distribution and genetic diversity of begomoviruses infecting tomato and pepper plants in the...

Annals of Applied Biology ISSN 0003-4746

R E S E A R C H A R T I C L E

Distribution and genetic diversity of begomoviruses infectingtomato and pepper plants in the PhilippinesW.S. Tsai1,2, S.L. Shih1, S.G. Venkatesan1, M.U. Aquino3, S.K. Green1, L. Kenyon1 & F.-J. Jan2

1 AVRDC-The World Vegetable Center, Shanhua, Tainan 74199, Taiwan

2 Department of Plant Pathology, National Chung Hsing University, Taichung 402, Taiwan

3 Department of Agriculture, Bureau of Agricultural Research, Quezon City, The Philippines

KeywordsBegomovirus; Geminiviridae; genetic

recombination; PCR; phylogenetic analysis.

CorrespondenceDr F.-J. Jan, Department of Plant Pathology,

National Chung Hsing University,

250, Kuo-Kuang Road, Taichung 402, Taiwan.

Email: [email protected]

Received: 10 October 2010; revised version

accepted: 20 December 2010.

doi:10.1111/j.1744-7348.2011.00462.x

Abstract

Begomoviruses were found to be the major viruses infecting tomato plantsin the Philippines based on the surveys conducted from 2005 to 2006.Pepper-infecting begomoviruses were also detected. Isolates of four distinctbegomovirus species, Ageratum yellow vein virus (AYVV), Tomato leaf curl Cebu

virus (ToLCCeV), Tomato leaf curl Mindanao virus (ToLCMiV) and Tomato leafcurl Philippines virus (ToLCPV), were characterised at the DNA sequencelevel by comparing 20 DNA-As from tomato samples comprising 13 fromLuzon, 2 from Cebu and 5 from Mindanao Islands, along with 3 DNA-As frompepper samples, 1 each from Luzon, Cebu and Mindanao Islands. Two ofthese species (ToLCCeV and ToLCMiV) were distinct novel begomoviruses,while AYVV was detected for the first time in the Philippines. By geographicdistribution, two tomato begomoviruses (ToLCPV and ToLCCeV) were detectedin Luzon and Cebu Islands. The ToLCMiV was also detected in LuzonIsland. The three tomato begomoviruses, AYVV, ToLCCeV and ToLCMiV,were detected in Mindanao Island. A ToLCPV isolate infecting pepper wasalso detected in Luzon Island, while ToLCCeV was detected in peppersamples from Cebu and Mindanao Islands. The diversity of viruses andtheir distinct geographic distribution need to be taken into considerationin the development and deployment of resistance against begomoviruses inthe Philippines. Strategies for the use of post-transcriptional gene silencingfor the control of tomato-infecting begomoviruses in the Philippines arediscussed.

Introduction

The genus Begomovirus, family Geminiviridae, comprises

more than 178 species of whitefly-transmitted and

dicotyledon-infecting geminiviruses (Stanley et al., 2005;

Fauquet et al., 2008). Their genome is circular single-

stranded DNA and may be bipartite (containing both

genomic DNA-A and -B) or monopartite (containing

DNA-A-like genome only) (Stanley et al., 2005). Bego-

moviruses cause severe damage in several economically

important crops, including cassava, cotton, cucurbits,

legumes, pepper, potato and tomato (Makkouk et al.,

1979; Legg, 1999; Varma & Malathi, 2003; Shih et al.,2007). Incidence and yield losses as high as 100% havebeen reported in vegetable crops such as tomato (Saikia& Muniyappa, 1989; Pico et al., 1996; Polston & Ander-son, 1997) and pepper (Sulandari et al., 2006; De Barroet al., 2008).

In the Philippines, severe crop damage caused bytomato leaf curl begomovirus diseases was documented,especially in the summer season (Soriano et al., 1989;Dolores & Bajet, 1995). The presence of a tomato-infecting begomovirus was first reported in the Philippinesin 1971, but the virus could not be distinguished from

Ann Appl Biol 158 (2011) 275–287 © 2011 The Authors 275Annals of Applied Biology © 2011 Association of Applied Biologists

Tomato- and pepper-infecting begomoviruses in the Philippines W.S. Tsai et al.

Tomato leaf curl virus (ToLCV) and Tobacco leaf curlvirus by its host range comparison (Retuerma et al.,1971). Twenty years later, the virus was confirmed asa geminivirus by a positive reaction with monoclonalantibodies against Tomato yellow leaf curl virus (TYLCV)and the hybridisation with TYLCV nucleic acid probes(Dolores & Bajet, 1995). In 1997, a tomato-infectingbegomovirus from the Philippines was characterised asa new distinct begomovirus based on a partial DNA-Asequence (Shih et al., 1997; Zeidan et al., 1998). In 2002,a monopartite tomato-infecting begomovirus (GenBankAccession No. AB050597) isolated from diseased plantsin Luzon Island was shown to have a low DNA-Anucleotide identity (<79%) with other begomoviruses(Kon et al., 2002). On the basis of the criteria forclassification of begomovirus species, including DNA-A nucleotide sequence identity of less than 89%, itwas classified as a distinct begomovirus species Tomatoleaf curl Philippines virus (ToLCPV) (Stanley et al., 2005;Fauquet et al., 2008). The host range of ToLCPV includesDatura stramonium, Nicotiana glutinosa, Nicotiana sylvestris,Nicotiana tabacum and Solanum lycopersicum (Dolores &Bajet, 1995). A bipartite squash-infecting begomovirusbelonging to Squash leaf curl Philippines virus (SLCPHV)was also found to cause severe crop damage in thePhilippines (Benigno, 1979; Dolores & Valdez, 1988;Kon et al., 2003). However, Solanaceae crops includingtomato (S. lycopersicum), N. glutinosa and N. tabacum werenot hosts of SLCPHV (Dolores & Valdez, 1988). Thepresence of begomoviruses on pepper and weeds wasonly confirmed by nucleic acid hybridisation using a DNAprobe produced from Tomato yellow leaf curl Thailand virus(Dolores & Pissawan, 1994).

Knowledge of virus diversity and epidemiology isimportant for disease management, especially in develop-ing virus-resistant cultivars. So far, begomoviruses haveonly been identified by DNA sequence comparison intomato and cucurbit plants from Luzon Island of thePhilippines (Shih et al., 1997; Kon et al., 2002, 2003;Matsuda et al., 2008). Since the first molecular identifica-tion of a tomato-infecting begomoviruses was reported,several full-length begomoviral DNA-A sequences (Gen-Bank Accession Nos. AB050597, AB307731, AB377111,AB377112, AB377113, AF136222 and DQ092867) havebeen identified from tomato samples collected in LagunaProvince, Luzon Island (Shih et al., 1997; Kon et al., 2002;Matsuda et al., 2008). They were all isolates of ToLCPV(Fauquet et al., 2008). However, molecular identificationof pepper-infecting begomoviruses has not been reportedin the Philippines. In this article, the genetic identity anddiversity of tomato- and pepper-infecting begomoviruseswas assessed for samples obtained from throughout thePhilippines.

