Genetic Diversity of Cowpea Mild Mottle Irus on Soybean in Several

Region in Indonesia

Mimi Sutrawati

, Sri Hendrastuti Hidayat

, Bonny Purnomo Wahyu Sukarno

, Gede Suastika

and

Ali Nurmansyah

University of Bengkulu, WR Supratman Street, Bengkulu, Indonesia

Bogor Agricultural University, Bogor, Indonesia

Keywords: DAS-ELISA, homology, nucleotide sequencing, PCR, phylogeny

Abstract: Soybean is one of the most important food commodities in Indonesia. Virus infection on soybean has been

reported worldwide as factors affecting yield loss. This study was aimed to detect Cowpea mild mottle virus

(CPMMV) from several soybean cultivation areas in Java, Sumatra and Southeast Sulawesi; and further

characterize their genetic variation based on nucleotide sequences of their coat protein. Several virus

infection was detected using double antibody sandwhich enzyme-linked immunosorbent assay (DAS-

ELISA),including CPMMV, Cucumber mosaic virus (CMV), and Soybean mosaic virus (SMV). Generally,

the symptoms caused by CPMMV, CMV, and SMV are similar, involving mottle, rugose, and vein banding.

Coat protein gene of 5 CPMMV isolates (Bantul, Musi Banyuasin, Cirebon, Kendari, Cianjur) was

successfully amplified and cloned. Sequence of this 5 clones of CPMMV showed high similarity, ranging

from 88.2 to 99.8%; whereas their sequence homology to those of Taiwan and China ranging from 88.2 to

98.6%. Phylogenetic analysis showed different clusters of CPMMV Indonesian isolates: isolates from

Bantul,Cirebon, Musi Banyuasin (Palembang) is clustered with Taiwan isolate (JX020701); isolate from

Cianjur is clustered with China isolate (KX534092); isolate from Kendari is clustered with Puerto Rico

(GU191840),Brazil (KC884247), and USA (KC774020) isolates.

1 INTRODUCTION

Several types of viruses reported to infect soybean

plants are Alfalfa mosaic virus (AlMV), Bean

common mosaic virus (BCMV), Bean yellow

mosaic virus (BYMV), Blackeye cowpea mosaic

virus (BlCMV), Cucumber mosaic virus (CMV),

Pea enation mosaic virus (PEMV), Peanut mottle

virus (PeMoV), Soybean mosaic virus (SMV),

Tobacco mosaic virus (TMV), Tobacco ringspot

virus (TRSV), Tobacco streak virus (TSV), Tomato

ringspot virus (ToRSV), and Spotted Tomato wilt

virus (TSWV) (Golnaraghi et al. 2004). In

Indonesia, several viruses have been reported in

soybean plants: Cowpea mild mottle virus

(CPMMV) (Iwaki et al. 1986), SMV (Andayanie

2012), CMV soybean strain (CMV-S) and Pepper

yellow leafcurl virus (PYLCV) ( Rahim et al. 2015).

CPMMV infection in soybean has caused endemic

diseases in Java and Sumatra (Jumanto et al. 1999).

CPMMV infection in soybeans in Lampung caused a

decrease in dry weight with soybean crop weight

between 15.5-53.4% and a decrease in soybean seed

weight between 11.5-51.6% and a decrease in seed

quality cause an abnormal seed shape of 7.6-54.35%

(Akin 2003).

CPMMV is a member of the Genus Carlavirus,

Family Betaflexiviridae (Martelli et al. 2007).

Losses due to CPMMV infection were also reported

in several other countries, including in Argentina

and Iran CPMMV reportedly caused severe damage

to soybeans (Laguna et al. 2006; Tavassoli et al.

2009). In addition to infecting soybean CPMMV

was also reported in yard long bean (Brito et al.

2012), tomatoes, beans, Bambara peanuts (Almeida

et al. (2005). Offei and Albrechtsen (2005) reported

CPMMV infection in bambara peanuts (Vigna

subterranea L.) causes the leaf area index to be

reduced by 70%, and decreases the number of pods

and seeds and the weight of the seeds CPMMV

infection in peanuts causes dwarf plants, reduction

in length of internodes and size of leaves, stripes on

leaves, chlorosis and leaf rolling CPMMV infection

has been reported to be a serious disease in peanuts

Sutrawati, M., Hidayat, S., Sukarno, B., Suastika, G. and Nurmansyah, A.

Genetic Diversity of Cowpea Mild Mottle Irus on Soybean in Several Region in Indonesia.

DOI: 10.5220/0009935718151819

In Proceedings of the 1st International Conference on Recent Innovations (ICRI 2018), pages 1815-1819

ISBN: 978-989-758-458-9

1815

in Sudan with a disease incidence of up to 50% even

in some regions up to 100% (El-Hassan et al. 1997).

Surveys on long bean plants in Venezuela show the

incidence of diseases ranging from CPMMV

infections. 15-40% (Brito et al. 2012). In India,

CPMMV infection in soybean causes systemic,

mosaic, and leaf deformation with 25.1-71 disease

incidence. % (Yadav et al. 2013).

