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Virus Genes ISSN 0920-8569 Virus GenesDOI 10.1007/s11262-012-0832-8

Characterization of a new begomovirus anda beta satellite associated with the leaf curldisease of French bean in northern India

Naimuddin Kamaal, MohammadAkram, Aditya Pratap & Prashant Yadav

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Characterization of a new begomovirus and a beta satelliteassociated with the leaf curl disease of French beanin northern India

Naimuddin Kamaal • Mohammad Akram •

Aditya Pratap • Prashant Yadav

Received: 12 July 2012 / Accepted: 20 September 2012

� Springer Science+Business Media New York 2012

Abstract Begomoviruses are emerging as serious threat

to many crops throughout the world particularly in tropical

and sub-tropical regions. A leaf curl disease with symp-

toms typical of infection by many begomoviruses was

observed in French bean (Phaseolus vulgaris) at Kanpur,

India, during 2010–2012. The disease caused downward

leaf curling and made the plants unproductive. The disease

was transmitted from infected to healthy plants through

whitefly (Bemisia tabaci). The products of five samples

digested with EcoRI yielded DNA fragments of about

2.7 kb. The complete sequence of the Fb1 sample com-

prised 2,741 nucleotides with genome organization typical

of begomoviruses having two ORFs in virion-sense and

five ORFs in complementary-sense separated by an inter-

genic region with begomovirus conserved nonanucleotide

sequence, TAATATTAC. The complete DNA-A sequence

homology was most closely related to Cotton leaf curl

Bangalore virus with 80 % nucleotide sequence identity.

Based on the demarcation criteria for identifying a

begomovirus species, Fb1 is considered as a distinct

begomovirus species, named French bean leaf curl virus

and designated as FbLCV-[IN:Knp:12]. The complete

sequence of associated satellite DNA-b comprises 1,379

nucleotides with single ORF and has 80 % identity with

Papaya leaf curl beta satellite. There was no evidence of

recombination in DNA-A of FbLCV and associated beta

satellite DNA molecule.

Keywords Begomovirus � DNA-A �Rolling circle amplification � Beta satellite �French bean leaf curl virus � Bemisia tabaci

Introduction

A large number of viruses belonging to the family

Geminiviridae have been described from different parts of

the globe. They are classified into four genera viz, Mas-

trevirus, Curtovirus, Topocuvirus, and Begomovirus, which

differ from each other with respect to host range, vector

(insect), and genome organization [1]. With the advent of

rolling circle amplification (RCA) technology, there has

been a rapid progress in the identification of new begom-

oviruses species from different plant species [2–7]. Of the

four genera of the family Geminiviridae, Begomovirus is

the largest and comprises viruses transmitted by whitefly

(Bemisia tabaci Genn.) that are economically important

plant pathogens. Begomoviruses are assumed to have been

coevolving with their hosts for a long time; however, it is

in the past two decades, these viruses have become eco-

nomically important plant pathogens [8–10]. Based on

genome organization, three types of begomoviruses are

recognized viz, Type I: with bipartite genome (DNA-A and

DNA B), Type II: with monopartite genome (DNA-A), and

Type III: with monopartite genome and a satellite DNA

component (DNA-A and DNA-b) [11].

N. Kamaal � M. Akram (&) � P. Yadav

Division of Crop Protection, Indian Institute of Pulses Research,

Kanpur 208024, India

e-mail: akram23859@gmail.com

N. Kamaal

e-mail: naimk@rediffmail.com

P. Yadav

e-mail: prashantnduat@gmail.com

A. Pratap

Division of Crop Improvement, Indian Institute of Pulses

Research, Kanpur 208024, India

e-mail: adityapratapgarg@gmail.com

123

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DOI 10.1007/s11262-012-0832-8