Materials and methods

Collection of symptomatic samples and virus detection

Eighty-seven samples from tomato (S. lycopersicum) and18 from pepper (Capsicum sp.) plants showing symptomsof virus infection including leaf yellowing, mosaic, curl-ing, blistering and plant stunting were collected fromacross the Philippines (Table 1). For begomovirus detec-tion, viral DNAs were extracted from a leaf disk (0.5 cmdiameter) and analysed by PCR with a degenerate primerpair – PAL1v1978B/PAR1c715H – which amplified anapproximately 1.5-kb DNA-A product (including the 5′

of C1 gene, intergenic region, the V2 gene and the 5′ ofCP gene) (Gilbertson et al., 1991; Tsai et al., 2011). ThePCR amplication was carried out as previously describedwith 0.8 mM dNTP and 1 U Taq DNA polymerase (Invit-rogen, Carlsbad, CA, USA) (Rojas et al., 1993). Samplesfound to contain begomoviral DNA-A were also testedfor the presence of begomoviral DNA-B using the DNA-B-specific degenerate primer pairs DNABLC1/dNAVBL1and DNABLC2/DNABLV2 (Green et al., 2001). The pres-ence of infections with isolates of Ageratum yellow vein

virus (AYVV), Tomato leaf curl Cebu virus (ToLCCeV),Tomato leaf curl Mindanao virus strain N (ToLCMiV-N),ToLCMiV strain S (ToLCMiV-S) and ToLCPV was indi-vidually analysed in the begomovirus-positive samples byusing primer pairs AYVVSP-V/PAR1c715H, ToLCCeVSP-V/PAR1c715H, ToLCMiVNSP-V/PAR1c715H, ToLCMiVSSP-V/PAR1c715H and ToLCPVSP-V/C, respectively(Table 2). These virus-specific primers were designedbased on the sequence alignment of tomato and pepper-infecting begomoviruses including the newly identifiedand reported Philippine isolates listed in Fig. 1. All col-lected samples were also tested for the presence ofCucumber mosaic virus (CMV), Potato virus Y (PVY) andTomato mosaic virus (ToMV) by double antibody sand-wich enzyme-linked immunosorbent assay (DAS-ELISA)(Clark & Adams, 1977). CMV polyclone antibodies, pro-duced from NT9 isolate and generated by Dr F.-J. Jan’sLaboratory, National Chung Hsing University, Taichung,Taiwan, were used for CMV detection. PVY and ToMVantisera were purchased from the Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH (DSMZ,Braunschweig, Germany).

Cloning and sequencing of begomoviral DNAs

Begomoviral DNAs for sequencing were selected basedon the crops and locations (Table 1). The 1.5-kb DNA-Afragments amplified by PCR using general DNA-Adetection primers (Table 2) were cloned into pGEM-TEasy vector (Promega, WI, USA) as per supplier’sinstructions and then used for sequencing. On the basis

276 Ann Appl Biol 158 (2011) 275–287 © 2011 The AuthorsAnnals of Applied Biology © 2011 Association of Applied Biologists

W.S. Tsai et al. Tomato- and pepper-infecting begomoviruses in the Philippines

Tab

le1

Vir

usd

etec

tion

ofsa

mp

les

colle

cted

inth

eP

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pin

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Spec

ific

Det

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nof

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No.

ofV

irus

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cted

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ple

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fect

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olat

esin

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ecie

s

Mix

edIn

fect

ion

by

Isol

ates

inTw

o

Spec

ies

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ndP

rovi

nce

Loca

tion

Year

of

Col

lect

ion

Cro

ps

No.

of

Sam

ple

s

Col

lect

edB

GV

CM

VP

VY

ToM

VD

NA

-Asb

ToLC

CeV

ToLC

PV

ToLC

MiV

-N

ToLC

MiV

-N+

ToLC

MiV

-S

AYV

V+

ToLC

MiV

-N

ToLC

PV

+To

LCC

eV

ToLC

PV

+To

LCM

iV-N

Nor

thLu

zon

Ben

guet

Bag

uio

2006

Tom

ato

1812

06

17P

36,P

410

120

00

00

Cen

tral

Luzo

nN

ueva

Ecija

Mun

oz20

06To

mat

o9

91

00

P2-

1,P

2-2,

P7

07

00

02

0

Wes

t-ce

ntra

lLuz

onP

anga

sina

nP

anga

sina

n20

06To

mat

o6

51

00

P20

05

00

00

0

Sout

hwes

tLu

zon

Lagu

naLo

sB

anos

2006

Tom

ato

1717

10

0P

77,P

93,P

96,

P10

1,P

102

014

00

00

3

2006

Pep

per

51

31

0P

108

01

00

00

0

Bat

anga

sLi

pa

2006

Tom

ato

99

00

0P

115,

P11

80

90

00

00

Ceb

uC

ebu

Ceb

u20

06To

mat

o6

60

00

P13

4,P

135

50

00

01

0

2006

Pep

per

121

70

0P

152

10

00

00

0

Min

dan

aoN

orth

Cot

abat

oM

akila

la20

06To

mat

o5

30

04

GSD

1,G

SD6

00

30

00

0

Sout

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ato

Gen

eral

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os20

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mat

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511

64

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bal

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05To

mat

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30

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PL9

30

00

00

0

Man

olo

Fort

ich

2005

Pep

per

11

10

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L31

00

00

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Tota

lP

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311

10

32

10

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Tom

ato

8769

1412

2520

847

34

13

3

aN

um

ber

of

sam

ple

sposi

tive

for

bego

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ng

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ith

the

gen

eral

pri

mer

pai

r–

PA

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978B

/PA

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,an

dby

DA

S-E

LIS

Afo

rC

ucu

mbe

rm

osai

cvi

rus

(CM

V),

Pot

ato

viru

sY

(PV

Y)

and

Tom

ato

mos

aic

viru

s(T

oM

V).

bIs

ola

tes

for

wh

ich

com

ple

teD

NA

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ucl

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de

sequ

ence

sw

ere

obt

ain

ed.

c Nu

mbe

rin

dic

ates

sam

ple

sth

atw

ere

posi

tive

tosp

ecifi

cdet

ecti

on

of

Age

ratu

mye

llow

vein

viru

s(A

YV

V),

Tom

ato

leaf

curl

Ceb

uvi

rus

(ToLC

CeV

),T

omat

ole

afcu

rlM

inda

nao

viru

sst

rain

N(T

oLC

MiV

-N),

ToLC

MiV

stra

inS

(ToLC

MiV

-S)

and

Tom

ato

leaf

curl

Ph

ilip

pin

esvi

rus

(ToLC

PV

).ToLC

CeV

+ToLC

PV

=sa

mple

sth

atw

ere

posi

tive

for

ToLC

CeV

and

ToLC

PV

;ToLC

MiV

-N+

ToLC

PV

=sa

mple

sth

atw

ere

posi

tive

for

ToLC

MiV

-Nan

dToLC

PV

;A

YV

V+T

oLC

MiV

-N=

sam

ple

sth

atw

ere

posi

tive

for

AY

VV

and

ToLC

MiV

-N;

ToLC

MiV

-N+T

oLC

MiV

-S=

sam

ple

sth

atw

ere

posi

tive

for

ToLC

MiV

-Nan

dToLC

MiV

-S.



Table 2 Primer sequences used in this study

Primers Sequence (5′ to 3′)a Purpose

PAL1v1978Bb GCATCTGCAGGCCCACATBGTYTTHCCNGT General DNA-A detection of begomovirusesPAR1c715Hb GATTTCTGCAGTTDATRTTHTCRTCCATCCA General DNA-A detection of begomovirusesGSD1-FV ACCGGATCCATTAGTTAATGAGTTTCC Full-length amplification of the DNA-A sequences of GSD1 and GSD6GSD1-FC TCCGGATCCCACATGTTTGGATATCA Full-length amplification of the DNA-A sequences of GSD1 and GSD6P2-FV AACGGATCCTCTGGTACATCCA Full-length amplification of the DNA-A sequences of P2-2, P7, P20, P36, P41, P108

and P118P2-FC TTGGGATCCCACATTCTTAGT Full-length amplification of the DNA-A sequences of P2-2, P7, P36, P41, P108

