti oleh „New Jaliteng‟ dan „Wulung‟ masingmasing sebesar 40% (2/5). Masa inkubasi Begomovirus pada semua varietas
adalah 14 hari.
Diagnosis penyebab penyakit mosaik kuning kacang panjang berdasarkan
deteksi molekuler dan uji penularan membuktikan Mungbean yellow mosaic India
begomovirus sebagai agens utama penyebab penyakit. Strategi pengendalian
untuk menekan kejadian, keparahan dan penyebaran penyakit harus segera
diupayakan.
Kata kunci: Common Region, ELISA, MYMIV, PCR, Sekuensing DNA
©2014, Bogor Agricultural University
All Right Reserved
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citing the author or the copyright holder. Quotation is allowed as long as for
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No part of this thesis may be reproduced or transmitted in any forms or by any
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Bogor Agricultural University.
MOLECULAR CHARACTERIZATION OF BEGOMOVIRUS INFECTING
YARD LONG BEAN (Vigna unguiculata subsp. sesquipedalis L.) AND
CONSTRUCTION OF ITS SPECIFIC PRIMERS
SARI NURULITA
Thesis
in partial fulfillment of the requirements for the degree of
Master of Science
at the
Phytopathology Study Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
External Examiner on Thesis Defense: Dr Ir Gede Suastika, MSc
Thesis title : Molecular Characterization of Begomovirus Infecting Yard Long
Bean (Vigna unguiculata subsp. sesquipedalis L.) and Construction
of Its Specific Primers
Name
: Sari Nurulita
SIN
: A352110081
Approved by
Committee members
Prof Sri Hendrastuti Hidayat, PhD
Supervisor
Kikin Hamzah Mutaqin, PhD
Co-Supervisor
Acknowledged by
Head of Phytopathology
Study Program
Dean of Graduate School
Prof Sri Hendrastuti Hidayat, PhD
Dr Ir Dahrul Syah, MScAgr
Date of Examination: July 14th, 2014
Date of Submission:
PREFACE
I am very grateful to Allah Subhanallahuwataala for the mercy, so I can
finish this thesis. From this moment I would also like to thank the people who
specially contribute to my education.
I would like to express my thankfulness to my committee members, Prof.
Sri Hendrastuti Hidayat and Dr. Kikin Hamzah Mutaqin for their guidance and
support, both in scientific and personal matters. Their patience and excellent
advice made this work enjoyable and rewarding. I am very thankful to Dr. Gede
Suastika for his suggestions during thesis defense.
I am very thankful to Dr. John Thomas and Matthew Webb, Department of
Agriculture, Forestry and Fisheries (DAFF) Queensland – Australia, for their
technical assistance especially for teaching me laboratory technique and providing
molecular material in Plant Virology Laboratory, Australia. I also thank Dr.
Cherie Gambley for the guidance on methodology to design pecific primers. I
thank Tuti Legiastuti, SSi for her assistance in Plant Virology Laborator, IPB. I
would also like to thank Dr. Sri Sulandari, Gadjah Mada University, for helping
me during the survey.
I am very thankful to AusAID Funded Economic Cooperation Work
Program of the ASEAN – Australia – New Zealand Free Trade Agreement for
funding my research. I am very thankful to I-MHERE B.2C IPB for granting me a
master scholarship.
I would also like to thank my classmates “Phytopathology class 2011”,
Plant Virology Laboratory members, especially Dwiwiyati Nurul Septariani for
their supports and cooperations. My appreciation goes to my family who always
support and care me from far away.
Partial of this thesis has been presented in International Workshop on
Collaborative Educations for Sustainability of Agriculture and Environment,
Ibaraki University – Japan 2012; Indonesian Phytopathology Society, Padang
2013; also has been submitted to Biotropia Journal – SEAMEO Biotrop.
Bogor, August 2014
Sari Nurulita
TABLE OF CONTENTS
LIST OF TABLES
vi
LIST OF FIGURES
vi
LIST OF APPENDIX
vi
INTRODUCTION
Background
Hypothesis
Research Objectives
Research Advantages
1
1
3
3
3
LITERATURE REVIEWS
Importance of Begomovirus Infection on Agricultural Production
Disease Control Strategy for Begomovirus Infection
Legume – Infecting Begomovirus
Biological Characteristics of Begomovirus
Organization and Molecular Characteristics of Begomovirus
Diagnosis and Detection of Begomovirus
5
5
5
6
7
8
9
MATERIALS AND METHODS
Field Survey and Samples Collection
Detection and Identification of Viruses from Infected Leaves
Virus Detection by I-ELISA
Begomovirus Detection by PCR
Cloning Strategy of DNA-A of Begomovirus
DNA Sequencing
Construction of Specific Primers
Evaluation of Primer Specificity
Koch's Postulate
11
11
11
11
12
12
14
14
16
16
RESULTS AND DISCUSSION
Identification of Begomovirus from Field Samples
Cloning of DNA-A Begomovirus
Sequence Identity Analysis of Begomovirus Infecting Yard Long
Bean
Analysis of Common Region of Begomovirus Infecting Yard Long
Bean
Specificity of MYMIV Specific Primers
Koch‟s Postulate
General Discussion
17
17
19
CONCLUSION
31
REFERENCES
32
20
21
27
28
29
APPENDIXES
40
BIOGRAPHY
49
LIST OF TABLES
1 Genome organization of Begomovirus
2 Degenerate primers for amplification of Begomovirus using polymerase
chain reaction
3 Specific primers for amplification of Begomovirus infecting yard long
bean
4 Detection of CMV, Potyvirus, and Begomovirus from leaf samples
collected from various locations using I-ELISA and PCR
5 Nucleotide sequence homology (%) of Begomovirus infecting yard long
bean in Java with other Begomoviruses reported earlier in GenBank
6 Comparison of repetitive sequences (iteron) between MYMIV and
MYMV on common region
7 Nucleotide sequence homologies (% identity) of common region among
Begomovirus
8
12
15
19
20
24
26
LIST OF FIGURES
1 Research flow chart: “Molecular Characterization of Begomovirus
Infecting Yard Long Bean (Vigna unguiculata subsp. sesquipedalis L.)
and Construction of Its Specific Primers”
2 Genome organization of Begomovirus: bipartite (above) and
monopartite (below)
3 Hair pin loop structure of Begomovirus
4 pCR®4-TOPO® vector for cloning
5 Alignment of conserved region to design specific primer for
Begomovirus infecting yard long beans
6 Construction of specific primer using primer BLAST program at NCBI
7 Various symptoms found associated with yellow mosaic disease on
yard long bean
8 Visualization of PCR products of Begomovirus amplification from leaf
samples using universal primers PAL1v1978/ PAR1c715 on 1 %
agarose gel
9 Visualization of PCR products of Begomovirus amplification from leaf
samples using universal primers PBL1v2040/ PCR1c on 1 % agarose
gel
10 Visualization of PCR product from Begomovirus clone (pTgl)
11 Phylogenetic analysis based on alignment of partial nucleotide
sequences of the DNA-A of Begomoviruses using Mega 6.06
(Algorithm Neighbour Joining with 1000 bootstraps replicates)
12 Alignment of nucleotide sequences of the CR of Mungbean yellow
mosaic India virus (MVMIV) isolates from Java with other reported
MYMIV in Genbank
13 Hairpin loop structures of several Begomovirus isolates
14 Amplification of partial DNA-A Begomovirus genome using universal
primer PAL1v1978/PAR1c715 (A), specific primers MYF/MYR (B),
and MY1/MY2 (C)
4
9
10
13
15
16
17
18
18
20
21
22
25
27
LIST OF APPENDIXES
1 Sequence alignment of all MYMIV isolates
2 Description of yard long bean varieties
40
48
INTRODUCTION
Background
Begomovirus is an important plant viral pathogen group causing serious
diseases in several tropical and sub-tropical countries (Mansoor et al. 2003;
Varma and Malathi 2003). Infection of Begomovirus resulted in crop damage and
yield loss up to 20% or equal to US$ 140 million economic losses in tomato
production in Florida, US (Moffat 1999; Polston et al. 1999). Mosaic disease of
cassava in East and Central Africa was also the reason for US$ 1.9 – 2.7 billion
yield loss (Patil and Fauquet 2009). Similar situation was reported from cucurbit
production in India which was suffered from yellow mosaic disease (Varma and
Malathi 2003; Varma et al. 1992). In Indonesia, serious infection of Begomovirus
caused disease epidemic on some chilli production regions in Central Java and
West Java Province, and Special District of Jogyakarta (Sulandari et al. 2006).
