molecular characterization of begomovirus infecting sweet...

Indian Journal of Biotechnology

Vol 6, January 2007, pp 45-51

Molecular characterization of Begomovirus infecting sweet pepper in Oman

Akhtar J Khan*, Nadiya A Al-Saady, Madleen S Al-Mahruki, Muna Al-Oufi and Ali M Al-Subhi

Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box-34, Al-

Khod 123, Sultanate of Oman

Received 18 October 2005; revised 16 January 2006; accepted 22 March 2006

Whitefly transmitted tomato yellow leaf curl is one of the most devastating viral disease of cultivated sweet pepper

(Capsicum frutescens grossum) and other vegetables in Oman. Infected sweet pepper plants showed typical begomovirus

symptoms as upward leaf curling, interveinal and leaf chlorosis, and growth stunting. Begomovirus infecting sweet pepper in

Oman was detected by polymerase chain reaction (PCR) using begomovirus specific degenerate primers

(PAL1v1978/PAR1c496 and AV494/AC1048). Core region (74-604 bp) of coat protein gene of the begomovirus was

amplified by PCR with tomato yellow leaf curl virus (TYLCV) specific degenerate primers (TycpV369/TycpC1023). Core

region of coat protein gene contains highly conserved regions and is used to identify the begomovirus infecting sweet

peppers. Virus identification was performed by percent sequence identity and parsimony analysis using core coat protein

gene sequences of sweet pepper virus with complete genome, core region of coat protein and coat protein gene sequences

from reference begomoviruses. The core region sequence identity of coat protein gene of sweet pepper virus from Oman

was 92.2, 96.5, 94.0, 93.8, and 96.5% with TomGV-Lebanon, TYLCV-Guadeloupe, TYLCV-Israel, TYLCV-Kuwait, and

TYLCV-Mexico, respectively. Phylogenetic trees and percent sequence identity with reference to begomoviruses permitted

the identification of sweet pepper virus as TYLCV based on tree position and extent of sequence identity. Phylogenetic

analysis revealed that sweet pepper tomato yellow leaf curl virus clustered with its closest relatives from Middle East

regions but formed a separate strain.

Keywords: sweet pepper, begomovirus, core region of coat protein gene

IPC Code: Int. Cl.8 C12N15/11, 15/34

Introduction Diseases of many vegetables, including sweet

pepper, caused by whitefly-transmitted geminivirus

are known worldwide1,2

. The typical symptoms of

sweet pepper disease caused by begomovirus are leaf

curl, leaf crumple, foliar mosaic, and mottle among

others. However, symptoms of a given virus-host

combination may vary depending on cultivars,

environmental conditions and virus strain. Infection of

a single host by two different viruses causing similar

symptoms is often noticed, which can complicate the

diagnosis of the disease and make the phenotype

symptoms an unreliable criteria for viral

identification. While DNA sequence comparison and

phylogenetic analysis with sequences of known

begomoviruses can evidently provide a reliable viral

identification3.

In Oman, exotic cultivars of sweet pepper are

cultivated each year during October-March in the

northern coastal region of Al-Batinah. The crop is

grown to meet the high domestic demand for fresh

produce. However, the major constraint in sweet

pepper cultivation is a disease, resulting in leaf

curling, mottling and growth stunting of plants,

caused by whitefly-transmitted geminivirus.

Geminiviruses are a group of plant DNA viruses

characterized by the geminate shape and particle size

of 18-25 nm4. Whitefly transmitted geminiviruses are

classified into the genus Begomovirus of the family

Geminiviridae, and are transmitted by Bemisia

tabaci5. The majority of the Begomoviruses are

known to have bipartite genome but single genome

(DNA-A) monopartite viruses are being reported, in

increasing numbers, mainly from the old world6.