and P118P20-FC TCGGGATCCCACATTCTTAATTCA Full-length amplification of the DNA-A sequence of P20P77-FC TCGGGATCCCACATTTTTAGTACA Full-length amplification of the DNA-A sequence of P77P96-FV TCCGGATCCTTTGGTACATCCATTTCCT Full-length amplification of the DNA-A sequence of P96P96-FC CCCGGATCCCACATTCTTAGTACA Full-length amplification of the DNA-A sequence of P96P102-FV AACGGATCCTCTGGTACATCCCTTTCCT Full-length amplification of the DNA-A sequences of P77, P93, P101, P102 and P115P102-FC CCCGGATCCCACATTTTTTAGTACA Full-length amplification of the DNA-A sequences of P93, P101, P102 and P115P134-FV AACGGATCCTCTAGTTCATCCTTTCCCT Full-length amplification of the DNA-A sequences of P2-1, P134, P135 and P152P157-FV AAAGGATCCACTGCTCAACGAGT Full-length amplification of the DNA-A sequence of P157P157-FC CCCGGATCCCACATGTTAATAATTGT Full-length amplification of the DNA-A sequence of P157P162-FV CCCGGATCCGTTAGTTAATGAGTTTCC Full-length amplification of the DNA-A sequence of P162P162-FC TGGGGATCCCACATGTTTGGGAAACA Full-length amplification of the DNA-A sequence of P162PL3-FV AACGGATCCTCTAGTTCACCCCTTCCCT Full-length amplification of the DNA-A sequences of PL3 and PL9PL3-FC TCTGGATCCCACATGTTCGCCATTACCT Full-length amplification of the DNA-A sequences of P2-1, P134, P135, P152, PL3

and PL9FGSD1-FC1 CGTCAAGTCCTATATCGACA Sequencing of the DNA-A sequences of GSD1 and GSD6FGSD1-FV1 ATAGAAGGCCCTTTGGTACT Sequencing of the DNA-A sequences of GSD1 and GSD6FP2-FC1 TCTGCTAGAGGGGGTCAACA Sequencing of the DNA-A sequences of P2-1, P2-2, P96, P108 and P152FP2-FV1 TAATGAGCCTAGTACTGC Sequencing of the DNA-A sequences of P2-1, P2-2, P96, P108 and P152FP7-FC1 TAAATCAAGCTCTGACGTCA Sequencing of the DNA-A sequence of P7FP20-FC1-1 TAAATCAAGCTCCGACGTCA Sequencing of the DNA-A sequences of P20, P36, P41, P77, P93, P101, P102, P115,

P118, P134 and P135FP20-FV1 AGTTCGTGATAGGAGACCCT Sequencing of the DNA-A sequences of P7, P20, P36, P41, P77, P93, P101, P102,

P115 and P118FP134-FV1 AGTGCGTGATAGGAGGCCAT Sequencing of the DNA-A sequences of P134 and P135FP157-FC1 ACCAGATCAGCACATTTCCA Sequencing of the DNA-A sequence of P157FP157-FV1 AGACCCTTTGGTACTGCTAT Sequencing of the DNA-A sequence of P157FP162-FC1 AGATCGACGCACGATCTGCA Sequencing of the DNA-A sequence of P162FP162-FV1 TATGGAATTTGGTCAGGTGT Sequencing of the DNA-A sequence of P162FPL3-FC1 AAGTTCCGATGTCAAATCCT Sequencing of the DNA-A sequences of PL3 and PL9FPL3-FV1 TGATAGGAGGCCATATGGGA Sequencing of the DNA-A sequences of PL3 and PL9AYVVSP-V TAACAAATGTCCCCCACTCA Specific detection of AYVVToLCCeVSP-V TCATATTGGACCTTGACAGCT Specific detection of ToLCCeVToLCMiVNSP-V GAGATTTAATTTCCGTCGTC Specific detection of ToLCMiV-N strainToLCMiVSSP-V CGCATCGAAGGTACGTCGTC Specific detection of ToLCMiV-S strainToLCPVSP-V AYCAYACAGAGAACGCTTTAC Specific detection of ToLCPVToLCPVSP-C AAAGAGTTTATGGGGGCCCA Specific detection of ToLCPV

aB = G, T, C; D = A, G, T; H = A, T, C; N = A, T, G, C; R = A, G; Y = C, T.bPrimers used were published by Tsai et al. (2011).

of the comparison of 1.5 kb DNA-A sequences, abuttingprimers were designed to amplify full-length DNA-Asby PCR (Table 2). The PCR reaction was conductedas described above with a PfuUltra™ high-fidelity DNApolymerase (Stratagene, La Jolla, CA, USA) and 3 min forDNA elongation. Amplified full-length DNA-As were alsocloned and sequenced as described above. Sequencingprimers were also designed to allow sequencing of the

complete full-length DNA-As (Table 2). These sequencesof full-length DNA-As were used for further sequenceanalysis.

Sequence analysis

The sequences were compared by using BLAST searchingGenBank and by using MegAlign software (DNASTAR,



98

Cluster 1/ToLCPV

Cluster 2/AYVV

Cluster 4/ToLCMiV

LB2 strain

Lag strain LB1 strain

Cluster 3/ToLCCeV

P93, Lu, T, EU487034 P101, Lu, T, EU487036

P102, Lu, T, EU487037 P115, Lu, T, EU487039 ToLCPV-LB2[PH:Lag2], AB377112 ToLCPV-LB2[PH:Lag1], AB377111 ToLCPV-LB2[PH:LB2], AB050597 P20, Lu, T, EU487028 P96, Lu, T, EU487035 P108, Lu, P, EU487038 P2-2, Lu, T, EU487026 P118, Lu, T, EU487040 P41, Lu, T, EU487030 P36, Lu, T, EU487029

ToLCPV-LB2[PH:BL1], DQ092867 P77, Lu, T, EU487032 P7, Lu, T, EU487027

ToLCPV-LB1[PH:Lag3], AB377113 ToLCPV-LB1[PH:LB1], AF136222

ToLCPV- Lag[PH:Lag], AB307731 AFPL9, Mi, T, EU487009 AFPL3, Mi, P, EU487008

P134, Ce. T, EU487042 P152, Ce, P, EU487044 P2-1, Lu, T, EU487025 P135, Ce, T, EU487043

TYLCKaV, AF511529 PepYLCIDV, DQ083765

P157, Mi, T, EU487045 AYVV-Gx[CN:Gx13], AJ558120 AYVV-Gx[CN:Gx68], AJ849916 AYVV-ID[ID:Tom], AB100305

AYVV-SG[TW:Tao], DQ866134 AYVV-SG[SG], X74516

AYVV-TW[TW:PD], AF327902 AYVV-TW[TW:Tai], AF307861

AYVV-Hn[CN:Hn2.19], AJ564744 AYVV-Hn[CN:Hn2], AJ495813 P162, Mi, T, EU487046 GSD1, Mi, T, EU487047 GSD6, Mi, T, EU487048

ToLCLV, AF195782 ToLCMYV, AF327436

ToLCJV, AB100304 TbLCYnV, AJ566744 ToLCGxV, AM236784 ALCuV, AJ851005

PaLCuCNV, AJ558116 PepLCV, AF414287

ToLCTWV, U88692 ToLCGuV, AY602165

TYLCGuV, AY602166 HYVV, AB055009

TbLCJV. AB055008 VeYVV, AM182232

SLCYNV, AJ420319 TYLCTHV, AY514631

TYLCCNV, AF311734 ToLCGV, AF449999 ToLCCNV, AJ558118

ToLCVV, AF264063 ToLCV, S53251

TYLCAxV, AY227892 TYLCSV, Z25751

TYLCMalV, AF271234 TYLCMLV, AY502934

TYLCV, X15656 ToLCMGV, AJ865339

ToLCUV, DQ127170 ToLCKMV, AJ865341

ToLCYTV, AJ865340 PepYVMV, AY502935

ToLCArV, DQ519575 WmCSV, AJ245652

SLCCNV, AM260205 SLCPHV, AB085793 ToLCNDV, U15015

ToLCRaV, DQ339117 BYVMV, AF241479

MaYVV, AJ744881 ToLCKeV, DQ852623

ToLCBV, Z48182 ToLCPuV, AY754814

ToLCSLV, AF274349 ToLCPKV, DQ116884

PepLCBDV, AF314531 ToLCJoV, AJ875159

ChiLCV, DQ629103 ToLCKV, U38239 TbCSV, AJ457986 ToLCBDV, AF188481

TYLCIDV, AF189018 MYMIV, AF126406

MYMV, DQ400848 ToMoV, L14460

WDV, X02869

100

100

100

100

100

100

98

100

100

100

100

100

100

100

100

6398

100

100

100

100

87100

81

100

67

94

94

98

100

98

100

100

100

96

82

92

98

100

100

100

99

100

99

100

75

100

99

78

73

100

95

91

91

94

89

98

88

7361

77

60

68

99

1

1

0.05

S strain N strain

Figure 1 Legend on next page.