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All the genes required for important viral functions like

replication, control of gene expression, encapsidation, and

vector transmission lie in DNA-A [12, 13], whereas DNA

B contains two ORFs that codes for two proteins which are

said to be involved in virus movement between cell to cell

and within the cells [14]. In bipartite begomoviruses, both

DNA-A and DNA B components are essential for infec-

tivity [15, 16]. Monopartite begomoviruses such as Tomato

yellow leaf curl virus [17] and Tomato leaf curl virus [18]

consist of only a single genomic component functionally

equivalent to the DNA-A and DNA-B and homologous to

DNA-A of bipartite begomoviruses [19] and is alone

capable of causing disease in host. Third type of begom-

oviruses possess monopartite genome but require associa-

tion of a beta satellite DNA for induction of typical disease

symptoms, such as in case of Bhendi yellow vein mosaic

virus [20], Tomato yellow leaf curl China virus [21],

Cotton leaf curl Multan virus [22], and Okra leaf curl virus

[23]. Beta satellite is a circular, single-stranded DNA

molecule of *1.4 kb with a highly conserved organization

for DNA-b molecules consisting of a single conserved

open-reading frame, an adenine-rich region, and a region of

high sequence conservation [the satellite conserved region

(SCR)]. The SCR contains a potential hairpin structure

with the loop sequence TAA/GTATTAC similar to the

origins of replication of Gemini viruses [24].

All the Gemini viruses species known to infect French

bean (Phaseolus vulgaris) belong to the genus Begomovi-

rus except Bean yellow dwarf virus [25] that is a Mastre-

virus. Begomoviruses— Bean calico mosaic virus, Bean

dwarf mosaic virus, Bean golden mosaic virus, Bean

golden yellow mosaic virus, Bean mosaic florida virus,

Horsegram yellow mosaic virus (HgYMV), Mungbean

yellow mosaic India virus (MYMIV), Lima bean golden

mosaic virus [11], Macroptilium yellow mosaic Florida

virus, Macroptilium mosaic Puerto Rico virus [26] are

known to infect P. vulgaris naturally. These viruses

broadly cause green–yellow mosaic symptoms in the foli-

age. There are, however, several other viruses that infect

bean and cause severe leaf curling, stunting, and chlorosis

in bean. These viruses include: Chinodel tomate virus [27,

28], Cotton leaf crumple virus [29], Tomato yellow leaf

curl virus [30, 31]. In India, members of the Begomovirus

are known to infect many different plant species viz.,

tomato [5], papaya [32], radish [3], okra [20, 33], mung-

bean and urdbean [34], pigeonpea [35], soybean [36],

weeds like Ageratum conyzoides, Croton bonpladianum,

Malvastrum coromandelianum [37], Calendula [38], chilli

[39, 40], cotton [41], mesta [42], and tobacco [43].

French bean is grown in many parts of the India for both

green pods and grains. This crop is reported to be infected

by MYMIV and HgYMV, both species of begomovirus and

cause yellow mosaic symptoms [11]. A leaf curl disease of

bean was observed in the experimental fields of Indian

Institute of Pulses Research (IIPR), Kanpur for last many

years. However, during 2010–12 some plants showed

downward leaf curling, thickening of veins, distortion of

growing stem, and overall stunting. Diseased plants bore

flowers, but the pods formed were reduced in size and

deformed. Symptoms were typical of begomovirus infec-

tion reported in many hosts from different parts of the

world. The present article describes characterization of a

new monopartite begomovirus species and of a satellite

DNA-b associated with leaf curl disease of French bean at

Kanpur.

Materials and methods

Transmission

Mechanical sap inoculation was attempted from field

infected plants onto healthy plants of French bean. Infected

leaf tissue was homogenized in chilled 0.1 M neutral

phosphate buffer (1:1 w/w) and the homogenate was used

as inoculum. Ten plants of each of the two French bean

varieties (Amber and Utkarsh) were inoculated and main-

tained in insect proof growth chamber. Whiteflies associ-

ated with diseased plants were collected using an aspirator

and released on ten healthy plants of French bean variety

Utkarsh and 20–30/plant kept in a net house and covered

with plastic cages and allowed to feed for 48 h. Inoculated

plants were maintained for 30 days and observed for the

development of symptoms, if any.

Sample collection and DNA extraction

Naturally infected French bean leaves showing leaf curling

symptoms (Fig. 1) were collected from the Main Farm of

the IIPR, Kanpur, India. Total DNA was extracted from

five samples using DNeasy Plant Mini Kit (QIAGEN

GmbH, Hilton).