Infection of Begomovirus in chilli pepper produced 30-100% disease incidence
since 1999 (Hidayat et al. 2006).
Yellow mosaic disease outbreak has been reported on yard long bean
(Vigna unguiculata subsp. sesquipedalis L.) growing area in Java since 2008.
Bean common mosaic virus (BCMV) and Cucumber mosaic virus (CMV) were
identified from leaf samples showing yellow mosaic symptom collected from yard
long bean growing area in Java (Damayanti et al. 2009). In the meantime similar
yellow mosaic disease was first reported from Pakistan and Nepal on grain
legume, mungbean, cowpea, soybean, kidney bean and Rhynchosia capitata.
Further identification showed the involvement of bipartite Begomovirus on this
disease (Ilyas et al. 2010; Shahid et al. 2012).
Begomovirus is the largest genus of the Family Geminiviridae. Its natural
spread relies on its specific insect vector, Bemicia tabaci (Hemiptera :
Aleyrodidae) which transmit the virus in circulative persistent manner. Most
Begomovirus is not transovarially transmitted, except TYLCV from Israel
(Ghanin et al. 1998). Since B. tabaci is a polyphagous insect, Begomovirus has a
high chance to infect many plant species. For instance, tomato leaf curl disease
(ToLCD) caused by Begomovirus has been observed on many countries. Tomato
leaf curl New Delhi virus (ToLCNDV) was investigated as new emerging virus
causing yellow mosaic disease on eggplant in India (Pratap et al. 2011).
ToLCNDV was also reported infecting cucumber plant in Central Java, Indonesia
(Mizutani et al. 2011). Another ToLCD caused by Tomato leaf deformation virus
was characterized as a new world monopartite Begomovirus infecting tomato in
Peru and Ecuador (Melgarejo et al. 2013). A new Begomovirus associated with
tomato yellow leaf curl and eggplant yellow mosaic disease, Tomato yellow leaf
curl Kanchanaburi virus was found in Thailand (Green et al. 2003), Indonesia
(Nurul and Tsai 2009), and Laos (Tang et al. 2014). Three Begomoviruses were
identified associated with bean golden mosaic disease in Nicaragua i.e., Bean
golden yellow moaic virus (BGYMV), Squash yellow mild mottle virus
(SYMMoV), and Calopogonium golden mosaic virus (CalGMV) (Karkashian et
al. 2011).
2
Molecular characteristics of Begomovirus genome is very unique; it posses
twin particles, each consisting of single stranded DNA (2.6 – 2.7 kb), bipartite or
monopartite (Hull 2002). DNA-A and DNA-B of Begomovirus have different
genome organization and function except their common region (CR). Among
other important function encodes by genome of DNA-A are virus replication and
insect transmission, whereas those encodes by DNA-B are systemic infection and
symptom development. Common region of both DNA-A and DNA-B each
consists of about 200 nucleotides and its sequence is highly identical for one
species of Begomovirus. In contrast, common region of each species of
Begomovirus is distinctinve, except for loop region on hairpin structure, i.e. 5‟TAATATTAC -3‟ which is known as the conserved nona-nucleotide region
(Lazarowitz 1987). There are three unique characteristics of common region
which is observed on every Begomovirus species, i.e. TATA box, repeated motifs
(iterons), and hairpin-loop structure (Hidayat et al. 2008; Lazarowitz 1987; Trisno
et al. 2009). Begomovirus possesing >92% similarity on their hairpin loop
structure can be grouped as one species (Fauquet et al. 2005).
The most common method for virus detection is using serological-based
technique, but different situation occurred for Begomovirus. Cross reaction and
low specificity are the reasons why serological method is not preferred for
detection of Begomovirus (Kushwaha et al. 2010). Method for detection of
Begomovirus is commonly based on polymerase chain reaction (PCR) technique
using universal primers. Several universal primers has been reported to work well
for different species of Begomovirus. Rojas et al. (1993) used degenerate primer
PAL1v1978/ PAR1c715 to detect several geminivirus from infected plants belong
to Solanaceae, Leguminosae, Euphorbiaceae, and Malvaceae from South America.
Li et al. (2004) used a pair of degenerate primer SPG1/ SPG2 to detect
geminivirus infecting sweet potato and other Ipomoea sp. Another pair of
degenerate primer, AV494/ AC1048, was used to detect coat protein of subgroup
III geminivirus (Wyatt and Brown 1996).
Despite the benefit of using universal primers, specific primers might
increase efficiency and effectiveness of detection method. Specific primer is used
to positively identify and determine the species involved (Ye et al. 2012). TorresPacheco et al. (1996) used three sets of specific primers to detect intergenic region
(IR) of DNA-A of Pepper jalapeño virus (PJV)/ Texas pepper geminivirus (TPV),
IR of DNA-B Chino del tomato virus (CdTV), and IR of DNA-A Pepper huasteco
virus (PHV) which infecting pepper in Mexico. Pratap et al. (2011) developed
specific primers to confirm agroinfectious clone of DNA-A and DNA-B
ToLCNDV infecting eggplant. Primer pair of HOG1/ HOG2 was specifically
designed to amplify full length DNA-A MYMIV using rolling circle replication
method (Shahid et al. 2012). Availability of specific primer for specific virus will
be very useful for early diagnosis especially when immediate control strategy is
required to take place.
This research is intended to determine the association of Mungbean yellow
mosaic India virus (MYMIV) with yellow mosaic disease on yard long bean in
Java and further characterization based on its specific genome property of
common region.
3
Hypothesis
(i)
There are multiple viruses associated with yellow mosaic disease on yard
long bean, one of them is Begomovirus.
(ii) Begomovirus infecting yard long bean is different from those previously
reported in Indonesia.
(iii) Begomovirus infecting yard long bean in Indonesia has close genetic
relationship with other Begomoviruses infecting Leguminosae in Asia.
(iv) Specific primer for Begomovirus infecting yard long bean will specifically
amplify virus target.
Research Objectives
Objective of this research is to determine the importance of Begomovirus
infection on yard long bean, to identify species of Begomovirus on yard long bean
and to analyse its molecular character, and to design specific primers for detection
of Begomovirus species infecting yard long bean.
Research Advantages
Basic information regarding molecular and biological characteristics of the
virus obtained from this research will be useful for further study on disease
ecology and for establishment of disease control strategy.