Madison, WI, USA) for pairwise comparisons. For phylo-genetic analysis, a neighbour-joining tree was generatedby using the Molecular Evolutionary Genetics Analy-sis software version 4.0 using the Clustal W alignmentwith 1000 bootstrap replications (Tamura et al., 2007).The p-distance model was used to test the evolutionarydistances. The Recombination Detection Program ver-sion 2 (RDP2) was used to test the potential recombinantsequences by setting the highest acceptable probabil-ity value (P value) as 0.00001 and the window size at20 (Martin et al., 2005). For recombination detection,all sequences listed in Fig. 1 were used and tested bythe following methods: bootscanning (Salminen et al.,1995), chimaera (Posada and Crandall, 2001), maximumchi-square (Maynard Smith, 1992), original RDP (Mar-tin & Rybicki, 2000) and sister-scanning (Gibbs et al.,2000). The geminiviral DNA-A sequences listed in Fig. 1were retrieved from the NCBI-GenBank and used for theanalyses.

Results

Virus detection in symptomatic samples

The results of virus detection in the collected symp-tomatic samples are summarised in Table 1. Symptomatictomato and pepper plants exhibited leaf curling, yellow-ing, mosaic and blistering and/or stunting symptoms.

Begomoviral DNA-As were detected in 69 of 87 tomatoand 3 of 18 pepper samples. No DNA-B was detected inany of the DNA-A-positive tomato or pepper samples.CMV was detected in 14 tomato and 11 pepper sam-ples, PVY in 12 tomato and 1 pepper samples, and ToMVin 25 tomato samples. When specific primer pairs wereused to detect single or mixed begomovirus infection inDNA-A-positive samples, the results showed that 62 ofthe 69 tomato samples and all 3 pepper samples werepositive for single begomovirus infection, whereas mixedinfections by isolates in two begomovirus species weredetected in seven tomato samples (Table 1).

Cloning and sequencing of begomoviral DNA-As

Twenty-three begomoviral DNA-As were completelysequenced (Table 1, Fig. 1). Twenty of these were from 19symptomatic tomato samples that were collected from theislands of Luzon, Cebu and Mindanao. Two distinct bego-moviral DNA-As (P2-1 and P2-2) with 87.9% nucleotidesequence identity were detected in the same tomato sam-ple. All 20 tomato begomoviral DNA-A sequences rangedfrom 2722 to 2761 nucleotides (nt) in length. ThreeDNA-A sequences from pepper samples, one each fromthe Luzon, Cebu and Mindanao Islands, were studiedand their length ranged from 2722 to 2755 nt. A hair-pin structure with the geminivirus-conserved sequence

Figure 1 Phylogenetic tree obtained from the alignment of the full-length DNA-A nucleotide sequences of begomoviruses. Isolates identified in this study

are in bold and their names are followed by islands (Lu: Luzon; Ce: Cebu; Mi: Mindanao), crops (T: tomato; P: pepper) and accession numbers. Horizontal

distances are proportional to evolutionary distances. The numbers at each branch indicate the percentage of 1000 bootstraps. New begomovirus

species Tomato leaf curl Cebu virus (ToLCCeV) and Tomato leaf curl Mindanao virus (ToLCMiV) were identified in this study. Abbreviations of the

virus names are Ageratum leaf curl virus (ALCuV), Ageratum yellow vein virus Gx13 isolate from China (AYVV-Gx[CN:Gx13]), AYVV Gx68 isolate from

China (AYVV-Gx[CN:Gx68]), AYVV Hn2 isolate from China (AYVV-Hn[CN:Hn2]), AYVV Hn2.19 isolate from China (AYVV-Hn[CN:Hn2.19]), AYVV Pingdong

isolate from Taiwan (AYVV-TW[TW:PD]), AYVV Singapore isolate (AYVV-SG[SG]), AYVV Tainan isolate from Taiwan (AYVV-TW[TW:Tai]), AYVV Taoyuan

isolate from Taiwan (AYVV-SG[TW:Tao]), AYVV tomato isolate from Indonesia (AYVV-ID[ID:Tom]), Bhendi yellow vein mosaic virus (BYVMV), Chilli leaf curl

virus (ChiLCV), Honeysuckle yellow vein virus (HYVV), Malvastrum yellow vein virus (MaYVV), Mungbean yellow mosaic India virus (MYMIV), Mungbean

yellow mosaic virus (MYMV), Papaya leaf curl China virus (PaLCuCNV), Pepper leaf curl Bangladesh virus (PepLCBDV), Pepper leaf curl virus (PepLCV),

Pepper yellow leaf curl Indonesia virus (PepYLCIDV), Pepper yellow vein Mali virus (PepYVMV), Squash leaf curl China virus (SLCCNV), Squash leaf curl

Philippines virus (SLCPHV), Squash leaf curl Yunnan virus (SLCYNV), Tobacco curly shoot virus (TbCSV), Tobacco leaf curl Japan virus (TbLCJV), Tobacco

leaf curl Yunnan virus (TbLCYnV), Tomato leaf curl Arusha virus (ToLCArV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Bangladesh

virus (ToLCBDV), Tomato leaf curl China virus (ToLCCNV), Tomato leaf curl Comoros virus (ToLCKMV), Tomato leaf curl Guangdong virus (ToLCGuV),

Tomato leaf curl Guangxi virus (ToLCGxV), Tomato leaf curl Gujarat virus (ToLCGV), Tomato leaf curl Java virus (ToLCJV), Tomato leaf curl Joydebpur

virus (ToLCJoV), Tomato leaf curl Karnataka virus (ToLCKV), Tomato leaf curl Kerala virus (ToLCKeV), Tomato leaf curl Laos virus (ToLCLV), Tomato leaf

curl Madagascar virus (ToLCMGV), Tomato leaf curl Malaysia virus (ToLCMYV), Tomato leaf curl Mayotte virus (ToLCYTV), Tomato leaf curl New Delhi

virus (ToLCNDV), Tomato leaf curl Pakistan virus (ToLCPKV), Tomato leaf curl Philippines virus Laguna isolate from the Philippines (ToLCPV-Lag[PH:Lag]),

ToLCPV Laguna 1 isolate from the Philippines (ToLCPV-LB2[PH:Lag1]), ToLCPV Laguna 2 isolate from the Philippines (ToLCPV-LB2[PH:Lag2]), ToLCPV

Laguna 3 isolate from the Philippines (ToLCPV-LB1[PH:Lag3]), ToLCPV Los Banos 1 isolate from the Philippines (ToLCPV-LB1[PH:LB1]), ToLCPV Los Banos

2 isolate from the Philippines (ToLCPV-LB2[PH:LB2]), ToLCPV San Leonardo isolate from the Philippines (ToLCPV-LB2[PH:BL1]), Tomato leaf curl Pune virus

(ToLCPuV), Tomato leaf curl Rajasthan virus (ToLCRaV), Tomato leaf curl Sri Lanka virus (ToLCSLV), Tomato leaf curl Taiwan virus (ToLCTWV), Tomato leaf

curl Uganda virus (ToLCUV), Tomato leaf curl Vietnam virus (ToLCVV), Tomato leaf curl virus (ToLCV), Tomato mottle virus (ToMoV), Tomato yellow leaf

curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf

curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow

leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl

virus (TYLCV), Vernonia yellow vein virus (VeYVV) and Watermelon chlorotic stunt virus (WmCSV). Wheat dwarf virus (WDV) was used as an out-group.



