Arch Virol (2008) 153:533–539
DOI 10.1007/s00705-007-0017-2
BRIEF REPORT
Infectivity of the cloned components of a begomovirus: DNA beta
complex causing chilli leaf curl disease in India
B. Chattopadhyay Æ A. K. Singh Æ T. Yadav Æ
C. M. Fauquet Æ N. B. Sarin Æ S. Chakraborty
Received: 19 July 2007 / Accepted: 22 November 2007 / Published online: 4 January 2008
Ó Springer-Verlag 2007
Abstract The full-length genome of a begomovirus and
its cognate DNA-b satellite component associated with
chilli leaf curl disease (ChLCD), originating from Vara-
nasi, India, were cloned. Sequence analysis revealed that
the viral genome (EF190217) is 2,750 bp and the DNA-b
satellite (EF190215) is 1,361 bp in length. Agroinoculation
with partial tandem repeats of the viral genome along with
the satellite induced symptoms typical of ChLCD in chilli
and Nicotiana benthamiana. However, symptom expres-
sion was delayed and milder when the viral genome was
agroinoculated alone in these hosts. Sequence comparisons
revealed that the genome had the highest sequence identity
(95%) with that of chilli leaf curl virus-PK[PK:Mul:98].
The DNA-b satellite shared maximum sequence identity
(88%) with a DNA-b satellite associated with tomato leaf
curl disease from Rajasthan (ToLCBDB-[IN:Raj:03]).
These results demonstrate that ChLCD is caused by a
complex consisting of the monopartite chilli leaf curl virus
and a DNA-b satellite component. This is the first experi-
mental demonstration of Koch’s postulates using cloned
DNA molecules associated with chilli leaf curl disease.
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-007-0017-2) contains supplementary
material, which is available to authorized users.
B. Chattopadhyay
A. K. Singh
T. Yadav