4
Field Survey and Virus Detection
West Java Province (Bogor and Subang);
Central Java Province (Tegal, Klaten, and Magelang);
Special District of Jogyakarta (Bantul and Sleman)
Molecular
detection
Serology
detection
Virus isolates from
the field
Potyvirus and CMV
Begomovirus
Nucleotide sequence
Clone
Molecular characterization:
bipartite or monopartite
hair pin loop structure
Primer
construction
Identity of Begomovirus
Specific primer
DNA probe
: process
: future process
: result
: future prospective result
Fig 1 Research flow chart: “Molecular Characterization of Begomovirus Infecting
Yard Long Bean (Vigna unguiculata subsp. sesquipedalis L.) and
Construction of Its Specific Primers”
5
LITERATURE REVIEWS
Importance of Begomovirus Infection on Agricultural Production
Begomovirus was reported to cause serious problems in the sub-tropical
and tropical region and its infection may potentially cause yield lost (Varma et al.
2011). Tomato production in Florida, USA was suffered from tomato yellow leaf
curl disease (TYLCD) which caused 20% yield lost or US$ 140 million economy
losses (Moffat 1999; Polston et al. 1999). TYLCD was also reported causing yield
loss up to 99% of tomato production in Central America, Europe, Middle East,
and South East Asia (Polston and Anderston 1993; Rochester et al. 1994).
Legume-infecting Begomovirus, Bean golden mosaic virus, caused 20 – 95% and
36.7% disease incidence in Nicaragua and Argentina, respectively (Karkashian et
al. 2011; Alemandri et al. 2012).
Similar condition was also reported from India and Africa, in which up to
50% yield loss of cucurbit in Northern India was occurred in 2001 due to Tomato
leaf curl virus (ToLCV) infection (Varma and Malathi 2003). Disease incidence
caused by Tomato leaf curl New Delhi virus (ToLCNDV) reached 60 – 66% and
damaged eggplant production in India (Pratap et al. 2011). Serious infection of
Begomovirus on cassava caused economy losses of US$ 1.9 – 2.7 billion in East
and Central Africa (Patil and Fauquet 2009). Yellow mosaic disease on legume
was a very serious problem in India because it caused up to US$ 300 million
(Varma et al. 1992). MYMV infection in India ranged from 4 to 40% depend on
crop variety and management (Bashir et al. 2006). Another species, MYMIV, was
reported infecting Phaseolus vulgaris in Pakistan (Naimuddin et al. 2011) and
Vigna mungo in Nepal (Shahid et al. 2011) with disease incidence reached 70 –
100% and 70 – 80%, respectively.
Incidence of Begomovirus was also reported as serious problem in Indonesia.
Pepper yellow leaf curl Indonesia virus (PepYLCIV) was the main disease
problem on chili pepper in Central Java, Yogyakarta, and West Java with 70 –
100% and 10 – 35% disease incidence for Capsicum frutescence and C. annuum,
respectively (Sulandari et al. 2006). Tomato leaf curl virus (ToLCV) infected
tomato in Java and Sulawesi with 100% disease incidence (Aidawati et al. 2005;
Tsai et al. 2009). Infection of Tobacco leaf curl virus Begomovirus on tobacco in
East Java reached 30% disease incidence (Trisusilowati 1990).
Disease control strategy for Begomovirus infection
Various control methods has been applied and evaluated in order to suppress
disease incidence caused by Begomovirus. Most of the control strategy
recommended was based on insect vector management. Preventive strategy in the
seedling and young plant using seedbeds with plastic polyethylene sheets was
common and was able to avoid Begomovirus transmission by whitefly (Hidayat et
al. 2010). Utilization of UV-absorbing plastic films as barrier and neem extract
can control whitefly population in the greenhouse (Antignus 1996; Senguttuvan et
al. 2005). Combination of screen and insecticide sprays in the greenhouse can
reduce TYLCV incidence up to 99% (Antignus 1996; Berlinger et al. 2002).
6
Cultural practice using border plants, such as maize, yard long bean, and
Crotalaria sp. as physical barrier around chili pepper plantation can delayed
PYLCIV for the first 30 days after transplanting (Hidayat et al. 2010). The maize
and cowpea intercrops on cassava plantation in southern Cameroon can reduce B.
tabaci population and cassava mosaic disease incidence up to 50% and 20%,
respectively (Fondong et al. 2002).
Treatment using bio-control agent such as PGPR (plant growth promoting
rhizobacteria) or PGPF (plant growth promoting fungi) was reported on several
studies in attempt to control Begomovirus infection. These treatments was
potentially delayed incubation period and decreased disease incidence of
PepYLCIV (Hidayat et al. 2010), induced systemic resistance of PepYLCIV
(Damayanti 2013) and CMV (Raupach et al. 1996), and reduced disease incidence
of ToMoV up to 40 days after treatment (Murphy et al. 2000).
Growing resistance varieties has been recommended as control strategy for
Begomovirus (Mason et al. 2000) and effort to develop resistance varieties has
been studied for various crops. Screening of germplasms from various sources is
important to identify source of resistance in the breeding program (Larsen and
Porter 2011). Evaluation of mungbean breeding lines was reported but all
germplasms was susceptible against MYMV based on observation on incubation
period and disease incidence (Shad et al. 2006). Recently, Akhtar et al. (2011)
reported there were 35 genotypes of mungbean showing moderate resistant toward
MMIV infection. Santoso (2008) reported F1-TYLCV which was crossed from
AVRDC germplasm and commercial seed (F1 FLA456/ Intan and FLA456/
CL6046) showed resistant response up to 68% and 66% toward TYLCV infection,
respectively.
The use of transgenic lines to reduce Begomovirus infection were reported.
RNA interference mechanism (RNAi/ silencing mechanism) can reduce viral
DNA replication of African cassava mosaic virus (ACMV) up to 99%
(Vanitharani et al. 2003). Transgenic cassava plant which resistant to cassava
mosaic disease was also observed by Zhang et al. (2005). Another mechanism,
intron-spliced hairpin construction was used to delay seven weeks of TYLCV
incubation period in tomato transgenic line (Zrachya et al. 2007) and recovery
MYMIV infection in blackgram (Vigna mungo) (Pooggin et al. 2003).
Legume–Infecting Begomovirus
Legume-infecting Begomovirus was reported as serious problem in subtropical and tropical countries (Varma and Malathi 2003). Symptom variation is
reported although it can be differentiated into two types, i.e. golden mosaic and
yellow mosaic. Golden mosaic disease was generally found in the new world such
as South and Central America and the Caribbean Basin. Bean golden mosaic virus
(BGMV) and Bean golden yellow mosaic virus (BGYMV) have been identified as
causal agent of bean golden mosaic disease in Brazil, Puerto Rico, and Nicaragua
(Gilbertson et al. 1993; Karkashian et al. 2011). Bean golden mosaic disease is
recognized by its typical symptoms including bright golden color of the leaves,
stunting, and smaller pods.
Yellow mosaic disease was more common in the old world, i.e. South and
South East Asia (Stanley et al. 2005; Shahid et al. 2012). Yellow mosaic disease
7
on legume characterized by bright yellow leaves with green spot or complete
yellowing, smaller pods and seeds was first reported from India (Nariani 1960).
Similar symptom was also reported in other Asian countries, such as Pakistan
(Qazi et al. 2007), Nepal (Shahid et al. 2012), and Thailand (Honda et al. 1983).
There are five species of Begomovirus causing yellow mosaic disease in Asia, i.e.
Mungbean yellow mosaic virus (MYMV), Mungbean yellow mosaic India virus
(MYMIV), Horsegram yellow mosaic virus (HgYMV), Dolichos yellow mosaic
virus (DoYMV) (Maruthi et al. 2006; Qazi et al. 2007), and a new species
proposed as Rhynchosia yellow mosaic virus (RhYMV) (Ilyas et al. 2009). These
species were distinct to other Begomovirus from new world which was identified
as causal agent of yellow mosaic disease on Leguminosae (Briddon and Stanley
2006).