Table 3 The sequence of the predicted hairpin structure of cloned begomovirus DNA-As

Sequence of the Hairpin Structure (5′ to 3′)a Crops Isolates

GCGGCCCACGACTATAATATTACCGTGGGCCGC Tomato P2-2, P7, P20, P36, P41, P77, P93, P96, P101, P102, P115 and P118Pepper P108

CGCGTCCCACGTATAGTTAATATTACCGTGGGACGCG Tomato P2-1, P134, P135 and PL9Pepper P152 and PL3

GCGGCCAACCGTATAATATTACCGGATGGCCGC Tomato P157GCGGCCATCCGTATAATATTACCGGATGGCCGC Tomato P162GCGGCCAGCCGTATAATATTACCGGATGGCCGC Tomato GSD1 and GSD6

aTAATATTAC is the geminivirus-conserved sequence in the loop of the hairpin structure.

TAATATTAC in the loop was present in the intergenicregion (IR) of all 23 begomoviral DNA-As. The sequencesof the hairpin structures are listed in Table 3. All viralDNA-A sequences contained six open reading frames(ORFs), two in virus sense (V1 and V2) and four in com-plementary sense (C1 to C4).

Sequence comparison and phylogenetic analysisof viral DNA-As

On the basis of the International Committee onTaxonomy of Viruses (ICTV) criteria for begomovirusspecies demarcation of <89% DNA-A nucleotide identityand phylogenetic analysis of the begomoviral DNA-Asequences, the 20 tomato and 3 pepper begomovirusisolates identified in this study were grouped into fourclusters (Fig. 1). Twelve tomato and one pepper isolatessharing 94.4–99.7% nucleotide sequence identity werein Cluster 1 and they had highest nucleotide sequenceidentity (94.4–99.4%) with ToLCPV-LB2[PH:LB2] fromthe Philippines. Only tomato isolate P157 was in Cluster 2and it had highest nucleotide sequence identity (91.2%)with AYVV-Gx[CN:Gx68] from China. Tomato isolatesP2-1, P134, P135 and PL9, and pepper isolates P152 andPL3 composed Cluster 3. They shared 95.3% to 99.9%nucleotide sequence identity with each other and hadthe highest nucleotide sequence identity (86.0–88.5%)with tomato isolate P7 in Cluster 1. The tomato isolatesP162, GSD1 and GSD6 made up Cluster 4. They shared88.4–99.7% nucleotide sequence identity with eachother and had the highest nucleotide sequence identity(85.2–87.8%) with tomato isolate P157 in Cluster 2.On the basis of the begomovirus sequences analyses,those grouped in Cluster 1 were isolates of ToLCPVand that in Cluster 2 was an isolate of AYVV. Thebegomoviruses in Clusters 3 and 4 should be consideredas new begomovirus species designated as ToLCCeVand ToLCMiV, respectively. Comparing all begomovirusisolates in the species AYVV and ToLCPV including thosenewly identified in this study and those listed in Fig. 1,the DNA-A nucleotide sequence identity of virus isolatesranged from 86.8% to 99.7% for ToLCPV (Cluster 1)

and 82.0% to 98.1% for AYVV (Cluster 2). Within each

begomovirus species including those identified in this

study and the Philippines isolates listed in Fig. 1, theCP ORF of isolates showed high nucleotide sequence

identity (96.7–100%), whereas the IR was more diverse

(36.9–100% nucleotide identity) (Table 4).

On the basis of the criteria for strain demarcation(85–93% of DNA-A sequence identity) in geminivirus

species (Fauquet et al., 2008), the species of ToLCPV

contained the three virus strains, LB1, LB2 and Lag

(Fig. 1). The two isolates in strain LB1 shared 97.3%nucleotide sequence identity. Isolates in strain LB2 shared

93.2–99.7% nucleotide sequence identity. ToLCPV-

Lag[PH:Lag] represents the strain Lag. Isolates in strain

LB1 shared 88.7–93.7% nucleotide sequence identitywith strain LB2 and 91.5–92.9% with strain Lag. Strains

LB2 and Lag shared 86.8% to 90.0% nucleotide sequence

identity. All the newly identified isolates in the speciesof ToLCPV belonged to strain LB2 based on their

highest nucleotide identity with ToLCPV-LB2[PH:LB2]

(Fig. 1). The tomato isolate P157 was a new strain

Philippines of AYVV which had 82.0–91.2% nucleotidesequence identity with other AYVV isolates. On the basis

of the high nucleotide sequence identity of ToLCCeV

isolates (95.3–99.9%), all six isolates composed a strain

Philippines of the species. The species ToLCMiV containedtwo virus strains, S and N (Fig. 1). Isolates GSD1 and

GSD6 were strain N and they shared 99.7% nucleotide

sequence identity with each other. Isolate P162 representsstrain S and had 88.4–88.5% nucleotide sequence

identity with both isolates in strain N.

On the basis of the RDP analysis, recombination was

detected in the IR region of ToLCPV isolates P2-2, P36,P77, P93, P101, P102, P115 from this study and in the

previously reported isolates ToLCPV-LB2[PH:Lag1] and

ToLCPV-LB2[PH:Lag2]. Similar possible recombination

was detected in the IR region of the ToLCCeV isolatesP134, PL3 and PL9, whereas that was also detected in the

CP and 3′ portion of the C3 ORF of the ToLCMiV isolates

GSD1 and GSD6 (Table 5). The recombination map of thementioned isolates is provided in Fig. 2.



Table 4 Percentage of nucleotide sequence identity among begomoviruses infecting tomato and pepper in the Philippines

Virus Speciesa ToLCPV ToLCeV AYVV ToLCMiV ToLCPV ToLCeV AYVV ToLCMiV

Complete DNA-A C1 ORFToLCPV 86.8–99.7 86.7–100ToLCCeV 83.0–88.4 95.3–99.9 85.5–90.7 94.9–99.9AYVV 72.7–73.9 73.2–73.5 — 83.7–87.5 84.9–85.2 —ToLCMiV 73.1–78.6 73.5–75.6 85.2–87.6 88.4–99.7 84.1–88.4 84.6–86.4 88.2–89.4 88.2–99.6

Intergenic region C2 ORFToLCPV 36.9–100 91.7–100ToLCCeV 27.0–62.1 78.6–99.6 91.7–94.1 97.1–100AYVV 29.3–60.5 25.8–30.6 — 77.0–80.1 79.9–80.1 —ToLCMiV 35.5–69.9 31.7–47.0 47.0–56.9 61.3–99.6 78.9–85.3 80.1–87.0 84.8–93.9 87.0–100

CP (V1) ORF C3 ORFToLCPV 96.7–100 93.5–100ToLCCeV 93.2–95.2 97.7–99.9 88.8–91.5 98.0–100AYVV 71.8–73.6 72.1–72.7 — 76.2–79.1 79.5–80.5 —ToLCMiV 70.8–73.1 71.3–73.1 94.7–95.6 97.1–99.7 77.2–83.6 80.0–86.2 85.9–92.6 88.6–100

PCP (V2) ORF C4 ORFToLCPV 90.7–100 87.6–100ToLCCeV 87.2–94.5 98.3–100 85.6–95.2 95.5–100AYVV 56.2–57.1 54.1–55.1 — 85.6–90.7 83.5–87.6 —ToLCMiV 54.4–57.4 54.2–54.8 85.8–86.6 94.3–99.4 85.3–89.0 82.5–88.0 90.0–90.4 87.6–99.7

AYVV, Ageratum yellow vein virus; ORF, open reading frame; ToLCCeV, Tomato leaf curl Cebu virus; ToLCMiV, Tomato leaf curl Mindanao virus;

ToLCPV, Tomato leaf curl Philippines virus.aVirus isolates in the species include those identified in this study and Philippine isolates listed in Fig. 1.