Full length genome amplification of the virus,

associated satellite DNA-b and cloning

Total DNA extracted from the diseased samples was used

as template to amplify the full length genome through the

RCA method using REPLI-g Mini Kit (QIAGEN GmbH,

Hilton) following the manufacturer’s instructions. The

RCA products were digested with restriction enzymes

(EcoRI, EcoRV, HindIII, and Ndel) to select an enzyme

that causes a single cut in circular DNA. The restriction

digested RCA product was observed in 1 % agarose gel to

select *2.7 kb linearized DNA band. The satellite DNA-bwas PCR-amplified using primers Beta01/Beta02 [44] and

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was observed in 1 % agarose gel. The *2.7 kb linearized

DNA and PCR amplified *1.4 kb DNA from one of the

samples randomly selected (referred as Fb1 hereafter) were

purified using HiYieldTM Gel/PCR DNA Mini Kit (RBC,

Banqiao City, Taipei County, Taiwan) and cloned into

pJET/1.2blunt vector using CloneJET PCR-Cloning Kit

(Fermentas Life Sciences, EU) and sequenced commer-

cially (First BASE, Malaysia).

Sequence analysis

Sequence data were assembled and analyzed with the aid of

Bio edit and the ORFs were determined by ORF finder avail-

able at NCBI site (http://www.ncbi.nlm.nih.gov/gorf/gorf.

html). In BLAST search, sequences that had maximum iden-

tity with the DNA-A of Fb1 at 100 % query coverage were

selected for comparison and phylogenetic relationship. These

sequences along with those used for comparison and phylo-

genetic analysis of betasetellite DNA molecule are given in

Table 1. Pairwise per cent nucleotide identity of Fb1 DNA-A

and associated beta satellite was worked out using ClustalW

software available at http://www.genome.jp/tools/clustalw/

using standard parameters. However, for analyzing phyloge-

netic relationship of the Fb1 DNA-A, besides begomoviruses,

few species of the other genera of the Geminiviridae were also

used (Table 1). The phylogram was constructed using the

Neighbor-joining method in MEGA 5 with bootstrap (1,000

replicates) and all positions containing gaps and missing data

were eliminated [45]. To detect the possible recombination

between Fb1 DNA-A and closely related viruses, they were

subjected to Recombination Detection Program (RDP v.3.44)

using default settings (http://darwin.uvigo.es/rdp/rdp.html).

Similarly, associated satellite DNA-b was also subjected to

RDP analysis.

Results and discussion

Symptomatology and transmission

Affected plants showed symptoms (Fig. 1) typical of

begomovirus infection and reported in this host under field

conditions from different parts of the world [31, 46, 47].

None of the plants inoculated mechanically developed any

symptom indicating that the causal virus was not

mechanically sap transmissible. Four of the ten plants

(40 %) inoculated with whiteflies developed leaf curling

symptoms similar to those observed in the field infected

plants. This indicated that the causal virus is transmitted by

whitefly. However, in India, leaf curl and leaf crinkle

symptoms in French bean have been shown to be caused by

agro inoculation with cloned DNA components of MYMV-

Bg using Agrobacterium tumifaciens strain C58 [48].

Amplification and genome organization

Rolling circle amplification products of all the five samples

digested with EcoRV, HindIII, and Ndel did not yield any

band indicating these enzymes did not linearize viral geno-

mic DNA, but EcoRI yielded *2.7 kb linearized DNA in

the agarose gel. The presence of only one DNA molecule in

all the samples coupled with the absence of any residual

(undigested) high molecular weight DNA led us to believe

that only one DNA molecule was present in all the five

samples indicating that the virus associated with the leaf curl

disease of French bean at Kanpur has a monopartite genome.

Sequence data revealed that the *2.7 kb DNA molecule of

Fb1 actually has 2,741 nucleotides (nt). The sequence data

was submitted at NCBI data base under the accession no.

JQ866297.

The genome (DNA-A) of Fb1 has an organization typ-

ical of old world begomoviruses encoding seven ORFs,

two (AV1 and AV2) in virion-sense and five (AC1, AC2,

AC3, AC4, and AC5) in the complementary-sense sepa-

rated by an intergenic region (IR) that is 276nt long and has

a putative stem-loop structure containing the conserved

nonanucleotide sequence.