Identification of Begomoviruses using serology is not

suitable, because high titre antisera are difficult to

prepare and lack sufficient specificity. Consequently,

DNA based molecular diagnostic techniques, such as

polymerase chain reaction (PCR) amplification, using

universal or specific primers and DNA sequencing

has supplemented serology for detection and

identification of begomoviruses. So far, classification

and phylogenetic relationships of begomoviruses have

been based upon complete monopartite viral genomes

___________

*Author for correspondence:

Tel: (+968) 24 141217; Fax: (+968) 24 413418

E-mail: [email protected]

INDIAN J BIOTECHNOL, JANUARY 2007

46

or the DNA-A component of the bipartite viruses,

coat protein (CP) and/or replication association

protein gene (Rep) trees7-9

. Although complete

genome sequences are desirable for identification and

classification of geminiviruses, this requires

considerable time and expense. Full-length CP gene

sequences are accepted by the International

Committee on the Taxonomy of Viruses (ICTV) for

the provisional classification of begomoviruses when

complete genome (monopartite virus) or DNA-A

component (bipartite virus) sequences are

unavailable10

. For sequences to be useful for

identification at both the species and strain/variant

levels, regions of the genome that contain both

conserved and variable elements should be targeted.

So far, CP gene has been proven useful for plant virus

detection and identification. PCR based studies have

established the diversity of tomato yellow leaf curl

virus in Sudan, showing that there are two distinct, yet

closely related, Begomovirus species associated with

tomato leaf curl disease11

. In this paper, authors

describe the molecular characterization of a tomato

yellow leaf curl virus (TLCV), a begomovirus

infecting sweet pepper in Oman.

Materials and Methods Sample Collection

To identify the causal agent(s), sweet pepper

samples showing typical leaf curl disease symptoms

were collected from different fields in Al-Batinah

region during 2004-2005. Samples were brought in

cool boxes from field and stored at -80oC until used

for DNA extraction.

Total DNA Extraction

Total DNA was extracted from symptomatic and

asymptomatic leaves of sweet pepper using

Dellaporta extraction buffer12

with some

modifications.

PCR

DNA samples extracted from symptomatic sweet

pepper plants were used as template for PCR. DNA

extracted from asymptomatic plants and sterile water

was used as negative control. Begomovirus specific

degenerate primer pairs PAL1v1978 (5’-

GCATCTGCAGGCCCACATYGTCTTYCCNGT-3)

and PAR1c496 (5’-AATACTGCAGGGCTTYCTR-

TACATRGG-3’)13

; and AV494 (5’-GCC(C/T)AT-

(G/A)TA(T/C)AG(A/G)AAGCC(A/C)AG-3’) and

AC1048 (5’-GG(A/G)TT(A/G/T)GA(G/A)GCATG-

(T/A/C)GTACATG-3’)14

were used to prime the

amplification of 1.1-1.4 kb and 500-600 bp product,

respectively. TYLCV core CP gene specific

degenerate primer pair TycpV369 (5’-

ACGCCCGYCTCGAAGGTTCG-3’) and TycpC1023

(5’-GTACAWGCCATATACAATAA-CAAGGC-3’)

was used to amplify 500-650 bp core CP region of the

virus genome15

. Fifty ng of each DNA preparation in

1 µL TE were added to ‘Ready-To-Go’ PCR beads

(Pharmacia Biotech, Sweden), followed by 1 µL of

each primer (10 pmoles) and 22 µL of sterile

deionized water for a final reaction mixture volume of

25 µL. The DNA was amplified by 35 cycles,

consisting of denaturation at 94oC for 30 sec (2 min

for cycle 1 and 45 sec for cycle 35), annealing at 55oC

for 1 min, and primer extension at 72oC for 2 min (5

min for cycle 35). After amplification, 4 µL aliquot

from each sample was electrophoresed in a 1%

agarose gel and visualized by staining with ethidium

bromide and UV illumination.

Cloning of PCR Amplified DNA

The PCR amplified DNA fragments using CP gene

specific primer pairs TycpV369/TycpC1023 were

purified using the Wizard PCR DNA purification

system (Promega Corp., Wisconsin, USA). The

purified DNA was cloned using Promega pGEM-T

Easy Vector System II cloning kit according the

manufacturer’s instructions (Promega Corp.,

Wisconsin, USA). Recombinant plasmids were

screened by blue/white color screening on indicator

plates and PCR amplification using

TycpV369/TycpC1023 primers.