Discussion

The survey conducted in 2005 and 2006 in the Philippinesindicated that begomoviruses were the most commontomato-infecting viruses, followed by ToMV, CMV andPVY (Table 1). CMV was the most common virusdetected in pepper samples. However, pepper-infectingbegomoviruses were also detected. On the basis of theanalysis of 20 DNA-A sequences of tomato-infectingand 3 of pepper-infecting begomoviruses, 4 distinctbegomoviruses (AYVV, ToLCCeV, ToLCMiV and ToLCPV)were found to infect tomato and 2 of these (ToLCCeVand ToLCPV) were also detected in infected pepperin the Philippines. The tomato-infecting ToLCPV hasbeen reported previously (Kon et al., 2002; Matsudaet al., 2008). In this study, three tomato-infectingbegomoviruses (AYVV, ToLCCeV and ToLCMiV) werethe newly reported viruses in the Philippines and twoof these (ToLCCeV and ToLCMiV) represent the firstidentification of these respective begomovirus species.The identification of ToLCCeV and ToLCPV that couldinfect peppers in the Philippines indicates that pepperplants could serve as natural alternative hosts for thetomato-infecting begomoviruses.

Recombination has been reported to contribute togenetic diversification of tomato-infecting begomovirusesin nature (Padidam et al., 1999; Navas-Castillo et al., 2000;Rojas et al., 2005; Moriones et al., 2007; Moriones andNavas-Castillo, 2008). In the Philippines, recombination

has been reported in the IR and 5′ C1 ORF of tomato-infecting ToLCPV-Lag[PH:Lag] (Matsuda et al., 2008). Inthis study, recombination events were also detected in theIR of nine ToLCPV and three ToLCCeV isolates (Table 5).This might reflect the observation that the IR is the hotspot of recombination of begomoviruses (Green et al.,2005; Seal et al., 2006). This might also explain why theIR is the region of greatest diversity in the ToLCPV andToLCCeV isolates. Recombination analysis results for theIRs of isolates in ToLCPV strain LB2 might simply suggestthat all of them share closely related ancestors. The sameseems to apply to ToLCCeV isolates P134, PL3 and PL9.The level of nucleotide sequence identity among the IRs ofToLCCeV isolates may reflect their geographic dispersal,with greatest identity between isolates within an island(e.g. 96.8% of Mindanao PL3 and PL9; 97.2–99.6%of Cebu P134, P135 and P152 and Luzon P2-1) andlower identity between isolates from different islands(e.g. 78.6–83.1% of isolates from Mindanao, Cebu andLuzon). Recombination may occur when there is a mixedinfection of begomoviruses in a plant. In this study,mixed infections by two begomoviruses were detectedin seven tomato samples covering Luzon, Cebu andMindanao. This highlights the risk of further occurrenceof recombination events and the possibility that thesenew recombinants will alter virulence or host specificitywhich should probably be monitored for in the futurethroughout the Philippines.



Table 5 Results of recombination detection

Virusesa Regionb Major Parent Minor Parent

Detection Method, Average

P value

GSD1 408-1079 ToLCPV-Lag[PH:Lag] SLCYNV RDP, 1.091 × 10−6;

MaxChi, 5.165 × 10−35;

Chimaera, 1.964 × 10−39;

SiScan, 1.538 × 10−21

GSD6 408-1079 ToLCPV-Lag[PH:Lag] SLCYNV RDP, 1.091 × 10−6;

MaxChi, 3.969 × 10−35;

Chimaera, 1.421 × 10−39;

SiScan, 1.538 × 10−21

P2-2 2720-28 P41 P36 RDP, 5.392 × 10−7;

MaxChi, 9.125 × 10−10;

SiScan, 3.818 × 10−13

P36 2722-30 P96 P77 RDP, 1.183 × 10−6;

MaxChi, 1.251 × 10−10;

SiScan, 5.014 × 10−11

P77 2721-29 P41 P36 RDP, 1.117 × 10−6;

MaxChi, 7.612 × 10−11;

SiScan, 5.433 × 10−14

P93 2722-141 P96 ToLCPV-LB2[PH:Lag1] RDP, 2.660 × 10−7;

MaxChi, 4.051 × 10−22;

Chimaera, 2.716 × 10−64;

SiScan, 2.182 × 10−6


MaxChi, 4.051 × 10−22;

Chimaera, 1.266 × 10−68;

SiScan, 5.587 × 10−11


MaxChi, 4.053 × 10−22;

Chimaera, 5.644 × 10−67;

SiScan, 5.587 × 10−11

P115 47-141 P118 P77 RDP, 6.615 × 10−7;

BootScan, 1.320 × 10−92;

MaxChi, 1.236 × 10−19;

Chimaera, 8.931 × 10−20;

SiScan, 7.369 × 10−28

ToLCPV-LB2[PH:Lag1] 2721-2754 P41 P36 RDP, 2.842 × 10−7;

BootScan, 4.779 × 10−35;

SiScan, 8.875 × 10−14

ToLCPV-LB2[PH:Lag2] 2721-2754 P41 P36 RDP, 9.901 × 10−7;

Bootscan, 1.361 × 10−35;

SiScan, 8.875 × 10−14

P134 2649-2681 PL9 SLCCNV RDP, 4.559 × 10−9;

Bootscan, 2.195 × 10−15

PL3 2663-2722 P134 P2-1 RDP, 5.828 × 10−7;

Bootscan, 2.777 × 10−46;

SiScan, 1.651 × 10−14

PL9 2663-2689 P134 P2-1 RDP, 4.559 × 10−9;

Bootscan, 4.452 × 10−17;

SiScan, 4.059 × 10−6

aThe abbreviated virus names are listed in Fig. 1.bPotential recombination regions are selected and detected by at least two of the following methods: bootscanning (Bootscan), chimaera, maximum

chi-square (MaxChi), original RDP (RDP) and sister-scanning (SiScan) in the Recombination Detection Program version 2.

Luzon and Mindanao Islands are the major tomato

production areas in the Philippines [CountrySTAT Philip-

pines (http://countrystat.bas.gov.ph)]. Some tomatoes

are also produced in Cebu Island, which is located

between Luzon and Mindanao. Two tomato bego-

moviruses ToLCPV and ToLCCeV were found in Luzon



Figure 2 Recombination linearised map of the pepper- and tomato-infecting begomovirus isolates from the Philippines. Each horizontal line represents

a virus isolate. Different colours and patterns represent the tentative origins of the putative recombinant fragments. The genome organisation is shown

at the bottom and started at the nicking site of the geminivirus-conserved sequence TAATATT/AC. The open reading frames in virus sense (V1 and V2),

in complementary sense (C1 to C4), right part of intergenic region (RIR) and left part of intergenic region (LIR) are also indicated. The abbreviated virus

names are listed in Fig. 1.

and Cebu Islands. The ToLCMiV was also detected inLuzon Island. The tomato begomoviruses were morediverse in Mindanao Island where isolates of three distinctspecies (AYVV, ToLCCeV and ToLCMiV) were detected.Isolates of ToLCCeV were identified from throughout thePhilippines. This information is important for the devel-opment of disease management strategies, including thebreeding of resistant cultivars. As part of an AVRDC-The World Vegetable Center network, several ‘wild’species such as Solanum hirsutum LA1777, Solanum peru-vianum INRA sel., Solanum chilense LA1969 and LA1932and S. lycopersicum FL699sp were screened for resis-tance to tomato leaf curl disease in Los Banos, Lagunaprovince, Luzon Island (Green & Shanmugasundaram,2007). However, in this area the majority of the iden-tified tomato-infecting begomovirus isolates belonged toToLCPV, including isolates tested in this study and thosefrom previous reports (Kon et al., 2002). Therefore, beforea nation-wide resistance breeding programme for tomatoleaf curl diseases is initiated, the potential resistancesources also need to be tested in other provinces ofLuzon, Cebu and Mindanao, where at least three otherdistinct tomato begomovirus species were found to bepresent.