MYMV and MYMIV were reported as the most destructive agent for some
Leguminosae crops in India and Pakistan. MYMV was found to infect
Leguminosae in Southern and Western India (Morinaga et al. 1993) whereas
MYMIV was reported to infect Leguminosae in Northern and Central region
(Mandal et al. 1997), and in Pakistan (Ilyas et al. 2010). MYMV and MYMIV
have close relationship based on phylogenetic analysis except in common region
(CR). CR of MYMIV has >95% identity with the same isolate but only
YARD LONG BEAN (Vigna unguiculata subsp. sesquipedalis L.) AND
CONSTRUCTION OF ITS SPECIFIC PRIMERS
SARI NURULITA
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
AUTHOR’S STATEMENT ON THESIS AND ITS SOURCE OF
INFORMATION AND DELEGATION OF COPYRIGHT
I declare that this thesis entitled Molecular Characterization of Begomovirus
Infecting Yard Long Bean (Vigna unguiculata subsp. sesquipedalis L.) and
Construction of Its Specific Primers is my own and authentic work under
supervision of a thesis commitee and it is not yet submitted to any universities for
any degree fulfillment. Source of information, both published or unpublished by it
authors, used for quotations in this thesis is already cited appropriately and
present in thesis‟s Literature Cited chapter.
I hereby delegate my thesis copyright to the Bogor Agricultural University.
Bogor, August 2014
Sari Nurulita
Student ID number: A352110081
Copyright delegation of scientific paper from research collaboration with other
organization must be referred on its collaboration agreement.
SUMMARY
SARI NURULITA. Molecular Characterization of Begomovirus Infecting Yard
Long Bean (Vigna unguiculata subsp. sesquipedalis L.) and Construction of Its
Specific Primers. Supervised by SRI HENDRASTUTI HIDAYAT and KIKIN
HAMZAH MUTAQIN.
Begomovirus is an important plant viral pathogen group causing serious
diseases in several tropical and sub-tropical countries. Infection of Begomovirus
was reported to cause economic losses up to million dollars. Infection of
Begomovirus on horticultural crops in Indonesia was first reported on chilli pepper
in 1999 with disease incidence reached 30 to 100%. Infection of Begomovirus on
chili pepper is obviously recognized by its typical symptoms involving leaf
curling and yellowing.
Yellow mosaic disease outbreaks have also been reported on yard long bean
(Vigna unguiculata subsp. sesquipedalis L.) growing area in Java since 2008. At
the same time, similar yellow mosaic disease was first reported in Pakistan and
Nepal on Leguminosae. The causal agent of yellow mosaic disease in South Asia
was identified as a member of Begomovirus. Yellow mosaic disease on yard long
bean in Java was considered to be caused by Begomovirus.
Begomovirus is the largest genus of the Family Geminiviridae. Molecular
characteristics of Begomovirus genome is very unique, comprising twin circular
particles, each particle is consisting of single stranded DNA (2.6 – 2.7 kb),
bipartite or monopartite. DNA-A and DNA-B of bipartite Begomovirus have
different genome organization and function except for their common region. On
the other hand, monopartite Begomovirus has all combination function of DNA-A
and DNA-B in one genome. There are three unique characteristics of common
region which is observed on every Begomovirus species, i.e. TATA box, repeated
motives (iterons), and hairpin-loop structure.
Begomovirus is only transmitted in nature by insect vector, B. tabaci
(Hemiptera: Aleyrodidae). Studies on virus – vector relationship indicates that
Begomovirus is transmitted in circulative persistent manner. Begomovirus can not
be transmitted by mechanical nor seed manner. The nature of Begomovirus of
being insect transmissable and having a wide host range may contribute to its
potential to cause high disease incidence and being difficult to control.
Common method for detection of Begomovirus is based on polymerase
chain reaction (PCR). PAL1v1978/PAR1c715 and AV494/AC1048 primer pairs
have been reported to work well for different species of Begomovirus. The former
pair amplifies common region (CR), whereas the latter amplifies coat protein gene
of Begomovirus. PAL1v1978/PAR1c715 was successfully used to detect several
geminivirus from Solanaceae, Leguminosae, Euphorbiaceae, and Malvaceae
plants.
The objective of this research is to determine the importance of
Begomovirus infection on yard long bean, to identify species of Begomovirus on
yard long bean and to analyse its molecular character, and to design specific
primers for detection of Begomovirus species infecting yard long bean.
Research was conducted in four activities: (i) field survey and sample
collection in yard long bean fields located in Central Java (Tegal, Magelang, and
Klaten), Jogyakarta (Sleman and Klaten), and West Java (Bogor and Subang)
provinces; (ii) virus detection using I-ELISA, PCR, cloning, and sequencing; (iii)
molecular characterization of Begomovirus using software BioEdit v.7.0.5, CLC
Sequence Viewer, and MEGA 6.06; (iv) construction and use of specific primer;
and (v) Koch‟s Postulate trial using whitefly transmission.
Yellow mosaic disease was found in almost all fields surveyed. Infection of
Potyvirus and Begomovirus was detected using I-ELISA and PCR, respectively.
Both viruses were detected as either single or mixed infection. Begomovirus
infections were detected more often than Potyvirus, which indicated that
Begomovirus is predominant in all locations. Begomovirus infecting yard long
bean in all samples has a bipartite genome based on detection using
PAL1v1978/PAR1c715 universal primer pair and PBL1v2040/PCR1c, i.e.
possessing DNA-A and DNA-B.
Common region sequence of DNA-A has identity >85% to that ofMungbean
yellow mosaic India virus (MYMIV). Further phylogenetic analysis to study their
relationship showed that all Begomoviruses infecting mungbean (MYMIV and
MYMV) are belong to similar cluster and they are separated from Begomoviruses
which have been reported previously. All MYMIV isolates from Java have three
unique characteristics, i.e. three repetitive sequences ATCGGTGT and one invert
sequence ACACCGAT, TATA box sequence, and hairpin loop structure sequence
which
consist
of
32
nucleotides
GGGCACTCAGCTATA
ATATTACCTGAGTGCCC. All CR components of all MYMIV isolates from
Java has similarity with MYMIV from India, Pakistan, and Nepal with >85%
homology; whereas homology among Java isolates reached >90 – 100%. Spesific
primer pairs MYF/MYR and MY1/MY2 were successfully amplified specific
DNA target of 1000 bp and 238 bp, respectively and can be used to distinguish
MYMIV from other Begomoviruses.
Transmission study to fulfill Koch‟s Postulateusing three yard long bean
varieties resulted in similar yellow mosaic symptom with those in the field.
„Parade‟ is the most susceptible variety with 100% (5/5) disease incidence,
followed by „New Jaliteng‟ and „Wulung‟ with both caused 40% (2/5) disease
incidence. Incubation period of Begomovirus in all varieties was 14 days in
average.
Diagnosis based on molecular detection and transmission study confirmed
that Mungbean yellow mosaic India begomovirus is the main causal agent of
yellow mosaic disease of yard long bean. Disease control strategy to suppress
incidence, severity and dissemination of the disease should be undertaken
promptly.
Keywords: common region, DNA sequencing, ELISA, MYMIV, PCR
RINGKASAN
SARI NURULITA. Karakterisasi Molekuler Begomovirus yang Menginfeksi
Kacang Panjang (Vigna unguiculata subsp. sesquipedalis L.) dan Pembuatan
Primer Spesifik. Dibimbing oleh SRI HENDRASTUTI HIDAYAT dan KIKIN
HAMZAH MUTAQIN.