N. B. Sarin
S. Chakraborty (&)
Molecular Virology Laboratory, School of Life Sciences,
Jawaharlal Nehru University, New Delhi 110067, India
e-mail: supriyachakra@yahoo.com
C. M. Fauquet
Danforth Plant Science Center, 975 North Warson Road,
Saint Louis, MO 63132, USA
Introduction
The geminiviruses are plant-infecting viruses with genomes
consisting of circular, single-stranded DNA (ssDNA) en-
capsidated within geminate particles [17, 28]. Members of
the family Geminiviridae have been grouped into four
genera (Begomovirus, Curtovirus, Mastrevirus and Topoc-
uvirus) based on genome organization, host range and
insect vector [13, 18, 28]. The majority of the geminivi-
ruses belong to the genus Begomovirus, are transmitted by
whiteflies (Bemisia tabaci Gennadius), and infect dicoty-
ledonous plant species [28]. Some begomoviruses possess a
genome of two components, designated as DNA-A and
DNA-B, of approximately *2.7 kb each. DNA-A encodes
a replication-associated protein (Rep), which is essential
for viral replication, a replication enhancer protein (REn), a
transactivator protein (TrAP) that controls late gene
expression, and the coat protein (CP) [13]. DNA-B encodes
a nuclear shuttle protein (NSP) and a movement protein
(MP), both of which are essential in efficient systemic
spread and symptom expression [13]. However, many
begomoviruses have a single genomic component resem-
bling DNA-A, which is capable of autonomous replication
and movement within the plant [7, 10, 16, 20]. Recently, a
novel satellite molecule, called DNA-b, has been found to
be associated with some monopartite begomoviruses
[2, 3, 14, 22, 23]. DNA-b depends on the helper virus for
replication and encapsidation, and its presence is essential
for symptom expression [2].
During the past several years, leaf curl disease of chilli
(ChiLCD) has become a serious problem in most of the
chilli-growing areas of India, including Varanasi. Symp-
toms of mild yellowing, severe leaf curling, leaf distortion,
stunting and blistering, and shortening of internodes have
been observed in chilli fields around Varanasi. Although
123
534
chilli leaf curl etiology has been established during the
1960s in the country [9, 19], pathogenicity of the cloned
DNA components has not been demonstrated. Based on
partial DNA-A sequences, tomato leaf curl New Delhi
virus (ToLCNDV) and chilli leaf curl virus (ChiLCV) were
reported to be associated with ChiLCD [15, 24]. Although
full-length genome sequence suggested association of chilli
leaf curl virus—India [India::05] (GenBank accession
number DQ673859) with ChiLCD in India [26], no
detailed information is available. Here we demonstrate the
association of chilli leaf curl virus (ChiLCV) with a
DNA-b satellite from leaf curl infected chilli samples from
Varanasi, which together cause typical leaf disease
symptoms in chilli.
Leaves from chilli plants, cultivar Kashi Anmol, show-
ing yellowing and leaf curling were collected from a
severely affected field in the experimental farm of the
Indian Institute of Vegetable Research, Varanasi, Uttar
Pradesh, India, in 2005. Total DNA from symptomatic
leaves was isolated by the cetyl-trimethylammonium bro-
mide method [27] followed by concentration of the super-
coiled DNA by the alkaline lysis method [1]. The viral
replicative form (RF) was used as a template for poly-
merase chain reaction using degenerate primers
(PAL1v1978/PAR1c496) used for detection of begomovi-
ruses [21], and begomovirus infection was initially
detected. RF DNA was digested with restriction endonu-
cleases (BamHI, EcoRI, HindIII, PstI) and cloned into
pUC18 previously linearized with the same enzymes. Four
full-length clones were obtained. Cross-hybridization
among the four ChiLCV isolates, partial sequencing of
several clones, and sequences comparisons with other
begomoviruses revealed that all four clones comprised the
complete viral genome (at Bam HI site) and yielded similar
restriction patterns after digestion with BamHI, BglI, ClaI,
EcoRI, EcoRV, HindIII, KpnI, PstI and XhoI. All four
clones were obtained from the same sample. One clone was
selected arbitrarily for further analysis.
Table 1 Agroinfection of selected plant species with cloned tandemly repeated copies of ChiLCD components
Plant/constructs
Viral genome (pTChVA) alone
N. benthamiana
Chilli (Capsicum annuum cv. Kashi Anmol)
Viral genome (pTChVA) and DNA-b (pTChV b)
N. benthamiana
Tomato (Lycopersicon esculentum cv. Kashi Amrit)
Chilli (Capsicum annuum cv. Kashi Anmol)
a
Number of plants infected/inoculated
b
LC leaf curling, YL yellow leaf lamina, Bls blistering, LR leaf rolling, Chls chlorotic spots, LD leaf distortion, SL small leaves, MiLC mild leaf
curling
c
Time of first symptom appearance, in days post-inoculation
123
Arch Virol (2008) 153:533–539
For cloning of the DNA-b satellite, universal primers
located in the highly conserved region found in all DNA-b
satellite sequences were used for amplification [5]. An
approximately 1.3-kb fragment was amplified from infec-
ted chilli plants and was cloned in the pTZ57R/T vector
(INSTA cloning kit, MBI Fermentas, USA). In spite of
several attempts, we could not isolate a DNA-B component
from the infected samples. PCR reactions using different
combinations of primers based on the DNA-A intergenic
region and DNA-B [21] did not yield a DNA-B fragment.
This shows that there is no DNA-B component for this
virus.
To assess infectivity, the viral genome and DNA-b
satellite were subcloned as partial tandem repeats in the
binary vector, pCAMBIA2301. The BamHI (147)—PstI
(1,444) fragment of the ChiLCV genome (pBCVA8) was
first cloned into pCAMBIA2301 and the full-length BamHI
fragment was then inserted to produce a 1.5mer tandem
repeat named pTChVA. For the DNA-b satellite
(pBCVb4), the KpnI (1,111)-BamHI (1,148) fragment was
cloned into pCAMBIA2301, followed by ligation at the
KpnI site to produce a 1.9mer tandem repeat named
pTChVb. Insert orientation within partial tandem repeats
was confirmed by restriction digestion with the appropriate
enzymes. The partial tandemly repeated inserts of clones of