Virus-derived transgenic resistance provides anotherpossibility for disease control. It can be generated

based on the post-transcriptional gene silencing (PTGS)mechanism (Tenllado et al., 2004). Broad-spectrumresistance against cassava-infecting geminiviruses hasbeen developed based on the transgene-specific siRNAs(short interfering RNAs) (Chellappan et al., 2004). Thisspecific siRNA strategy provides one possible routefor controlling tomato-infecting begomoviruses in thePhilippines. The coat protein (CP) genes of isolates ofToLCCeV and ToLCPV can be the targets to generatetransgenic tomato resistant to both viruses. Their CP geneshad close nucleotide sequence identity (93.2–99.9%) and57 predicted siRNAs (1 with perfect identity, 19 with onemismatched and 37 with two mismatched) are conservedin all isolates of both virus species (Yuan et al., 2004).The other two tomato-infecting begomoviruses, AYVVand ToLCMiV, are also expected to be controlled bythis strategy. The CP genes of all isolates in bothbegomovirus species also had high nucleotide sequenceidentity (95.0–99.7%) and 124 predicted siRNAs (48 withperfect identity, 44 with one mismatched and 32 withtwo mismatched) were conserved in all isolates (Yuanet al., 2004). Recently, silencing of different viral genomesthrough PTGS was achieved by chimeric gene constructsderived from two distinct virus species (Jan et al., 2000;Praveen et al., 2006; Lin et al., online first). This providesthe possibility of generating transgenic tomato resistant to



all four identified tomato-infecting begomoviruses in thePhilippines using a chimeric transgene, which combinesCP gene fragments effective against both ToLCCeV andToLCPV, as well as against AYVV and ToLCMiV.

Acknowledgements

We thank Dr Wen-Hsiung Ko, Professor Emeritus of theUniversity of Hawaii at Manoa and Dr Chung-Jan Chang,Professor of the University of Georgia at Griffin Campusfor their critical review of this manuscript. This work wassupported by the National Science Council, ExecutiveYuan, Taiwan (project no. 95-2317-B-125-001), and theGesellschaft fur Technische Zusammenarbeit (GermanAgency for Technical Cooperation), Germany (Interna-tional Agricultural Research Project No. 2001.7860.8-001.00). We are grateful to Dr C.G. Kuo, AVRDC-TheWorld Vegetable Center, Shanhua, Tainan, Taiwan, forproviding his contacts to initiate and assist in the sur-vey. We acknowledge the help of the following personswith the sample collection: Ms Yen-Wei Wang, AVRDC-The World Vegetable Center, Shanhua, Tainan, Taiwan,Dr A.C. Roxas, Central Luzon State University, ScienceCity of Munzo, Nueva Ecija, Ms D.L. Sandoval, Depart-ment of Agriculture, Bureau of Agricultural Research,Dilliman, Quezon City, and Ms B.D. Acabal JR, Depart-ment of Agriculture, Regional Crop Protection Center,Magulkay, Mandaue City, Cebu. We appreciate the tech-nical assistance provided by Ms Li-Mei Lee, Ms Jin-TehWang and Mr Yung-Chia Huang, AVRDC-The World Veg-etable Center, Shanhua, Tainan, Taiwan.

References

Benigno D.A. (1979) Leaf curl disease of squash. Philippine

Agriculturalist, 61, 304–305.Chellappan P., Masona M.V., Vanitharani R., Taylor N.J.,

Fauquet C.M. (2004) Broad spectrum resistance to ssDNAviruses associated with transgene-induced gene silencingin cassava. Plant Molecular Biology, 56, 601–611.

Clark M.F., Adams A.N. (1977) Characteristics of themicroplate method of enzyme-linked immunosorbentassay for the detection of plant viruses. Journal of General

Virology, 34, 475–483.De Barro P.J., Hidayat S.H., Frohlich D., Subandiyah S.,

Ueda S. (2008) A virus and its vector, pepper yellow leafcurl virus and Bemisia tabaci, two new invaders ofIndonesia. Biological Invasions, 10, 411–433.

Dolores L.M., Bajet N.B. (1995) Isolation and transmissionof tomato leaf curl virus in the Philippines. Philippine

Phytopathology, 31, 40–51.Dolores L.M., Pissawan C. (1994) Detection of leaf curl

virus in pepper by using non-productive DNA probe.Philippine Journal of Crop Science, 19, 80.

Dolores L.M., Valdez R.B. (1988) Identification of squash

viruses and screening for resistance. Philippine

Phytopathology, 24, 43–52.

Fauquet C.M., Briddon R.W., Brown J.K., Moriones E.,

Stanley J., Zerbini M., Zhou X. (2008) Geminivirus strain

demarcation and nomenclature. Archives of Virology, 153,783–821.

Gibbs M.J., Armstrong J.S., Gibbs A.J. (2000)

Sister-scanning: a Monte Carlo procedure for assessing

signals in recombinant sequences. Bioinformatics, 16,

573–582.

Gilbertson R.L., Rojas M.R., Russell D.R., Maxwell D.P.

(1991) Use of the asymmetric polymerase chain reactionand DNA sequencing to determine genetic variability of

bean golden mosaic geminivirus in the Dominican

Republic. Journal of General Virology, 72, 2843–2848.

Green S.K., Shanmugasundaram S. (2007) AVRDC’s

international networks to deal with the tomato yellow

leaf curl disease: the needs of developing countries. In

Tomato Yellow Leaf Curl Virus Disease: Management, Molecular

Biology, Breeding for Resistance, pp. 417–439. Ed.

H. Czosnek. Dordrecht, the Netherlands: Springer.

Green S.K., Tsai W.S., Shih S.L., Black L.L., Rezaian A.,

Rashid M.H., Roff M.M.N., Myint Y.Y., Hong L.T.A.

(2001) Molecular characterization of begomoviruses

associated with leaf curl disease in Bangladesh, Laos,

Malaysia, Myanmar, and Vietnam. Plant Disease, 85,1286.

Green S.K., Tsai W.S., Shih S.L., Huang Y.C., Lee L.M.

(2005) Diversity of begomoviruses of tomato and weeds

in Asia. In Proceeding of the International Seminar on

Whitefly Management and Control Strategy, pp. 19–66.

Eds T.-Y. Ku, C.-L. Wang and C.-A. Chang. Taipei,

Taiwan, ROC: Food and Fertilizer Technology Center forthe Asian and Pacific Region.

Jan F.-J., Fagoaga C., Pang S.Z., Gonsalves D. (2000) A

single chimeric transgene derived from two distinct

viruses confers multi-virus resistance in transgenic plants

through homology-dependent gene silencing. Journal of

General Virology, 81, 2103–2109.

Kon T., Dolores L.M., Murayama A., Bajet N.B., Hase S.,Takahashi H., Ikegami M. (2002) Genome organization of

an infectious clone of Tomato leaf curl virus (Philippines), a

new monopartite Begomovirus. Journal of Phytopathology,

150, 587–591.