Comparison of DNA-A with other begomoviruses

In pair wise alignment, the DNA-A sequence of the Fb1

(JQ866297) was found to have highest nucleotide identity

(80 %) with CLCuBV-GU112003 and ToLCPV-DQ116884.

However, the genome organization of the Fb1 is similar to

that of CLCuBV-GU112003 as both of these viruses contain

the ORF AC5 in addition to the AV1, AV2, AC1, AC2, AC3,

and AC4, which was absent in ToLCPV-DQ116884. With

other viruses used for comparison, the present virus had

75–79 % nucleotide similarity. Complete nucleotide sequence

Fig. 1 Naturally infected French bean plant showing leaf curl

symptoms

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of DNA-A of Fb1 has less than 89 % identity with the pre-

viously described begomoviruses available in the data base,

well below the begomovirus species demarcation limit [1].

The virus isolate (Fb1) infecting French bean at Kanpur is,

therefore, considered as a new begomovirus species for which

the name French bean leaf curl virus-[India:Kanpur:2012] is

proposed and is designated as FbLCV-[IN:Knp:12]. Genes of

FbLCV isolate showed highest identity with the correspond-

ing genes of different begomoviruses (Table 2). AV1 gene is

771/774nt long in all the begomoviruses used for comparison;

however, it was 774nt long in FbLCV. AV1 gene of FbLCV

had highest nt identity (82 %) with the corresponding gene of

PepLCBDV-HM007097 followed by 81 % identity with

CLCuBV-GU112003,-AY705380, ToLCJoV-DQ673859,

Table 1 Details of viruses and beta satellite DNA molecule sequences used in the study for comparison and phylogenetic relationship

Abbreviationb Accession number

Viruses

French bean leaf curl virus-[India:Kanpur:2012]a FbLCV-[IN:Knp:12] JQ866297

Bean yellow dwarf virus-[South Africa:Mpumalanga:1994] BeYDV-[ZA:Mpu:94] NC_003493

Beet mild curly top virus-[United States of America:Worland4] BMCTV-[US:Wor4] NC_004753

Beet severe curly top virus-[United States of America:Cfh] BSCTV-[US:Cfh] NC_004754

Bhendi yellow vein mosaic virus-[India:Pandarahalli:OY173:2006] BYVMV-[IN:Pan:OY173:06] GU112073

Bhendi yellow vein mosaic virus-[India:Tirupathi:OY99:2005] BYVMV-[IN:Tir:OY99:05] GU112071

Chickpea red leaf virus-[Australia:Queensland22:2010] CpRLV-[AU:Qld22:10] NC_014739

Cotton leaf curl Bangalore virus-[India:Bangalore:2004] CLCuBV-[IN:Ba:04] AY705380

Cotton leaf curl Bangalore virus-[India:Bangalore:2004] CLCuBV-[IN:Ba:05] GU112003

Maize streak virus-G-[Mali-Mic25-1987] MSV-G[ML-Mic25-1987] EU628631

Malvastrum yellow vein Baoshan virus-[China:Baoshan:2009] MaYVBV-[CN:Bn:09] FN386459

Mimosa yellow leaf curl virus-[Vietnam:Binhduong:2005] MiYLCV-[VN:Bin:05] DQ641695

Mungbean yellow mosaic India virus-[India:Varanasi:Dolichos:2005 MYMIV-[IN:Var:Dol:05] AY547317

Mungbean yellow mosaic virus-[Thailand:Mungbean1] MYMV-[TH:Mg1] NC_001983

Pedilanthus leaf curl virus-[Pakistan:Multan:2004] PeLCV-[PK:Mu:04] NC_012118

Pepper curly top virus-[United States of America:New Mexico:2005] EF501977 PepCTV-[US:NM:05] NC_009518

Pepper leaf curl Bangladesh virus-India[India:Ghazipur:2007] PepLCBDV-IN[IN:Gha:2007] HM007097

Spinach curly top virus-[United States of America:Spinach3:1996] SpCTV-[US:Sp3:96] NC_005860

Tomato leaf curl China virus-Nannig1[China:Guangxi16:2002] ToLCCNV-Nan1[CN:Gx16:02] AJ704602

Tomato leaf curl Joydebpur virus-India[India:Ludhiyana:2007] ToLCJoV-IN[IN:Lud:07] DQ673859