Sequencing and Phylogenetic Analysis

A cloned TycpV369/TycpC1023 fragment from

infected pepper virus was sequenced to verify

phylogenetic relationships; the sequence was

deposited in NCBI Gene Bank under the Accession

Number DQ132859.

Automatic sequencing was performed on an ABI

Prism Model 377 (PE Applied Biosystem, CA, USA)

with TycpV369/TycpC1023 primers using Big Dye

terminator chemistry16

at DNA sequencing facility of

Sultan Qaboos University.

DNA sequence of sweet pepper virus CP gene was

analyzed in BLAST on NCBI nucleotide database to

obtain information on sequence similarity. CP and

complete genome sequences of 31 geminiviruses

(Table 1) were aligned by the CLUSTAL W method

with MegAlign suite of LaserGene 6.0 software

KHAN et al: IDENTIFICATION OF BEGOMOVIRUS FROM SWEET PEPPER IN OMAN

47

(DNASTAR, Inc. Madison, WI, USA). The sequences

were visually inspected for logical placement of gaps

and manually adjusted where necessary. Fifteen CP

sequences belonging to geminivirus were separately

aligned including Oman strain to analyze CP

sequence identity using MegAlign suit of Lasergene

5.01 (Table 2). Cladistic analyses were performed

with PAUP (Phylogenetic Analysis Using Parsimony,

version 4.0b10)17

. Uninformative characters were

excluded from analyses. A phylogenetic tree was

constructed by a heuristic search via random stepwise

addition, implementing the tree bisection and

reconnection algorithm to find the optimal

phylogenetic tree(s). Beet curly top virus (Curtovirus),

a geminivirus was designated as outgroup to root the

tree. The analysis was replicated 1000 times.

Bootstrap analysis was performed to estimate stability

and support for the inferred clades18

.

Results Survey of Disease

Ten sweet pepper grown agricultural farms,

averaging 10-15 acres in size, were surveyed in Al-

Batinah, northern region of the Sultanate of Oman.

Mainly green cultivars of Sweet peppers were grown

on farms either under shade house or open field

during September to February. All the farms surveyed

were 5-10 km apart from each other and had varying

degree of leaf curl disease incidence. The disease

incidence ranged from 5 to 45% in older crops (data

not shown). Further, the incidence on sweet peppers

was high on field grown plants as compared to those

grown under shade house.

Symptoms in Diseased Plants

Typical leaf curl symptoms were observed on sweet

peppers infected with TYLCV. Peppers exhibited

upward curling of leaf margins with interveinal and

marginal chlorosis, and thickening of veins (Fig. 1).

In severe infection, yellow of the leaves was evident.

Infected leaves were often found thicker than the

normal ones. Detection of TYLCV by PCR Assays

A total of sixty samples (6 sample from each

farm) collected from infected sweet pepper yielded

Table 1—List of viruses compared in the present study, their origin and NCBI gene accession numbers

Virus strains Acronyms Origin Gene Accession No.