Kon T., Dolores L.M., Bajet N.B., Hase S., Takahashi H.,

Ikegami M. (2003) Molecular characterization of a strain

of Squash leaf curl China virus from the Philippines. Journal

of Phytopathology, 151, 535–539.

Legg J.P. (1999) Emergence, spread and strategies for

controlling the pandemic of cassava mosaic virus

disease in east and central Africa. Crop Protection, 18,

627–637.

Lin C.-Y., Ku H.-M., Tsai W.S., Green S.K., Jan F.-J. (2011)Resistance to a DNA and a RNA virus in transgenic plants



by using a single chimeric transgene construct. Transgenic

Research. doi: 10.1007/s11248-010-9412-7.

Makkouk K.M., Shehab S., Majdalani S.E. (1979) Tomato

yellow leaf curl: incidence, yield losses and transmission

in Lebanon. Journal of Phytopathology, 96, 263–267.

Martin D., Rybicki E. (2000) RDP: detection ofrecombination amongst aligned sequences. Bioinformatics,

16, 562–563.

Martin D.P., Williamson C., Posada D. (2005) RDP2:

recombination detection and analysis from sequence

alignment. Bioinformatics, 21, 260–262.

Matsuda N., Sharma P., Bajet N.B., Ikegami M. (2008)

Molecular characterization of a new strain of tomato leafcurl Philippines virus and its associated satellite DNAβ

molecule: further evidence for natural recombination

amongst begomoviruses. Archives of Virology, 153,

961–967.

Maynard Smith J. (1992) Analyzing the mosaic structure of

genes. Journal of Molecular Evolution, 34, 126–129.

Moriones E., Garcıa-Andres S., Navas-Castillo J. (2007)Recombination in the TYLCV complex: a mechanism to

increase genetic diversity. Implications for plant resistance

development. In Tomato Yellow Leaf Curl Virus Disease:

Management, Molecular Biology, Breeding for Resistance,

pp. 119–138. Ed. H. Czosnek. Dordrecht, the

Netherlands: Springer.

Moriones E., Navas-Castillo J. (2008) Review. Rapidevolution of the population of begomoviruses associated

with tomato leaf curl disease after invasion of a new

ecological niche. Spanish Journal of Agricultural Research, 6,

147–159.

Navas-Castillo J., Sanchez-Campos S., Noris E., Louro D.,

Accotto G.P., Moriones E. (2000) Natural recombination

between Tomato yellow leaf curl virus-Is and Tomato leaf curl

virus. Journal of General Virology, 81, 2797–2801.

Padidam M., Sawyer S., Fauquet C.M. (1999) Possible

emergence of new geminiviruses by frequent

recombination. Virology, 165, 218–225.

Pico B., Dıez M.J., Nuez F. (1996) Viral diseases causing the

greatest economic losses to the tomato crop. II. The

tomato yellow leaf curl virus – a review. Scientia

Horticulturae, 67, 151–196.

Polston J.E., Anderson P.K. (1997) The emergence of

whitefly-transmitted geminiviruses in tomato in the

Western Hemisphere. Plant Disease, 81, 1358–1369.

Praveen S., Mishra A.K., Antony G. (2006) Viral

suppression in transgenic plants expressing chimeric

transgene from Tomato leaf curl virus and Cucumber mosaic

virus. Plant Cell, Tissue and Organ Culture, 84, 47–53.

Posada D., Crandall K.A. (2001) Evaluation of methods for

detecting recombination from DNA sequences: computer

simulations. Proceedings of the National Academy of Sciences of

the United States of America, 98, 13757–13762.

Retuerma M.L., Pableo G.O., Price W.C. (1971) Preliminarystudy of the transmission of Philippine tomato leaf

curl virus by Bemisia tabaci. Philippine Phytopathology, 7,

29–34.

Rojas M.R., Gilbertson R.L., Russell D.R., Maxwell D.P.

(1993) Use of degenerate primers in the polymerase chain

reaction to detect whitefly-transmitted geminiviruses.

Plant Disease, 77, 340–347.Rojas A., Kvarnheden A., Marcenaro D., Valkonen J.P.T.

(2005) Sequence characterization of Tomato leaf

curl Sinaloa virus and Tomato severe leaf curl virus:

phylogeny of New World begomoviruses and detection

of recombination. Archives of Virology, 150,

1281–1299.

Saikia A.K., Muniyappa V. (1989) Epidemiology andcontrol of tomato leaf curl virus in southern India. Tropical

Agriculture, 66, 350–354.

Salminen M.O., Carr J.K., Burke D.S., McCutchan F.E.

(1995) Identification of breakpoints in intergenotypic

recombinants of HIV type 1 by Bootscanning. AIDS

Research and Human Retroviruses, 11, 1423–1425.

Seal S.E., vandenBosch F., Jeger M.J. (2006) Factorsinfluencing begomovirus evolution and their increasing

global significance: implications for sustainable control.

Critical Reviews in Plant Sciences, 25, 23–46.

Shih S.L., Dolores L.M., Nakhla M.K., Maxwell D.P.,

Green S.K. (1997) A new geminivirus associated with leaf

curl disease of tomato in the Philippines. Plant Protection

Bulletin (Taiwan), 39, 394–395.Shih S.L., Tsai W.S., Green S.K., Singh D. (2007) First

report of tomato leaf curl Joydebpur virus infecting Chilli

in India. Plant Pathology, 56, 341.

Soriano J.M., Villareal R.L., Roxas V.P. (1989) Tomato and

pepper production in the Philippines. In Proceedings of the

International Symposium on Integrated Management Practices,

pp. 549–560. Shanhua, Tainan, Taiwan, ROC:AVRDC-The World Vegetable Center.

Stanley J., Bisaro D.M., Briddon R.W., Brown J.K.,

Fauquet C.M., Harrison B.D., Rybicki E.P., Stenger D.C.

(2005) Geminiviridae. In Virus Taxonomy. VIIIth Report of

ICTV, pp. 301–326. Eds C.M. Fauquet, M.A. Mayo,

J. Maniloff, U. Desselberger and L.A. Ball. New York:

Academic Press.Sulandari S., Suseno R., Hidayat S.H., Harjosudarmo J.,

Sosromarsono S. (2006) Detection and host range study

of virus associated with pepper yellow leaf curl disease.

HAYATI Journal of Biosciences, 13, 1–6.

Tamura K., Dudley J., Nei M., Kumar S. (2007) MEGA4:

molecular evolutionary genetics analysis (MEGA)

software version 4.0. Molecular and Evolution, 24,1596–1599.

Tenllado F., Llave C., Dıaz-Ruız J.R. (2004) RNA

interference as a new biotechnological tool for

the control of virus diseases in plants. Virus Research,

102, 85–96.

Tsai W.S., Shih S.L., Kenyon L., Green S.K., Jan F.-J.(2011) Temporal distribution and pathogenicity of the



predominant tomato-infecting begomoviruses in Taiwan.Plant Pathology, doi: 10.1111/j.1365-3059.2011.02424.x

Varma A., Malathi, V.G. (2003) Emerging geminivirusproblems: a serious threat to crop production. Annals of

Applied Biology, 142, 145–164.Yuan B., Latek R., Hossbach M., Tuschi T., Lewitter F.

(2004) siRNA selection server: an automated siRNA

oligonucleotide prediction server. Nucleic Acids Research,

32, W130–W134.Zeidan M., Green S.K., Maxwell D.P., Nakhla M.K.,

Czosnek H. (1998) Molecular analysis ofwhitefly-transmitted tomato geminiviruses fromSoutheast and East Asia. Tropical Agriculture Research and

Extension, 1, 107–115.


distribution and genetic diversity of begomoviruses infecting tomato and pepper plants in the...

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