Expansion of soybean cultivation areas in

Indonesia must anticipate the emergence of diseases

that have the potential to cause loss of results. The

current status of the area spread CPMMV on

soybeans in Indonesia needs to be known. Recently

on 2015, we conducted a field survey to collected

soybean’s leaves with typical symptom of virus-like

infection from several cultivation areas in Java,

Sumatra, and Sulawesi. In the earlier reports, the the

yield impact of CPMMV infection were studied, but

none of these were reported about genetic diversity

of CPMMV in Indonesia. Here, we reported the

genetic diversity of CPMMV from several soybean

cultivation areas in Indonesia.

2 MATERIAL AND METHODS

Sampling activities for collection of CPMMV

isolates were carried out during the 2014-2015

planting season in Cirebon, Cianjur, Bogor (West

Java Province); Bantul, (Yogyakarta); Ngawi (East

Java Province); Musi Banyuasin (South Sumatra

Province), Sungai Hitam (Bengkulu Province), Kota

Baru (Jambi Province); and Kendari (Southeast

Sulawesi Province). Virus detection and cloning

were carried out at the Plant Virology Laboratory,

Department of Plant Protection, Faculty of

Agriculture, IPB.

2.1 Samples Collection

Samples of soybean leaves taken from the field are

young leaves that show symptoms of viral infection

including mottle, vein clearing, chlorosis, dwarf,

leaves malformation. Leaf samples are put into a

plastic bag and stored in a box to be brought to the

laboratory. Symptoms of leafy leaves were then

separated into two groups, namely leaves with

symptomatic stripes, chlorosis, distortion of leaves

and dwarfs and a group of yellowing leaves. Each

sample is weighed 0.1 g each, labeled and stored at -

80 ˚C until it is used for the next stage.

2.2 Serological Detection

Serological detection of viruses was carried out with

DAS-ELISA following the Clark and Adams (1977)

protocol using 3 types of antiserum separately,

namely CMV, SMV, and CPMMV antiserum

(DSMZ, Germany).

2.3 Reverse Transcription-Polimerase

Chain Reaction (RT-PCR)

Total RNA extraction. Total RNA extraction was

carried out using RNeasy Plant Mini Kits (Qiagen,

Hilder, Germany) according to the Qiagen protocol

(Qiagen 2003). RNA extraction results were

synthesized into cDNA using reverse transcription

(RT) method. CDNA amplification was performed

using a specific primary pair for CP CPMMV gene.

The primary forward used was CPF (5'-

ATTAAGGATCCGAGTTGATTTAAATAAGT-3

') and the reverse CPR primer (5'-

ATTAAGAATTCCTTGTGATTGAAATTGCG-3')

with an expected amplification product measuring

958 bp. The composition of the PCR reactant

consisted of 12.5 µl Dream taq PCR master mix

(Thermo Scientific), 1 µl primer forward 10 µM, and

1 µl reverse primer 10 µM, 1 µl cDNA, and 9.51 µl

H2O. Amplification of cDNA with 94 ° C stages for

5 minutes, then followed by 35 cycles consisting of

denaturation of 94 ° C for 1 minute, annealing 45 °

C for 1 minute, and DNA synthesis (extension) 72 °

C for 2 minutes, then extension 72 ° C for 10

minutes and the cycle ends at 4 ° C. Amplification of

agarose gel follows the method described

previously. CPMMV isolates which showed a clear

and thick DNA band of ± 958 bp then continued to

cloning stage.

2.4 DNA Cloning and Sequences

Analysis

PCR products were cloned into pTZ57R/T aesy

vector system based on the protocol provided by

Thermo Scientific, USA. Plasmid DNA recombinant

was sequenced and analyzed. The nucleotide

sequences of the gene were aligned with those

corresponding virus sequences deposited in

GenBank database by using software Clustal-W

(www.ebi.ac.uk). Phylogenetic tree CPMMV was

constructed using the ClustalX Bio Edit version 7.05

program and the MEGA 5.0 program with the

neighbor-joining algorithm and 1000 repetition

bootstrap methods.

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3 RESULT AND DISCUSSION

3.1 Results

3.1.1 Symptoms on Infected Soybean Leaves

Symptoms of viral infection in soybean in the field

varied, namely systemic chlorosis and leaf

distortion, systemic leaf and curly leaf surface,

systemic stripes with green patches, striped with

lamina blisters, stripes and leaf distortion, chlorosis

in leaf lamina and vein banding, and some mixed

symptoms in one plant (figure 1). The symptom

variation was depending on the soybean cultivar and

climate on each location.

Figure 1: Symptoms of viral infection in soybean:

systemic chlorosis and leaf distortion (A), systemic stripes

and curly leaf surface (B), systemic stripes with green

spots (C), striped with lamina of blister (D) leaves, stripes

and leaf distortion (E), and chlorosis in the leaf lamina and

vein banding (F).