Begomovirus merupakan salah satu patogen penting yang menginfeksi
beberapa komoditas hortikultura utama di negara tropis dan sub-tropis. Kerugian
ekonomi akibat infeksi Begomovirus di beberapa negara dilaporkan mencapai
jutaan dolar. Infeksi Begomovirus di Indonesia pada tanaman hortikultura pertama
kali dilaporkan tahun 1999 pada tanaman cabai dengan kejadian penyakit 30 –
100%. Tanaman cabai yang terinfeksi Begomovirus mempunyai gejala yang khas,
yaitu daunnya mengeriting dan menguning.
Gejala mosaik kuning juga mulai ditemukan di sejumlah pertanaman kacang
panjang pada tahun 2008 dan kejadian penyakit semakin meningkat terutama di
beberapa daerah di Jawa. Gejala yang sama juga dilaporkan pada tanaman
Leguminosae di India, Bangladesh, Pakistan, dan Nepal. Penyakit mosaik kuning
di kawasan Asia Selatan ini disebabkan oleh infeksi Begomovirus. Penyakit
mosaik kuning di Jawa diduga juga disebabkan oleh Begomovirus.
Begomovirus merupakan salah satu genus terbesar dari famili
Geminiviridae. Begomovirus mempunyai karakter molekuler yang unik dengan
partikel kembar (bipartit) atau tunggal (monopartit). Partikel bipartit Begomovirus
terdiri dari DNA-A dan DNA-B, masing-masing berukuran 2.6 – 2.7 kb dengan
fungsi yang berbeda. Partikel monopartit mempunyai fungsi gabungan dari DNAA dan DNA-B. Identitas setiap spesies Begomovirus ditemukan pada bagian
common region (CR). Setiap CR Begomovirus mempunyai tiga komponen unik,
yaitu sekuen berulang (iteron), sekuen TATA box, dan struktur hair pin loop.
Penularan Begomovirus di lapangan sebagian besar melalui vektornya
Bemisia tabaci Gen. (Hemiptera: Aleyrodidae) secara persisten sirkulatif.
Begomovirus tidak dapat ditularkan baik secara mekanis maupun benih. Kisaran
inang yang luas dan penyebaran melalui vektor membuat kejadian penyakit
Begomovirus tinggi dan sulit dikendalikan.
Deteksi Begomovirus umumnya dilakukan dengan metode Polymerase chain
reaction (PCR). Metode PCR merupakan salah satu teknik deteksi yang sensitif
dan cepat. Primer merupakan salah satu komponen utama dalam proses PCR.
PAL1v1978/ PAR1c715 dan AV494/ AC1048 merupakan primer universal yang
banyak digunakan untuk mendeteksi Begomovirus. Primer PAL1v1978/
PAR1c715 dilaporkan dapat mendeteksi Begomovirus yang menginfeksi beberapa
tanaman dari famili Solanaceae, Leguminosae, Euphorbiaceae, dan Malvaceae.
Pasangan primer PAL1v1978/ PAR1c715 mengamplifikasi bagian CR, sedangkan
primer AV494/ AC1048 mengamplifikasi pada bagian protein selubung.
Penelitian ini bertujuan untuk melaporkan pentingnya infeksi Begomovirus
pada tanaman kacang panjang, mengidentifikasi spesies Begomovirus pada
tanaman kacang panjang, menganalisis karakter molekuler dan membuat primer
spesifik untuk mendeteksi spesies Begomovirus yang menginfeksi tanaman
kacang panjang.
Penelitian ini dilakukan dengan beberapa tahapan, yaitu: (i) survei penyakit
di beberapa lokasi pertanaman kacang panjang di provinsi Jawa Tengah (Tegal,
Magelang, dan Klaten), D.I. Yogyakarta (Sleman dan Kalasan), serta Jawa Barat
(Bogor dan Subang); (ii) deteksi beberapa virus yang menginfeksi tanaman
kacang panjang menggunakan I-ELISA serta deteksi Begomovirus menggunakan
PCR, cloning, dan sekuensing; (iii) analisis karakter molekuler Begomovirus
menggunakan perangkat lunak BioEdit v.7.0.5, CLC Sequence Viewer, dan
MEGA 6.06; (iv) membuat primer spesifik menggunakan perangkat lunak
Oligonucleotide Calculator dan PrimerBLAST; serta (v) uji postulat Koch
menggunakan penularan kutukebul dan inokulum dari lapangan untuk
membuktikan agen penyebab gejala mosaik kuning.
Gejala mosaik kuning ditemukan hampir di semua lokasi survei.
Berdasarkan hasil deteksi menggunakan I-ELISA dan PCR ditemukan adanya
infeksi Potyvirus tunggal, Begomovirus tunggal, serta campuran antara
Begomovirus dan Potyvirus. Hasil deteksi seluruh sampel menunjukkan dominasi
infeksi Begomovirus di beberapa lokasi. Hasil deteksi menggunakan primer
universal PAL1v1978/ PAR1c715 dan PBL1v2040/ PCR1c membuktikan bahwa
Begomovirus yang menginfeksi tanaman kacang panjang di Indonesia memiliki
genom bipartite, yaitu memiliki DNA-A dan DNA-B.
Sekuen bagian CR DNA-A isolat Begomovirus hasil survei mempunyai nilai
kemiripan lebih dari 85% dengan spesies Mungbean yellow mosaic India virus
(MYMIV). Semua isolat MYMIV asal Jawa pada analisis filogenetik
mengelompok bersama isolat MYMIV asal India, Banglades, Nepal, dan Pakistan.
Isolat MYMIV ini terpisah dari spesies-spesies Begomovirus Indonesia yang telah
dilaporkan sebelumnya.
Semua isolat MYMIV asal Jawa memiliki tiga karakter unik CR, yaitu tiga
sekuen berulang ATCGGTGT dan satu invert sequence ACACCGAT, sekuen
TATA box, dan struktur pembentuk hairpin loop yang terdiri dari 32 sekuen
nukleotida
GGGCACTCAGCTATAATATTACCTGAGTGCCC.
Semua
komponen CR isolat MYMIV asal Jawa memiliki kesamaan dengan CR isolat
MYMIV asal India, Pakistan, dan Nepal. Sekuen CR semua isolat MYMIV asal
Jawa juga memiliki nilai homologi >85% dengan isolat MYMIV asal Banglades,
India, Nepal, dan Pakistan serta memiliki homologi >90 – 100% antara sesama
isolat asal Indonesia. Primer spesifik MYF/ MYR dan MY1/ MY2 yang didesain
dalam penelitian ini berhasil mengamplifikasi MYMIV secara spesifik berturutturut pada 1000 bp dan 238 bp.
Hasil penularan (uji postulat Koch) menggunakan kutukebul B. tabaci pada
tiga varietas kacang panjang menghasilkan gejala mosaik kuning sama dengan
yang ada di lapangan. Varietas „Parade‟ merupakan varietas paling rentan dengan
kejadian penyakit 100% (5/5) diikuti oleh „New Jaliteng‟ dan „Wulung‟ masingmasing sebesar 40% (2/5). Masa inkubasi Begomovirus pada semua varietas
adalah 14 hari.
Diagnosis penyebab penyakit mosaik kuning kacang panjang berdasarkan
deteksi molekuler dan uji penularan membuktikan Mungbean yellow mosaic India
begomovirus sebagai agens utama penyebab penyakit. Strategi pengendalian
untuk menekan kejadian, keparahan dan penyebaran penyakit harus segera
diupayakan.
Kata kunci: Common Region, ELISA, MYMIV, PCR, Sekuensing DNA
©2014, Bogor Agricultural University
All Right Reserved
It is prohibited to make any quotations from part or whole of this thesis without
citing the author or the copyright holder. Quotation is allowed as long as for
education, research, scientific writing, scientific report, research proposal or
scientific review purposes only; those quotations should not produce any adverse
effects to Bogor Agricultural University.