Tomato leaf curl Joydebpur virus-India[India:Kolkata:2007] ToLCJoV-IN[IN:Kol:07] HM007109

Tomato leaf curl Karnataka virus-Janti[India:Janti:2005] ToLCKV-Jan[IN:Jan:05] AY754812

Tomato leaf curl Pakistan virus-[Pakistan:Rahaim Yar Khan1:2004] ToLCPV-[PK:RYK1:04] DQ116884

Tomato leaf curl Vietnam virus-[Vietnam:Hai Phong:2009] ToLCVV-[VN:Hai:09] GQ338765

Tomato pseudo curly top virus-[United States of America:Florida:1994] TPCTV-[US:FL:94] NC_003825

Beta satellite DNA molecules

Papaya leaf curl beta satellite-[India:Kanpur:Frenchbean:2012]a PaLCuB-[In:Knp:Fb:12] JQ866298

Papaya leaf curl beta satellite-[India:Panipat:Papaya:2008] PaLCuB-[In:Pan:Pap:08] HM143906

Tomato leaf curl Vietnam beta satellite-[Vietnam:Dang Xa2:2008] ToLCVB-[VN:Dx2:08] EU189146

Cotton leaf curl beta satellite-[India:Mohanpura:2012] CLCuB-[IN:Moh:12] HM146307

Tomato leaf curl China beta satellite-[China:Guangxi14:2002] ToLCCNB-[CN:Gx14:02] AJ704609

Tomato leaf curl China beta satellite-[China:Guangxi61:2003] ToLCCNB-[CN:Gx61:03] AJ704616

Cotton leaf curl Multan beta satellite-[Pakistan:Multan:2009] CLCuMB-[PK:Mul:09] EU384589

Papaya leaf curl beta satellite-[India:Chinthapalli:2005] PaLCuB-[In:Chi:05] DQ118862

Mimosa yellow leaf curl beta satellite-[VietnamBinhduong:2007] MiLCuB-[VN:Bin:07] DQ641710

Malvastrum yellow vein beta satellite-[China:Yunnan:2010] MaYLCuB-[CN:Yun:10] GU058336

Chilli leaf curl beta satellite-[Pakistan:Faisalabad:2004] ChLCuB-[PK:Fai:04] AM279672

Tomato leaf curl Karnataka beta satellite-[India:Janti:2004] ToLCuKB-[IN:Jan:04] AY754813

a This studyb As per Fauquet et al. [1] and Briddon et al. [50]

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and ToLCKV-AY754812. AV2 was 360nt long and differed

in size from all the begomoviruses used for comparison

among which its size varied between 339 and 405nt. AV2

gene had highest identity (81 %) with ToLCCNV-AJ704602.

AC1 gene was 1,089nt long like in many other viruses used

for comparison. It had highest identity (84 %) with corre-

sponding gene of CLCuBV-GU112003 and ToLCPV-

DQ116884. AC2 of FbLCV was 405nt long and similar in

size with corresponding gene of PepLCBDV-HM007097,

ToLCPV-DQ116884, and ToLCJoV-DQ673859, and had

highest nt identity (81 %) with corresponding gene of ToL-

CKV-AY754812. AC2 was 408 or 453nt long in other

viruses used for comparison. AC3 gene of FbLCV and of all

other viruses used for comparison was 450nt long and

had highest nt identity with PepLCBDV-HM007097,

ToLCJoV-DQ673859, ToLCCNV-AJ704602, and ToLCVV-

GQ338765. AC4 was 303nt long and similar in size with

corresponding gene of CLCuBV- GU112003, -AY705380,

and BYVMV-GU112073, and had highest nt identity (90 %)

with corresponding gene of BYVMV-GU112073. The other

begomoviruses used for the comparison size of AC4 varied

between 258 and 300nt. AC5 gene of FbLCV was 405 nt long

and was present in only four of the begomoviruses used for

comparison and in all of them its size was smaller than that of

the former. It has highest nt identity (84 %) with corre-

sponding gene of CLCuBV-GU112003. Intergenic region of

FbLCV was the shortest among all the begomoviruses used

for comparison and had highest similarity (83 %) with

CLCuBV-GU112003. It is evident from the information

summed up in the Table 2 that the coat protein gene (AV1) is

the most conserved and the IR is the most variable part

among the different begomoviruses. Based on the complete nt

sequence identity of DNA-A and AV1 gene of FbLCV, it

appears that the present virus, CLCuBV, ToLCPV, and

PepLCBDV had closely related ancestors and during the

course of evolution, would have diverged at some point of

time. Recombination detection program analysis carried out

for FbCLV and associated beta satellite DNA (PaLCuB),

however, did not reveal any signal of recombination.