viral genome and DNA-b satellite were agroinoculated into
chilli (Capsicum annuum cv. Kashi Anmol), N. benthami-
ana and tomato (Lycopersiscon esculentum cv. Kashi
Amrit). Seeds were surface sterilized after soaking in
HgCl2 for 1 min and sown on sterilized soil. Equimolar
concentrations of viral genome and DNA-b satellite were
mixed and agroinoculated onto 15-day-old test plants by
stem agroinoculation (by crown needle puncture). Symp-
toms were recorded periodically.
Upon agroinoculation, the viral genome could induce
mild curling and yellowing of leaves on chilli (Table 1). On
N. benthamiana, the viral genome alone could induce leaf
curling and yellowing of lamina. The tandemly repeated
Infected/inoculateda Symptomb Incubation periodc
4/14 YL, LC 12
3/12 MiLC, Chls 30
9/15 YL, LC, Chls, St 8
12/15 LC, Bls, SL, St, LD, LR 17
11/18 LC, Bls, LD, SL, LR 20
Arch Virol (2008) 153:533–539
construct of the DNA-b satellite (pTChVb) could not
induce symptoms on chilli plants when agroinoculated
alone. To investigate the role of the DNA-b satellite, test
plants were agroinoculated with pTChVA and pTChVb. On
chilli, systemic symptoms appear as downward leaf curling
within 20 days post-inoculation (dpi) when the viral
genome and DNA-b satellite were inoculated together
(Table 1). On tomato, symptoms appeared at 17 dpi as
yellowing of the leaf lamina, downward leaf curling and
leaf surface reduction (Table 1). On N. benthamiana,
yellowing of the leaf lamina, chlorotic spots and leaf curling
were also noticed (Table 1). Infectivity and symptom
severity increased when both the viral genome and DNA-b
satellite were co-inoculated. For example, when the viral
genome was inoculated alone, only 29% of N. benthamiana
plants and 25% of chilli plants were infected. However,
co-inoculation resulted in infectivity of up to 60% in these
two hosts (Table 1). The viral genome alone infected
N. benthamiana and chilli (Table 1), but symptoms were
milder compared with infection by the viral genome and
DNA-b satellite in combination, and took much longer to
develop (12 days for N. benthamiana, 30 days for chilli).
Total DNA from agroinoculated plants was extracted
using the method of Dellaporta et al. [8]. Viral genome-
specific oligonucleotide primers (ChAv2614, 50 ATG CCT
AGG GCT GGG AGA T30 and ChAc1526, 50 TCA ACG
CGT CGA CGC CTG 30 ) and DNA-b-specific primers [5]
were used for PCR analysis. Conditions for PCR amplifi-
cation in both cases were an initial strand separation at 94°C
for 2 min and then 30 cycles of 1 min at 94°C, 1 min at
55°C and 1 min at 72°C, followed by a final extension
period of 10 min at 72°C. PCR products were resolved on a
1.2% agarose gel. The presence of the viral genome and
DNA-b satellite in systemically infected plants was
confirmed by PCR amplification (Fig. 1). A viral-genome-
specific *1.0 kb amplicon was observed on agroinoculated
N. benthamiana and chilli plants (Fig. 1a). The presence
of DNA-b was detected in the form of a *1.3 kb
DNA-b-specific amplicon (Fig. 1b). In addition, a
*700-bp DNA band was also observed in agroinoculated
plants when DNA-b-specific primers were used.
Full-length sequence determination of the viral genome
and DNA-b satellite was carried out by the dideoxynu-
cleotide chain termination method using an ABI
automated sequencer with primers specific to either
cloning vectors or established viral sequences. Sequence
alignments were done with ClustalW (http://www.ebi.
ac.uk). Phylogenetic analyses were conducted on matrices
of aligned sequences (produced using ClustalW) using the
neighbor-joining and bootstrap options of MEGA version
4 [29]. Begomovirus genomes (or DNA-A components)
used for comparisons (abbreviations and GenBank
accession numbers) are followed as per Fauquet et al. [12]
535
Fig. 1 PCR analysis of ChiLCV and DNA-b from naturally infected
and agroinoculated plants using specific primers. PCR-amplified
fragments were separated on 1.5% agarose gels and stained with
ethidium bromide. a Reaction using the internal ChiLCV primer pair
(ChAv2614/ChAc1526). Lane 1 agroinoculated chilli plants (inocu-
lated with the viral genome and DNA-b); lane 2 N. benthamiana
plants (inoculated with the viral genome alone); lane 3 non-infected
chilli plants; lane 4 infected N. benthamiana plants (viral genome and
DNA-b); M molecular marker (1-kbp ladder). b Reaction using DNA-
b-specific primers (b01/b02): lane 1 agroinoculated N. benthamiana
plants, lane 2 naturally infected chilli plants, M molecular marker
(1-kbp ladder)
(Supplementary Table 1). DNA-b satellite abbreviations
and accesion numbers are given according to Briddon
et al. [6] (Supplementary Table 2).
The nucleotide sequences of the viral genome
(EF190217) had a genomic organization typical of Old
World begomoviruses. DNA-A encodes six ORFs, with
two in the virion sense (AV1 and AV2) and four in the
complementary-sense orientation (AC1, AC2, AC3, AC4)
separated by an intergenic region (IR). The IR contains
several features typical of begomoviruses, including a 33-
nucleotide potential stem-loop region, which includes the
conserve nonanucleotide sequence (TAATATTAC), two
TATA boxes at nucleotides 112–115 and 2,655–2,658, and
the iteron sequence GGTGG-AAT-GGGGG at nucleotides
2,610–2,614 and 2,638–2,650.
Sequence similarity searches were performed by
comparing the sequence to other begomovirus sequences in
the GenBank database (http://www.ncbi.nlm.nih.gov/
nucleotide). Results indicated that the viral genome has
higher sequence identity with begomoviruses isolated from
Asia and is most closely related to ChiLCV-PK[PK:
Mul:98] (95% identity), followed by ChiLCV-Kha[PK:
Kha:04] (88% identity) and ChiLCV-IN[IN:PRM:Tom:05]
(87% identity) (data not shown). The IR had the highest
sequence identity (90%) with ChiLCV-PK[PK:Mul:98] and
less sequence identity was found with other reported
begomoviruses. When individual encoded proteins were
compared, the highest amino acid sequence identity was
found with ChiLCV-PK[PK:Mul:98] for AV1 (98%),
ChiLCV-IN[IN:PRM:Tom:05] for AV2 (99%),
123
536 Arch Virol (2008) 153:533–539