No part of this thesis may be reproduced or transmitted in any forms or by any
means, electronic or mechanical, including photocopying, recording, or by any
information storage and retrieval system without express written permission from
Bogor Agricultural University.
MOLECULAR CHARACTERIZATION OF BEGOMOVIRUS INFECTING
YARD LONG BEAN (Vigna unguiculata subsp. sesquipedalis L.) AND
CONSTRUCTION OF ITS SPECIFIC PRIMERS
SARI NURULITA
Thesis
in partial fulfillment of the requirements for the degree of
Master of Science
at the
Phytopathology Study Program
GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2014
External Examiner on Thesis Defense: Dr Ir Gede Suastika, MSc
Thesis title : Molecular Characterization of Begomovirus Infecting Yard Long
Bean (Vigna unguiculata subsp. sesquipedalis L.) and Construction
of Its Specific Primers
Name
: Sari Nurulita
SIN
: A352110081
Approved by
Committee members
Prof Sri Hendrastuti Hidayat, PhD
Supervisor
Kikin Hamzah Mutaqin, PhD
Co-Supervisor
Acknowledged by
Head of Phytopathology
Study Program
Dean of Graduate School
Prof Sri Hendrastuti Hidayat, PhD
Dr Ir Dahrul Syah, MScAgr
Date of Examination: July 14th, 2014
Date of Submission:
PREFACE
I am very grateful to Allah Subhanallahuwataala for the mercy, so I can
finish this thesis. From this moment I would also like to thank the people who
specially contribute to my education.
I would like to express my thankfulness to my committee members, Prof.
Sri Hendrastuti Hidayat and Dr. Kikin Hamzah Mutaqin for their guidance and
support, both in scientific and personal matters. Their patience and excellent
advice made this work enjoyable and rewarding. I am very thankful to Dr. Gede
Suastika for his suggestions during thesis defense.
I am very thankful to Dr. John Thomas and Matthew Webb, Department of
Agriculture, Forestry and Fisheries (DAFF) Queensland – Australia, for their
technical assistance especially for teaching me laboratory technique and providing
molecular material in Plant Virology Laboratory, Australia. I also thank Dr.
Cherie Gambley for the guidance on methodology to design pecific primers. I
thank Tuti Legiastuti, SSi for her assistance in Plant Virology Laborator, IPB. I
would also like to thank Dr. Sri Sulandari, Gadjah Mada University, for helping
me during the survey.
I am very thankful to AusAID Funded Economic Cooperation Work
Program of the ASEAN – Australia – New Zealand Free Trade Agreement for
funding my research. I am very thankful to I-MHERE B.2C IPB for granting me a
master scholarship.
I would also like to thank my classmates “Phytopathology class 2011”,
Plant Virology Laboratory members, especially Dwiwiyati Nurul Septariani for
their supports and cooperations. My appreciation goes to my family who always
support and care me from far away.
Partial of this thesis has been presented in International Workshop on
Collaborative Educations for Sustainability of Agriculture and Environment,
Ibaraki University – Japan 2012; Indonesian Phytopathology Society, Padang
2013; also has been submitted to Biotropia Journal – SEAMEO Biotrop.
Bogor, August 2014
Sari Nurulita
TABLE OF CONTENTS
LIST OF TABLES
vi
LIST OF FIGURES
vi
LIST OF APPENDIX
vi
INTRODUCTION
Background
Hypothesis
Research Objectives
Research Advantages
1
1
3
3
3
LITERATURE REVIEWS
Importance of Begomovirus Infection on Agricultural Production
Disease Control Strategy for Begomovirus Infection
Legume – Infecting Begomovirus
Biological Characteristics of Begomovirus
Organization and Molecular Characteristics of Begomovirus
Diagnosis and Detection of Begomovirus
5
5
5
6
7
8
9
MATERIALS AND METHODS
Field Survey and Samples Collection
Detection and Identification of Viruses from Infected Leaves
Virus Detection by I-ELISA
Begomovirus Detection by PCR
Cloning Strategy of DNA-A of Begomovirus
DNA Sequencing
Construction of Specific Primers
Evaluation of Primer Specificity
Koch's Postulate
11
11
11
11
12
12
14
14
16
16
RESULTS AND DISCUSSION
Identification of Begomovirus from Field Samples
Cloning of DNA-A Begomovirus
Sequence Identity Analysis of Begomovirus Infecting Yard Long
Bean
Analysis of Common Region of Begomovirus Infecting Yard Long
Bean
Specificity of MYMIV Specific Primers
Koch‟s Postulate
General Discussion
17
17
19
CONCLUSION
31
REFERENCES
32
20
21
27
28
29
APPENDIXES
40
BIOGRAPHY
49
LIST OF TABLES
1 Genome organization of Begomovirus
2 Degenerate primers for amplification of Begomovirus using polymerase
chain reaction
3 Specific primers for amplification of Begomovirus infecting yard long
bean
4 Detection of CMV, Potyvirus, and Begomovirus from leaf samples
collected from various locations using I-ELISA and PCR
5 Nucleotide sequence homology (%) of Begomovirus infecting yard long
bean in Java with other Begomoviruses reported earlier in GenBank
6 Comparison of repetitive sequences (iteron) between MYMIV and
MYMV on common region
7 Nucleotide sequence homologies (% identity) of common region among
Begomovirus
8
12
15
19
20
24
26
LIST OF FIGURES
1 Research flow chart: “Molecular Characterization of Begomovirus
Infecting Yard Long Bean (Vigna unguiculata subsp. sesquipedalis L.)
and Construction of Its Specific Primers”
2 Genome organization of Begomovirus: bipartite (above) and
monopartite (below)
3 Hair pin loop structure of Begomovirus
4 pCR®4-TOPO® vector for cloning
5 Alignment of conserved region to design specific primer for
Begomovirus infecting yard long beans
6 Construction of specific primer using primer BLAST program at NCBI
7 Various symptoms found associated with yellow mosaic disease on
yard long bean
8 Visualization of PCR products of Begomovirus amplification from leaf
samples using universal primers PAL1v1978/ PAR1c715 on 1 %
agarose gel
9 Visualization of PCR products of Begomovirus amplification from leaf
samples using universal primers PBL1v2040/ PCR1c on 1 % agarose
gel
10 Visualization of PCR product from Begomovirus clone (pTgl)
11 Phylogenetic analysis based on alignment of partial nucleotide
sequences of the DNA-A of Begomoviruses using Mega 6.06
(Algorithm Neighbour Joining with 1000 bootstraps replicates)
12 Alignment of nucleotide sequences of the CR of Mungbean yellow
mosaic India virus (MVMIV) isolates from Java with other reported
MYMIV in Genbank
13 Hairpin loop structures of several Begomovirus isolates
14 Amplification of partial DNA-A Begomovirus genome using universal
primer PAL1v1978/PAR1c715 (A), specific primers MYF/MYR (B),
and MY1/MY2 (C)
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LIST OF APPENDIXES
1 Sequence alignment of all MYMIV isolates
2 Description of yard long bean varieties
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48
INTRODUCTION
Background
Begomovirus is an important plant viral pathogen group causing serious
diseases in several tropical and sub-tropical countries (Mansoor et al. 2003;
Varma and Malathi 2003). Infection of Begomovirus resulted in crop damage and
yield loss up to 20% or equal to US$ 140 million economic losses in tomato
production in Florida, US (Moffat 1999; Polston et al. 1999). Mosaic disease of
cassava in East and Central Africa was also the reason for US$ 1.9 – 2.7 billion
yield loss (Patil and Fauquet 2009). Similar situation was reported from cucurbit
production in India which was suffered from yellow mosaic disease (Varma and
Malathi 2003; Varma et al. 1992). In Indonesia, serious infection of Begomovirus
caused disease epidemic on some chilli production regions in Central Java and
West Java Province, and Special District of Jogyakarta (Sulandari et al. 2006).