Beet curly top virus BCTV USA M24597

Tomato begomovirus ToBV-MT Mayotte AJ620914

Tomato begomovirus ToBV-FR France AJ620916

Tomato geminivirus ToGV-DR Dominique Republic AF068640

Tomato geminivirus ToGV-LB Lebanon AF065823

Tomato geminivirus ToGV-SD Sudan AF058030

Tomato yellow leaf curl virus TYLCV-FR France AJ842306

Tomato yellow leaf curl virus TYLCV-GD Guadeloupe AY319646

Tomato yellow leaf curl virus TYLCV-IS Israel AF058020

Tomato yellow leaf curl virus TYLCV-KU Kuwait AF065822

Tomato yellow leaf curl virus TYLCV-MX Mexico U65089

Tomato yellow leaf curl virus TYLCV-OM Oman DQ132859

Tomato yellow leaf curl virus TYLCV-RU Reunion AJ842307

Tomato yellow leaf curl virus TYLCV-RU Reunion AJ842308

Tomato yellow leaf curl virus TYLCV-EG Egypt AY594174

Tomato yellow leaf curl virus TYLCV-PG Portugal AF105975

Tomato yellow leaf curl virus TYLCV-IS Israel AB110218

Tomato yellow leaf curl virus TYLCV-JO Jordan AY594175

Tomato yellow leaf curl virus TYLCV-SP Spain AF071228

Tomato yellow leaf curl virus TYLCV-JP Japan AB014346

Tomato yellow leaf curl virus TYLCV-AL Almeria AJ489258

Tomato yellow leaf curl virus TYLCV-DR Dominique Republic AF024715

Tomato yellow leaf curl virus TYLCV-CU Cuba AJ223505

Tomato yellow leaf curl virus TYLCV-PR Puerto Rico AY134494

Tomato yellow leaf curl virus TYLCV-IR Iran AJ132711

Tomato yellow leaf curl virus TYLCV-ML Mali AY502934

Cassava begomovirus CBM-MZ Mozambique AF329243

Cassava begomovirus CBM-SZ Swaziland AF329235

South African Cassava mosaic virus SACMV-SA South Africa AF155806

Tomato begomovirus ToBV-MYDB Mayotte:Dembeni AJ865341


Table 2--Comparison of nucleotide sequence of coat protein gene of begomovirus infecting sweet pepper from Oman with other begomoviruses

Coat protein gene sequence identity (%) Gemini virus strains* 1 2 3 4 5 6 7 8 9 10 11 12 13 14

SACMV-SA TomBV-MT TornBV-FR TomGV-DR TomGV-LB TomGV-SD TYLCV-FR TYLCV-GD TYLCV-IS TYLCV-KU TY LCV-MX TYLCV-OM TYLCV-RU TYLCV-RUSG

*Abbreviations of virus strains and descriptions are given in Table 1 I

I were aligned using Bio Edit program and analyzed in BLAST. NCBI BLAST results revealed that sequence - from symptomatic sweet pepper showed 98% identity with TYLCV reported from Egypt and Kuwait; 97%

I identity to TYLCV from Lebanon, Israel, and Jordan; 95% identity to TYLCV strains from Puerto Rico and Iran; 92% identity to TYLCV strain from Mali; 94% identity to Sudan strains; 85% identity to Cassava mosaic virus from South Africa; and 87% identity to tomato begomovirus from many countries (Table 2). Comparison of CP gene sequences of many begomoviruses from different parts of the world showed that TYLCV sequence from Oman is 96.5% identical to those reported from Guadeloupe and Mexico; 94% identical to strains from Israel; 93.8% identical to Kuwait; and more than 90% identical to

Fig. I-Sweet pepper plants infected with begomovirus tomato begomovirus strains from Dorninique Republic, Lebanon, and Sudan (Table 2).

begomovirus specific 1.2 kb products when amplified by PCR using the primer pair PALlv1978/PARlc496 Phylogenetic Analyses and 650 b~ with primer pair AV494 and AC1048 Results of phylogenetic analyses on core CP gene (Figs 2A & B). A 602 bp product was amplified by of T ~ C V from- sweet pepper in oman pig. 3) PCR using the TyLCV core CP gene specific primer revealed that sweet pepper virus clusters with TYLCV pairs T ~ c ~ V 3 6 9 and T ~ c ~ C 1 0 2 3 pig. 2C). PCR sbains from Kuwait, and Israel and TomGV from products were amp1ified plant Lebanon but formed a separate clade indicating a new samples unless otherwise mentioned. strain. The closest relative of TYLCV from Oman CP Gene Sequence Analysis was tomato begomovirus from Lebanon and Sudan.

PCR amplified DNA fragments of 602 b using These results support the conclusion that sweet pepper I P 9 . TYLCV CP gene specific primer pairs , were T K C V from Oman is closely related to other

sequenced. One PCR positive symptomatic sample TYLCV strains from Middle East region, but forms a. fiom each farm, i.e. a total of 10 samples, was separate strain and can be named as TYLCV-Oman sequenced for phylogenetic analysis. The sequences strain.