100 ChiLCV-Mul[PK:Mul:98];AF336806
100 ChiLCV-PK[IN:Var:06] ;EF190217
99 PepLCPKV-[PK:Kha1:04] ;DQ116878
ToLCJoV-BD[BD] ;AJ875159
35 100 ChiLCV-IN[IN:PRM:Tom:05];DQ629103
100 ChiLCV-IN[IN::05] ;DQ673859
ToLCBDV-[BD:2] ;AF188481
42 71 AEV-[NP:01] ;AJ437618
58 TbCSV-[CN:Yn35:01] ;AJ420318
CYVMV-[IN] ;AJ507777
100 PepLCBDV-PK[PK:Kha:04] ;DQ116881
100
24 PepLCBDV-BD[BD:Bog:99];AF314531
70 PepLCPKV-[PK:Kha2:04] ;DQ116879
46 PaLCuV-IN[IN:Luc] ;Y15934
100 PaLCuV-PK[PK:Cot:02] ;AJ436992
96
ToLCKV-Ban[IN:Ban:93] ;U38239
100 ToLCGV-[IN:Var:01] ;AY190290
EuLCV-[CN:Gx35:02] ;AJ558121
97
99 CLCuKV-Man[IN:Dab] ;AY456683
100 CLCuKV-Man[PK:M806b:96] :AJ002449
100
CLCuKV-Fai[PK:Fai1] ;AJ496286
ToLCSLV-[LK:Ban:97] ;AF274349
ToLCBV-[IN:Pun:05] ;AY754814
100
81 98 ToLCBV-A[IN:Ban1] ;Z48182
99
ToLCBV-B[IN:Ban5] ;AF295401
100 ToLCBV-C[IN:Ban4:97] AF165098
99 ToLCKeV-[IN:KerII:05] ;DQ852623
MaYVV-[CN:Yn47:01] ;AJ457824
100 CLCuMV-Fai[PK:Y62:95] ;AJ002447
100
CLCuMV-IN[IN:Sri:94] ;AF363011
100 CLCuAV-[PK:K802a:96] ;AJ002455
59
100 BYVMV-IN[IN:Mad] ;AF241479
100 OYVMV-[PK:Fai201:95] ;AJ002451
SLCCNV-IN[IN:Luc:Pum] ;DQ026296
100 ToLCNDV-IN[IN:ND:Svr:92] ;U15015
64 MaLCV-[CN:Gx87:04] ;AJ971263
100
TbLCYnV-[CN:Yn143:02] ;AJ512762
SiLCuV-[CN:Hn57:04] ;AM050730
SLCMV-LK[LK:Col:98] ;AJ314737
100 ICMV-Ker[IN:Ker2:02] ;AJ575819
100 ICMV-IN[IN:Mah:88] ;AJ314739
VeYVV-[IN:Mad:05] :AM182232
MYMIV-[IN:ND:Bg3:91] ;AF126406