Deduced amino acid sequences of different ORF enco-

ded proteins of DNA-A of the FbLCV revealed that the

coat protein(AV1) gene is the most conserved among the

different begomoviruses used for comparison. The highest

amino acid sequence identities of the deduced ORF prod-

ucts was found with CLCuBV-GU112003 for AV1 (93 %),

AC1 (88 %), and AC5 (60 %), ToLCCNV- AJ704602 for

AV2 (78 %), ToLCKV- AY754812 for AC2 (77 %), ToL-

CVV- GQ338765 for AC3 (75 %), and BYVMV- GU

Table 2 Comparison of complete DNA-A of FbLCV, its open reading frames (ORFs) and intergenic region(IR) with closely related

begomoviruses

aVirus Accession DNA-A AV1 AV2 AC1 AC2 AC3 AC4 AC5 IR

FbLCV JQ866297 100/2,741 b100/774 100/360 100/1,089 100/405 100/450 100/303 100/405 100/276c100/257 100/119 100/383 100/134 100/134 100/100 100/134

CLCuBV GU112003 80/2,758 81/771 71/366 84/1,089 76/453 71/450 89/303 84/357 83/444

93/256 60/121 88/362 64/150 64/134 73/100 60/118

CLCuBV AY705380 79/2,751 81/771 71/348 83/1,089 76/453 71/450 88/303 – 82/437

92/256 60/115 86/362 70/150 64/134 69/100 –

PepLCBDV HM007097 76/2,760 82/771 74/357 73/1,086 79/405 81/450 69/258 – 57/446

91/256 72/118 70/361 71/134 70/134 41/85 –

ToLCPV DQ116884 80/2,759 80/771 71/405 84/1,083 80/405 80/450 87/297 – 60/397

90/256 69/134 87/360 75/134 73/134 70/98 –

BYVMV GU112073 77/2,789 76/771 70/366 82/1,092 75/453 76/450 90/303 78/357 57/475

89/256 63/121 85/363 66/150 70/134 75/100 50/118

ToLCJoV DQ673859 78/2,767 81/771 73/366 78/1,086 79/405 81/450 73/300 82/849 58/447

90/256 71/121 81/361 73/134 69/134 43/99 57/282

ToLCCNV-Nan1 AJ704602 78/2,729 77/774 81/351 79/1,086 79/408 81/450 72/294 – 55/418

78/257 78/116 81/361 67/135 74/134 42/97 –

ToLCVV GQ338765 75/2,750 72/774 68/351 76/1,089 79/408 81/450 72/294 – 60/436

78/257 63/116 81/362 69/135 75/134 41/97 –

ToLCKV-Jan AY754812 77/2,757 81/771 72/357 76/1,086 81/405 80/450 72/294 – 65/449

92/256 69/118 78/361 77/134 69/134 42/97 –

a Expanded names are given in Table 1b First row in each column is of percentage nucleotide identity/total number of nucleotidesc Second row in each column is of percentage amino acid identity/total number of amino acids

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112073 for AC4 (75 %). The phylogenetic relationship of

the DNA-A nucleotide sequence of FbLCV and some be-

gomoviruses are shown in Fig. 2. French bean leaf curl

virus clustered with begomoviruses being closest to

CLCuBV-AY705380, -GU112003, BYVMV-GU112073,

BYVMV- GU112071, and MaYVBV- FN386459.