3.1.2 Serological Test

Based on serological test using three antisera, there

are three virures detetcted on samples with various

frequences and showed multiple infection occurred

naturally (data not shown). CPMMV detected on

samples in 8 regions from 9 regions of origin of

soybean leaf samples. Besides CPMMV also

detected several other types of viruses, namely CMV

and SMV. CMV infection is detected in 4 regions,

namely Cirebon, Cianjur, Bantul, Kendari; while

SMV is only detected in Kendari and Cirebon.

CPMMV was consider as endemic disease on

soybean in Java, further characterization of

biological, physical and nucleic acid characters are

necessary to identify to confirm its existence in

Indonesia.

3.1.3 Amplification and Nucleotide

Sequences Analysis

CPMMV specific DNA was successfully amplified

from 5 CPMMV isolates from Bantul (B),

Banyuasin Musi (MB), Cirebon (CB), Kendari

(KN), and Cianjur (CR) (figure 2). Furthermore, the

PCR product is used for cloning stages.

Figure 2: DNA amplification visualization of CP CPMMV

gene from Bantul (B), Banyuasin Musi (MB), Cirebon

(CB), Kendari (KN), and Cianjur (CR), M, 1 kb DNA

(Thermo Scientific) marker.

Isolates from 5 regions in Indonesia have

homology between isolates as much as 88.2-99.8%

(Table 1). Similarity of nucleotide sequences

between isolates in Indonesia and Taiwan and China

isolates with a range of 88.2-98.6% higher than

similarities with isolates from America; while the

lowest homology is with Ghana isolates. CPMMV

isolates in Indonesia showed more than 72%

homology with all isolates compared to GenBank.

Table 1: The nucleotide sequences of CPMMV isolates in

Indonesia with sequences of isolates from several other

countries in GenBank.

Sequence comparison CP

gene

(%)

Among Indonesian isolates

88.2-

99.8

Between Indonesian isolates with Taiwa

and China

8.2-

98.6

Between Indonesian isolates with

Amerika

6.9-

88.8

Between Indonesian isolates with Ghana

6.9-

79.2

Genetic Diversity of Cowpea Mild Mottle Irus on Soybean in Several Region in Indonesia

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3.1.4 Phylogenetic Analysis

Phylogenetic analysis showed that Bantul, Cirebon,

Musi Banyu asin (Palembang) isolates formed a

group with Taiwan isolates (accession number

JX020701); whereas Cianjur isolates were separated

from other isolates and closer to Chinese isolates

(accession number KX534092). Kendari isolates are

closer to Puerto Rico isolates (accession number

GU191840, Brazil (accession number KC884247),

and USA (accession number KC774020) (Figure 3).

Figure 3. Phylogeny tree isolates CPMMV from Bantul,

Cirebon, Musi Banyuasin, Cianjur, and Kendari based on

the CP CPMMV gene sequence from Asian, American

and African groups and CarMV as outgroup

3.2 Discussion

Jumanto et al. (1999) reported mottled disease in

soybeans associated with CPMMV infection in Java

and Sumatra. However, after the report, there is no

current information regarding the distribution of

CPMMV in Indonesia, even though the area of

soybean cultivation in Indonesia is increasingly

widespread. In this study it was found that CPMMV

was detected on soybeans in Bengkulu, Musi

Banyuasin (South Sumatra); Cianjur, Bogor and

Cirebon in West Java; Bantul (Yogyakarta); Ngawi

(East Java); and Kendari (Southeast Sulawesi).

The results of this study indicate that the

diversity of CPMMV not only occurs in virulence and

the type of symptoms caused but also in diversity its

molecular level. Based on nucleotide homology

analysis, CPMMV isolates obtained from Sumatra,

Java, and Kendari in this study, had a relationship with

isolates from Asia (China, Taiwan), and America

(USA, Brazil, Puerto Rico). Close relationship with

Asian and American isolates indicates the possibility

of CPMMV Indonesia coming from that country.

Nevertheless, CPMMV from various regions turned

out to show genetic variation at the level of its

nucleotide sequence. Genetic variation in viruses can

occur through two events, namely mutation and

recombination. The high rate of mutation in the RNA

virus indicates an evolutionary strategy. According to

Agrios (2005) the evolution of viruses occurs as a form

of adaptation to environmental suitability, such as host

plants, strains of viruses, insect vectors. Different

environmental conditions between regions in

Indonesia, and climate differences between Indonesia

and other countries may cause environmental stresses

that cause genetic changes in the virus.

4 CONCLUSIONS

The distribution of CPMMV on soybeans covering

several areas of soybean production field in Java,

Sumatra, and Sulawesi. CPMMV is the dominant

virus found in 8 soybean cultivation locations from 9

sampling locations. Besides CPMMV, CMV, and

SMV were also found. Based on phylogenetic

analysis CPMMV Bantul, Cirebon, and Musi

Banyuasin isolates formed a group with Taiwan

isolates; whereas Cianjur isolates are closer to

Chinese isolates; and Kendari isolates are closer to

isolates from Puerto Rico, Brazil, and USA.

ACKNOWLEDGEMENTS

If any, should be placed before the references

section without numbering.

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