0.15 0.10 0.05 0.00

Fig. 2 Phylogenetic tree based on complete nucleotide sequences of to calculated mutation distances. Numbers at nodes indicate percent-
43 geminiviruses isolates. Sequences were aligned using ClustalW. age bootstrap scores. GenBank accession numbers are indicated to the
Vertical distances are arbitrary, horizontal distances are proportional right of each virus name. Abbreviations are according to [12]

PepLCBDV-PK[PK:Kha:04] for AC1 (97%), PepLCBDV- for AC4 (96%), suggesting that different regions of the viral
BD[BD:Bog:99] for AC2 (94.8%), ChiLCV-IN[IN:PRM: genome have different origins, probably from chilli-
Tom:05] for AC3 (94.8%), and ChiLCV-PK[PK:Mul:98] infecting begomoviruses (data not shown). On the basis of

123
Arch Virol (2008) 153:533–539 537

100 PaLCuB-[IN:Chi:05] ;DQ118862

75 PaLCuB-[IN:Jab:03] ; AY230138
100
PaLCuB-[IN:ND:03] ;AY244706
100 PaLCuB-[IN:Mb:05] ;AY438557
ToLCMaB-[IN:Pu:04] ;AY838894

100 ToLCBB-[IN:Ban:03] ;AY428768

100 ToLCBB-[IN:Coi:03] ;AY438560
92
CroYVMB-[PK:Pun:06] ;AM410551
100 AYLCuB-[PK:Fai3:94] ;AJ316027
AYLCuB-[PK:BAH:97] ;AJ316031
100 ToLCB-[IN:ND:02] ;AJ542490
100
ToLCB-[IN:CP:04] ; AY728263

100 ToLCBDB-[IN:Luk:05] ; DQ343289

ToLCBDB-[IN:Raj:03] ;AY438558
100
100 ToLCBDB-[IN:Var:06] ;EF190215
ToYLCCNB-[CN:Yn261:02] ;AM260719
100 TbLCB-[PK:RYK:98] ;AJ316033
64 TbLCB-[PK:BAH:99] ;AJ316034
59 TbCSB-[CN:Yn115:02] ; AJ457822