49

Discussion Geminiviruses constitute an important group of

pathogen characterized by a circular single stranded

DNA genome and unique geminate particle

morphology19

. The TYLCV, a member of whitefly-

transmitted Geminiviruses, is a worldwide threat for

cultivated tomatoes and other agricultural crops3,20-22

.

Al-Batinah region is located on northern part of the

Sultanate of Oman along the coastal line and

comprises more than 80% of the total arable land.

During the survey in Al-Batinah region, 5-10%

incidence of leaf curl disease was found on sweet

peppers grown under shade house, while 35-45% on

those grown under open field. The low disease

incidence on plants under shade house could be due to

restricted movement of whiteflies, whereas in field

population of whiteflies was found in great

abundance. In addition, most of the field grown sweet

pepper crops were located adjacent to tomato field,

which showed more than 80% incidence of leaf curl

disease.

TYLCV was first recorded in Israel as a whitefly-

transmitted virus in tomato (Lycopersicon

esculentum) crops23-25

. It has been identified on the

basis of molecular data and placed in the Genus

Begomovirus, Family Geminiviridae. TYLCV occurs

in most eastern Mediterranean countries26

and parts of

sub-Saharan Africa, Asia, Australia, and the

Caribbean27

. In Europe two species of TYLCV are

present21

, comprising tomato yellow leaf curl virus

(TYLCV) syn TYLCV-IL, and tomato yellow leaf

curl Sardinia virus (TYLCSV) (using the most recent

taxonomic nomenclature described3).

All fragments obtained with TYLCV coat protein

gene specific PCR primers had a 90% or greater

nucleotide identity with their respective viruses. The

begomovirus specific primers yielded expected

product confirming the presence of virus in infected

sweet pepper samples14,28-31

. The CP gene is the most

highly conserved gene in the genus Begomovirus14

.

The CP gene sequence is used to predict strains,

species and taxonomic lineages of begomoviruses32

.

International Committee on Taxonomy of Viruses

(ICTV) accepts the classification of begomoviruses

based on CP sequence when full length genomic

sequences are not available9,33

.

Alignment of core CP sequences of different

begomoviruses with CP sequence of the present

Oman isolate and phylogenetic analysis of aligned

sequences clearly indicates that virus strain isolated

Fig. 2 (A-C)—PCR amplification of begomovirus isolated from

field grown sweet pepper in Oman: A. A product of 1.2 kb

amplified by using PAL1v1978 and PAR1c496; B. 620 bp

product amplified by primers AV494 and AC1048; & C.620 bp

product amplified by primers TycpV369 and TycpC1023 specific

to core CP region of TYLCV.


50

from sweet pepper in Oman is a TYLCV strain. CP

sequence analysis and phylogenetic tree

reconstruction permits a predition of relatedness

among begomoviruses that is highly similar to the

outcome from alignment of begomovirus CP gene

sequences8,10

. The core CP region has highly

conserved amino acid sequences interrelated with

variable bases in all members of Begomovirus. These

features provide useful molecular tool to perform

preliminary identification of begomoviruses. The core

CP sequence contains sufficient conserved and

variable regions that virtually mimic the overall

composition of the complete CP sequence32

. Padidam

et al8 have used highly variable 5’ end of the CP gene

sequence ( ~ 200 nt) as a molecular marker to reveal

differences between closely related strains of

begomoviruses. However, complete reliance on

highly variable region may complicate the

begomovirus identification that otherwise can be done

by the conserved region. Present results from core CP

gene sequence alignment and construction of

phyologenetic tree using CP sequences and complete

genome sequences have clearly established that the

virus infecting sweet pepper from Oman is a discrete

strain of TYLCV belonging to the genus Begomovirus

and the name tomato yellow leaf curl virus-Oman

(TYLCV-OM) is proposed for the same.

Acknowledgement

Authors are thankful to the Sultan Qaboos

University for financial support (AJK) to carry out

this work. We thank Dr David Dennison of SQU for

his help in DNA sequencing.

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