Sequence analysis of DNA-b associated with FbLCV

The presence of beta satellite DNA was confirmed by PCR

using universal primers (Beta01/Beta02). Amplified prod-

ucts observed in gel revealed the presence of *1.4 kb

band hinting at the amplification of full length genome of

beta satellite. The beta satellite associated with FbLCV was

found to have 1,379nt and the sequence was deposited at

NCBI data base under the accession no. JQ866298. It had

all the structural features reported to be present in beta

satellites like, single ORF (bC1), nonanucleotide sequence

TAATATTAC, satellite conserved region (SCR), and a

region of sequence rich in adenine (A-rich) [49]. The size

of the DNA-b satellite was approximately half the length of

helper virus (FbLCV) as has been reported for beta

satellites [50]. The complete nt sequence of beta satellite

associated with FbLCV shared highest identity (80 %) with

Papaya leaf curl beta satellite (PaLCuB-[IN:Pan6:Pap:08],

HM143906), which was higher than the 78 % identity

Fig. 2 Phylogenetic tree of

DNA-A of FbLCV and some

selected species of different

genera of Geminiviridae. The

analysis involved 25 nucleotide

sequences. The phylogram was

constructed using the Neighbor-

joining method in MEGA 5 with

bootstrap (1,000 replicates).

Details of the viruses are given

in Table 1

Fig. 3 Phylogenetic tree of beta satellite DNA (PaLCuB-French

bean) and some selected betasetellite species; and comparison of

nucleotide identities. The analysis involved 12 nucleotide sequences.

The phylogram was constructed using the Neighbor-joining method in

MEGA 5 with bootstrap (1,000 replicates). Details of the beta

satellites are given in Table 1. nt (%) = per cent nucleotide identity

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proposed as the species demarcation threshold for a distinct

DNA-b [50]. Phylogenetically also, it clustered with the

PaLCuB (Fig. 3). Therefore, the beta satellite associated

with FbLCV is an isolate of Papaya leaf curl beta satellite

and designated as Papaya leaf curl beta satellite-

[India:Kanpur:French bean:2012] with acronym PaLCuB-

French bean. PaLCuB-French bean had one ORF, bC1 that

was 330nt long and encoded for a 109-amino acid product.

Based on the amino-acid sequence of bC1 also, the PaL-

CuB-French bean had highest identity (81 %) with PaL-

CuB-HM143906.

In India, before the present report, French bean has not

been known to be a host of a monopartite Begomovirus.

The only begomoviruses reported to infect this host are

MYMV and HgYMV [11]. The leaf curling symptoms of

the FbLCV in French bean are commonly associated with

begomoviruses infection in many plant species. Also, in the

literature such symptoms in this host have been reported to

be caused by monopartite begomoviruses viz, Tomato

yellow leaf curl virus in China (JQ326957) [46] and Greece

(DQ144621) [31], Tomato yellow leaf curl virus-Israel in

Spain [30], Tomato yellow leaf curl China virus-(TYL-

CCV)-DQ256460 in China [47]. Restriction digestion of

RCA products yielded only one molecule of *2.7 kb

indicating the presence of only one DNA molecule (DNA-

A) and monopartite nature of the virus associated with leaf

curl disease of French bean at Kanpur. Since DNA beta

satellite is reported to be associated with many monopartite

begomoviruses causing leaf curl disease in different plant

species, we used universal primers beta01 and beta02 to

successfully amplify *1.4 kb DNA molecule that was

cloned and sequenced. Sequence data revealed it to be of

1,379 nt long with 80 % nt identity with PaLCuB-

[IN:Pan6:Pap:08]. Beta satellite DNA has been reported to

be associated with TYLCCNV that causes leaf crumple

disease of P. vulgaris in China [47]. Type of symptoms,

transmission by white fly, and restriction digestion of RCA

products showed that a monopartite begomovirus species is

associated with leaf curl disease of French bean at Kanpur,

India. DNA-A of FbLCV shared highest nt sequence

identity of 80 % with CLCuBV and ToLCPV. In phylo-

genetic relationship FbLCV clustered with begomoviruses.

As per the ICTV guidelines, there must be less than 89 %

nt sequence identity between two distinct begomoviruses

[1]. The present virus fulfills this criterion and is, therefore,

regarded a new begomovirus species distinct from all the

begomoviruses described so far. We propose to name it as

French bean leaf curl virus-[India:Kanpur:2012].

Acknowledgments Authors express their sincere thanks to

Dr. N. Nadarajan, Director, Indian Institute of Pulses Research,

Kanpur for providing the necessary facilities and his constant

encouragement.

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