100 ToLCB-[IN:Var:06] ;AY438559

100 TYLCTHB-[IN:Aur:06] ;EF095958
LuYVB-[CN:Gx37:04] ;AJ965541
AYVSLB-[IN:Mad:03] ;AJ557441
CLCuGB-[SD:Ok44-1:96] ; AY077799
100 BYVB-[PK:Goj:97] ; AJ316029
BYVB-[IN:Mut:00] ;AJ308425
100 CLCuMB-[IN:Sri:02] ; AY083590
100 CLCuMB-[IN:His2:02] ;DQ364230
100
CLCuMB-[PK:Fai3:96] ;AJ292769
100 CLCuMB-[PK:Fai1:96] ;AJ298903
100 CLCuMB-[IN:BG:04] ;AY817151
MaYVB-[CN:Yn160:03] ; AJ786712
61
CLCuMB-[IN:Bas:Hib:05] ;DQ298137
82 CLCuMB-[IN:Ban:Bar:04] ;AY705381

0.20 0.15 0.10 0.05 0.00

Fig. 3 Dendrogram of complete nucleotide sequences of DNA-b Numbers at nodes indicate percentage bootstrap scores. Accession
satellites. The tree was generated using the neighbor-joining method numbers indicated on the right of the virus name. Abbreviations are
in Phylip. Vertical distances are proportional to sequence distances. according to [6]

sequence identity of the viral genome, and in accordance Phylogenetic analyses were performed based on a mul-
with the ICTV Geminiviridae study group guidelines tiple alignment of the present isolate and 48 other well-
[11, 12], the present virus isolate is considered to be a strain characterized begomoviruses. The present virus isolate
of ChiLCV-PK and is named ChiLCV-PK[IN:Var:06]. clusters together with begomoviruses infecting chillies in