Infection of Begomovirus in chilli pepper produced 30-100% disease incidence
since 1999 (Hidayat et al. 2006).
Yellow mosaic disease outbreak has been reported on yard long bean
(Vigna unguiculata subsp. sesquipedalis L.) growing area in Java since 2008.
Bean common mosaic virus (BCMV) and Cucumber mosaic virus (CMV) were
identified from leaf samples showing yellow mosaic symptom collected from yard
long bean growing area in Java (Damayanti et al. 2009). In the meantime similar
yellow mosaic disease was first reported from Pakistan and Nepal on grain
legume, mungbean, cowpea, soybean, kidney bean and Rhynchosia capitata.
Further identification showed the involvement of bipartite Begomovirus on this
disease (Ilyas et al. 2010; Shahid et al. 2012).
Begomovirus is the largest genus of the Family Geminiviridae. Its natural
spread relies on its specific insect vector, Bemicia tabaci (Hemiptera :
Aleyrodidae) which transmit the virus in circulative persistent manner. Most
Begomovirus is not transovarially transmitted, except TYLCV from Israel
(Ghanin et al. 1998). Since B. tabaci is a polyphagous insect, Begomovirus has a
high chance to infect many plant species. For instance, tomato leaf curl disease
(ToLCD) caused by Begomovirus has been observed on many countries. Tomato
leaf curl New Delhi virus (ToLCNDV) was investigated as new emerging virus
causing yellow mosaic disease on eggplant in India (Pratap et al. 2011).
ToLCNDV was also reported infecting cucumber plant in Central Java, Indonesia
(Mizutani et al. 2011). Another ToLCD caused by Tomato leaf deformation virus
was characterized as a new world monopartite Begomovirus infecting tomato in
Peru and Ecuador (Melgarejo et al. 2013). A new Begomovirus associated with
tomato yellow leaf curl and eggplant yellow mosaic disease, Tomato yellow leaf
curl Kanchanaburi virus was found in Thailand (Green et al. 2003), Indonesia
(Nurul and Tsai 2009), and Laos (Tang et al. 2014). Three Begomoviruses were
identified associated with bean golden mosaic disease in Nicaragua i.e., Bean
golden yellow moaic virus (BGYMV), Squash yellow mild mottle virus
(SYMMoV), and Calopogonium golden mosaic virus (CalGMV) (Karkashian et
al. 2011).
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Molecular characteristics of Begomovirus genome is very unique; it posses
twin particles, each consisting of single stranded DNA (2.6 – 2.7 kb), bipartite or
monopartite (Hull 2002). DNA-A and DNA-B of Begomovirus have different
genome organization and function except their common region (CR). Among
other important function encodes by genome of DNA-A are virus replication and
insect transmission, whereas those encodes by DNA-B are systemic infection and
symptom development. Common region of both DNA-A and DNA-B each
consists of about 200 nucleotides and its sequence is highly identical for one
species of Begomovirus. In contrast, common region of each species of
Begomovirus is distinctinve, except for loop region on hairpin structure, i.e. 5‟TAATATTAC -3‟ which is known as the conserved nona-nucleotide region
(Lazarowitz 1987). There are three unique characteristics of common region
which is observed on every Begomovirus species, i.e. TATA box, repeated motifs
(iterons), and hairpin-loop structure (Hidayat et al. 2008; Lazarowitz 1987; Trisno
et al. 2009). Begomovirus possesing >92% similarity on their hairpin loop
structure can be grouped as one species (Fauquet et al. 2005).
The most common method for virus detection is using serological-based
technique, but different situation occurred for Begomovirus. Cross reaction and
low specificity are the reasons why serological method is not preferred for
detection of Begomovirus (Kushwaha et al. 2010). Method for detection of
Begomovirus is commonly based on polymerase chain reaction (PCR) technique
using universal primers. Several universal primers has been reported to work well
for different species of Begomovirus. Rojas et al. (1993) used degenerate primer
PAL1v1978/ PAR1c715 to detect several geminivirus from infected plants belong
to Solanaceae, Leguminosae, Euphorbiaceae, and Malvaceae from South America.
Li et al. (2004) used a pair of degenerate primer SPG1/ SPG2 to detect
geminivirus infecting sweet potato and other Ipomoea sp. Another pair of
degenerate primer, AV494/ AC1048, was used to detect coat protein of subgroup
III geminivirus (Wyatt and Brown 1996).
Despite the benefit of using universal primers, specific primers might
increase efficiency and effectiveness of detection method. Specific primer is used
to positively identify and determine the species involved (Ye et al. 2012). TorresPacheco et al. (1996) used three sets of specific primers to detect intergenic region
(IR) of DNA-A of Pepper jalapeño virus (PJV)/ Texas pepper geminivirus (TPV),
IR of DNA-B Chino del tomato virus (CdTV), and IR of DNA-A Pepper huasteco
virus (PHV) which infecting pepper in Mexico. Pratap et al. (2011) developed
specific primers to confirm agroinfectious clone of DNA-A and DNA-B
ToLCNDV infecting eggplant. Primer pair of HOG1/ HOG2 was specifically
designed to amplify full length DNA-A MYMIV using rolling circle replication
method (Shahid et al. 2012). Availability of specific primer for specific virus will
be very useful for early diagnosis especially when immediate control strategy is
required to take place.
This research is intended to determine the association of Mungbean yellow
mosaic India virus (MYMIV) with yellow mosaic disease on yard long bean in
Java and further characterization based on its specific genome property of
common region.
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Hypothesis
(i)
There are multiple viruses associated with yellow mosaic disease on yard
long bean, one of them is Begomovirus.
(ii) Begomovirus infecting yard long bean is different from those previously
reported in Indonesia.
(iii) Begomovirus infecting yard long bean in Indonesia has close genetic
relationship with other Begomoviruses infecting Leguminosae in Asia.
(iv) Specific primer for Begomovirus infecting yard long bean will specifically
amplify virus target.
Research Objectives
Objective of this research is to determine the importance of Begomovirus
infection on yard long bean, to identify species of Begomovirus on yard long bean
and to analyse its molecular character, and to design specific primers for detection
of Begomovirus species infecting yard long bean.
Research Advantages
Basic information regarding molecular and biological characteristics of the
virus obtained from this research will be useful for further study on disease
ecology and for establishment of disease control strategy.
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Field Survey and Virus Detection
West Java Province (Bogor and Subang);
Central Java Province (Tegal, Klaten, and Magelang);
Special District of Jogyakarta (Bantul and Sleman)
Molecular
detection
Serology
detection
Virus isolates from
the field
Potyvirus and CMV
Begomovirus
Nucleotide sequence
Clone
Molecular characterization:
bipartite or monopartite
hair pin loop structure
Primer
construction
Identity of Begomovirus
Specific primer
DNA probe
: process
: future process
: result
: future prospective result
Fig 1 Research flow chart: “Molecular Characterization of Begomovirus Infecting
Yard Long Bean (Vigna unguiculata subsp. sesquipedalis L.) and
Construction of Its Specific Primers”
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LITERATURE REVIEWS
Importance of Begomovirus Infection on Agricultural Production
Begomovirus was reported to cause serious problems in the sub-tropical
and tropical region and its infection may potentially cause yield lost (Varma et al.