123
538
Pakistan and India and is most closely related to
ChiLCV-PK[PK:Mul:98] and ChiLCV-Kha[PK:Kha1:05]
(Fig. 2). However, ChiLCV-PK[IN:Var:06] is distantly
related to chilli-infecting begomoviruses from Bangladesh,
such as PepLCBDV-BD[BD:Bog:99] and PepLCBDV-
PK[PK:Kha:04].
Sequence analysis demonstrated that the DNA-b satel-
lite isolated in the present investigation has 1,361
nucleotides (EF190215) and has structural features shared
by other DNA-b molecules, including an adenine-rich
region located at approximately nucleotides 716–878, a
satellite-conserved element (SCR) with 219 nucleotides
(1,157–13) and one conserved bC1 ORF encoding a
putative 13.7-kDa protein on the complementary-sense
DNA.
Nucleotide sequence comparisons show that the DNA-b
shares high nucleotide sequence identity with ToLCBDB-
[IN:Raj:03] and ToLCBDB-[IN:Luc:CH:05] isolated from
tomato and chillies, respectively. The highest nucleotide
sequence identity was found with ToLCB-[IN:Raj:03]
(87.9%). Since pBCVb4 shares nucleotide identity over
78% of the full-length sequence (a proposed Beta species
demarcation criterion; Briddon et al. [6]) with ToLCBDB-
[IN:Raj:03] and ToLCBDB-[IN:Luc:05], it has been
named tomato leaf curl Bangladesh beta—[India:Varana-
si:06] (ToLCBDB-[IN:Var:06]). A dendogram based on
the multiple alignment of DNA-b sequences demonstrated
that pBCVb4 clusters with ToLCBDB-[IN:Raj:03] and
ToLCBDB-[IN:Luk:CH:05] (Fig. 3).
Begomoviruses have been an emerging threat in chilli
for a long time, with disease incidence and severity greatly
increasing during the last decade. In India, although chilli
leaf curl disease was reported as early as 1960, information
was only available on host range and virus–vector rela-
tionship [9, 19]. Based on partial sequencing of a DNA-A-
like component, association of ToLCNDV and ChiLCV
was determined in chilli samples showing leaf curl disease
[15, 24]. However, as per ICTV guidelines, only the full-
length genomic sequence (DNA-A for bipartite begom-
oviruses) is considered for demarcation of any virus isolate
as belonging to a given strain or species [11, 12, 28]. The
only full-length sequence of a begomovirus isolated from
plants with ChLCD in India belongs to the species Chilli
leaf curl virus (ChiLCV-IN[IN::05]) [6, 26]. Based on
sequence similarity, ChiLCV-IN DNA-A was also detected
in naturally infected tomato (DQ629103) and papaya
(DQ989326) plants. However, infectivity analyses have not
been carried out for any of these viruses/strains. Associa-
tion of begomoviruses with ChLCD has also been reported
from Bangladesh and Pakistan [25]; however, ChiLCV-
PK[IN:Var:06] is distantly related to chilli-infecting
begomoviruses from Bangladesh.
123
Arch Virol (2008) 153:533–539
Association of a DNA-B was not observed, but the viral
genome was found to be associated with a DNA-b satellite.
Therefore, ChiLCV-PK[IN:Var:06] is a monopartite virus
which causes severe leaf curl disease of chillies in Varanasi
when associated with a DNA-b satellite. DNA-b satellites
are known to be required for induction of disease symptoms
in several host-virus combinations [2, 4, 14, 22, 23, 30].
Although the viral genome alone is infectious and produces
mild and delayed symptoms, association with a DNA-b
increases symptom severity and considerably shortens the
incubation period. Typical symptoms of ChiLCD (leaf
curling, yellowing of the leaf lamina, leaf distortion and leaf
size reduction) were observed only when both components
were present. This is the first demonstration of Koch’s
postulates using cloned DNA of ChiLCV and the associated
DNA-b satellite.
Acknowledgments This research was supported by the Department
of Science and Technology, Government of India.
References
1. Birnboim HC, Dolly J (1979) A rapid alkaline extraction proce-
dure for screening recombinant plasmid DNA. Nucleic Acids Res
7:1513–1523
2. Briddon RW, Stanley J (2006) Subviral agents associated with
plant single stranded DNA viruses. Virology 344:198–210
3. Briddon RW, Bull SE, Amin I, Idris AM, Mansoor S, Bedford ID,
Dhawan P, Rishi N, Siwatch SS, Abdel-Salam AM, Brown JK,
Zafar Y, Markham PG (2003) Diversity of DNA b: a satellite
molecule associated with some monopartite begomoviruses.
Virology 312:106–121
4. Briddon RW, Mansoor S, Bedford ID, Pinner MS, Saunders K,
Stanley J, Zafar Y, Malik KA, Markham PG (2001) Identification
of DNA components required for induction of cotton leaf curl
disease. Virology 285:234–243
5. Briddon RW, Bull SE, Mansoor S, Amin I, Markham PG (2002)
Universal primers for the PCR-mediated amplification of DNA-b:
a molecule associated with some monopartite begomoviruses.
Mol Biotechnol 20:315–318
6. Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou
X, Fauquet CM (2007) A proposal for the classification and
nomenclature of the DNA-b satellites of begomoviruses. Arch
Virol (accepted)
7. Chakraborty S, Pandey PK, Banerjee MK, Kalloo G, Fauquet CM
(2003) Tomato leaf curl Gujrat virus, a new Begomovirus species
causing a severe leaf curl disease of Tomato in Varanasi, India.
Phytopathology 23:1485–1495
8. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini-
preparation Version II. Plant Mol Biol Rep 1:19–21
9. Dhanraj KS, Seth ML (1968) Enation in Capsicum annum L
(Chilli) caused by a new strain leaf curl virus. Indian J Hort
25:70–71
10. Dry IB, Rigden JE, Krake LR, Mullineaux PM, Rezaian MA
(1993) Nucleotide sequence and genome organization of Tomato
leaf curl geminivirus. J Gen Virol 74:147–151
11. Fauquet CM, Stanley J (2003) Geminiviruses classification and
nomenclature, progress and problems. Ann Appl Biol 142:165–
189
Arch Virol (2008) 153:533–539
12. Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J,
Zerbini M, Zhou X (2007) Geminivirus strain demarcation and
nomenclature. Arch Virol (accepted)
13. Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robert-
son D (1999) Geminiviruses: models for plant DNA replication,
transcription, and cell cycle regulation. Crit Rev Plant Sci 18:71–
106
14. Jose J, Usha R (2003) Bhendi yellow vein mosaic disease in India
is caused by association of a DNA b satellite with a begomovirus.
Virology 305:310–317
15. Khan MS, Raj SK, Singh R (2006) First report of Tomato leaf
curl New Delhi virus infecting chilli in India. Plant Pathol 55:289
16. Kheyr-Pour A, Bendahmane M, Matzeit V, Accota GP, Crepsi S,
Gronerborn B (1991) The tomato yellow leaf curl virus from
Sardinia (TYLCV-S) has single genomic component. Nucleic
Acids Res 19:6763–6769
17. Lazarowitz SG (1992) Geminivirus: genome structure and gene
function. Crit Rev Plant Sci 11:327–349
18. Mayo MA, Pringle CR (1998) Virus taxonomy—1997. J Gen
Virol 79:649–657
19. Mishra MD, Raychaudhri SP, Jha A (1963) Virus causing leaf
curl of chilli (Capsicum annum L.). Indian J Microbiol 3:73–76
20. Navot N, Pichersky E, Zeidan M, Zamir D, Czosnek H (1991)
Tomato yellow leaf curl virus: a whitefly transmitted geminivirus
with a single genomic component. Virology 185:151–161
21. Rojas MR, Gilbertson RL, Russel DR, Maxwell DP (1993) Use of
degenerate primers in the polymerase chain reaction to detect
whitefly-transmitted geminiviruses. Plant Dis 77:340–347
22. Saunders K, Bedford ID, Briddon RW, Markham PG, Wong SM,
Stanley J (2000) A novel virus complex causes Ageratum yellow
vein disease. Proc Natl Acad Sci USA 97:6890–6895
539
23. Saunders K, Bedford ID, Yahara T, Stanley J (2003) The earliest
recorded plant virus disease. Nature 422:831
24. Senanayake DMJB, Mandal B, Lodha S, Varma A (2006) First
report of Chilli leaf curl virus affecting chilli in India. New
Disease Reports 13. http://www.bspp.org.uk/ndr/july2006/
2006-35.asp]
25. Shih SL, Tsai WS, Green SK, Khalid S, Ahmad I, Rezaian MA,
Smith J (2003) Molecular characterization of tomato and chilli
leaf curl begomoviruses from Pakistan. Plant Dis 87:200
26. Shih SL, Tsai WS, Green SK, Singh D (2006) First report of
Tomato leaf curl Joydebpur virus infecting chilli in India. New
Disease Reports 13. http://www.bspp.org.uk/ndr/jan2007/
2006-65.asp]
27. Srivastava KM, Hallan V, Raizada RK, Chandra G, Singh BP,
Sane PV (1993) Molecular cloning of Indian tomato leaf curl
virus genome following a simple method of concentrating the
supercoiled replicative form of viral DNA. J Virol Methods
51:297–304
28. Stanley J, Bisaro DM, Briddon RW, Brown JK, Fauquet CM,
Harrison BD, Rybicki EP, Stenger DC (2005) Geminiviridae. In:
Fauquet CM, Mayo MA, Maniloff J, Desselberger U, Ball LA
(eds) Virus taxonomy, VIIIth. Report of the ICTV. Elsevier/
Academic Press, London, pp 301–326
29. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0.
Mol Biol Evol 24:1596–1599
30. Zhou XP, Xie Y, Tao XR, Zhang ZK, Li ZH, Fauquet CM (2003)
Characterization of DNA beta associated with begomoviruses in
China and evidence for co-evolution with their cognate viral
DNA-A. J Gen Virol 84:237–247
123