2011). Tomato production in Florida, USA was suffered from tomato yellow leaf
curl disease (TYLCD) which caused 20% yield lost or US$ 140 million economy
losses (Moffat 1999; Polston et al. 1999). TYLCD was also reported causing yield
loss up to 99% of tomato production in Central America, Europe, Middle East,
and South East Asia (Polston and Anderston 1993; Rochester et al. 1994).
Legume-infecting Begomovirus, Bean golden mosaic virus, caused 20 – 95% and
36.7% disease incidence in Nicaragua and Argentina, respectively (Karkashian et
al. 2011; Alemandri et al. 2012).
Similar condition was also reported from India and Africa, in which up to
50% yield loss of cucurbit in Northern India was occurred in 2001 due to Tomato
leaf curl virus (ToLCV) infection (Varma and Malathi 2003). Disease incidence
caused by Tomato leaf curl New Delhi virus (ToLCNDV) reached 60 – 66% and
damaged eggplant production in India (Pratap et al. 2011). Serious infection of
Begomovirus on cassava caused economy losses of US$ 1.9 – 2.7 billion in East
and Central Africa (Patil and Fauquet 2009). Yellow mosaic disease on legume
was a very serious problem in India because it caused up to US$ 300 million
(Varma et al. 1992). MYMV infection in India ranged from 4 to 40% depend on
crop variety and management (Bashir et al. 2006). Another species, MYMIV, was
reported infecting Phaseolus vulgaris in Pakistan (Naimuddin et al. 2011) and
Vigna mungo in Nepal (Shahid et al. 2011) with disease incidence reached 70 –
100% and 70 – 80%, respectively.
Incidence of Begomovirus was also reported as serious problem in Indonesia.
Pepper yellow leaf curl Indonesia virus (PepYLCIV) was the main disease
problem on chili pepper in Central Java, Yogyakarta, and West Java with 70 –
100% and 10 – 35% disease incidence for Capsicum frutescence and C. annuum,
respectively (Sulandari et al. 2006). Tomato leaf curl virus (ToLCV) infected
tomato in Java and Sulawesi with 100% disease incidence (Aidawati et al. 2005;
Tsai et al. 2009). Infection of Tobacco leaf curl virus Begomovirus on tobacco in
East Java reached 30% disease incidence (Trisusilowati 1990).
Disease control strategy for Begomovirus infection
Various control methods has been applied and evaluated in order to suppress
disease incidence caused by Begomovirus. Most of the control strategy
recommended was based on insect vector management. Preventive strategy in the
seedling and young plant using seedbeds with plastic polyethylene sheets was
common and was able to avoid Begomovirus transmission by whitefly (Hidayat et
al. 2010). Utilization of UV-absorbing plastic films as barrier and neem extract
can control whitefly population in the greenhouse (Antignus 1996; Senguttuvan et
al. 2005). Combination of screen and insecticide sprays in the greenhouse can
reduce TYLCV incidence up to 99% (Antignus 1996; Berlinger et al. 2002).
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Cultural practice using border plants, such as maize, yard long bean, and
Crotalaria sp. as physical barrier around chili pepper plantation can delayed
PYLCIV for the first 30 days after transplanting (Hidayat et al. 2010). The maize
and cowpea intercrops on cassava plantation in southern Cameroon can reduce B.
tabaci population and cassava mosaic disease incidence up to 50% and 20%,
respectively (Fondong et al. 2002).
Treatment using bio-control agent such as PGPR (plant growth promoting
rhizobacteria) or PGPF (plant growth promoting fungi) was reported on several
studies in attempt to control Begomovirus infection. These treatments was
potentially delayed incubation period and decreased disease incidence of
PepYLCIV (Hidayat et al. 2010), induced systemic resistance of PepYLCIV
(Damayanti 2013) and CMV (Raupach et al. 1996), and reduced disease incidence
of ToMoV up to 40 days after treatment (Murphy et al. 2000).
Growing resistance varieties has been recommended as control strategy for
Begomovirus (Mason et al. 2000) and effort to develop resistance varieties has
been studied for various crops. Screening of germplasms from various sources is
important to identify source of resistance in the breeding program (Larsen and
Porter 2011). Evaluation of mungbean breeding lines was reported but all
germplasms was susceptible against MYMV based on observation on incubation
period and disease incidence (Shad et al. 2006). Recently, Akhtar et al. (2011)
reported there were 35 genotypes of mungbean showing moderate resistant toward
MMIV infection. Santoso (2008) reported F1-TYLCV which was crossed from
AVRDC germplasm and commercial seed (F1 FLA456/ Intan and FLA456/
CL6046) showed resistant response up to 68% and 66% toward TYLCV infection,
respectively.
The use of transgenic lines to reduce Begomovirus infection were reported.
RNA interference mechanism (RNAi/ silencing mechanism) can reduce viral
DNA replication of African cassava mosaic virus (ACMV) up to 99%
(Vanitharani et al. 2003). Transgenic cassava plant which resistant to cassava
mosaic disease was also observed by Zhang et al. (2005). Another mechanism,
intron-spliced hairpin construction was used to delay seven weeks of TYLCV
incubation period in tomato transgenic line (Zrachya et al. 2007) and recovery
MYMIV infection in blackgram (Vigna mungo) (Pooggin et al. 2003).
Legume–Infecting Begomovirus
Legume-infecting Begomovirus was reported as serious problem in subtropical and tropical countries (Varma and Malathi 2003). Symptom variation is
reported although it can be differentiated into two types, i.e. golden mosaic and
yellow mosaic. Golden mosaic disease was generally found in the new world such
as South and Central America and the Caribbean Basin. Bean golden mosaic virus
(BGMV) and Bean golden yellow mosaic virus (BGYMV) have been identified as
causal agent of bean golden mosaic disease in Brazil, Puerto Rico, and Nicaragua
(Gilbertson et al. 1993; Karkashian et al. 2011). Bean golden mosaic disease is
recognized by its typical symptoms including bright golden color of the leaves,
stunting, and smaller pods.
Yellow mosaic disease was more common in the old world, i.e. South and
South East Asia (Stanley et al. 2005; Shahid et al. 2012). Yellow mosaic disease
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on legume characterized by bright yellow leaves with green spot or complete
yellowing, smaller pods and seeds was first reported from India (Nariani 1960).
Similar symptom was also reported in other Asian countries, such as Pakistan
(Qazi et al. 2007), Nepal (Shahid et al. 2012), and Thailand (Honda et al. 1983).
There are five species of Begomovirus causing yellow mosaic disease in Asia, i.e.
Mungbean yellow mosaic virus (MYMV), Mungbean yellow mosaic India virus
(MYMIV), Horsegram yellow mosaic virus (HgYMV), Dolichos yellow mosaic
virus (DoYMV) (Maruthi et al. 2006; Qazi et al. 2007), and a new species
proposed as Rhynchosia yellow mosaic virus (RhYMV) (Ilyas et al. 2009). These
species were distinct to other Begomovirus from new world which was identified
as causal agent of yellow mosaic disease on Leguminosae (Briddon and Stanley
2006).
MYMV and MYMIV were reported as the most destructive agent for some
Leguminosae crops in India and Pakistan. MYMV was found to infect
Leguminosae in Southern and Western India (Morinaga et al. 1993) whereas
MYMIV was reported to infect Leguminosae in Northern and Central region
(Mandal et al. 1997), and in Pakistan (Ilyas et al. 2010). MYMV and MYMIV
have close relationship based on phylogenetic analysis except in common region
(CR). CR of MYMIV has >95% identity with the same isolate but only