Download 705_2013_1926_MOESM1_ESM - Springer Static Content Server

Identification of a disease complex involving a novel monopartite begomovirus with betaand alphasatellites associated with okra leaf curl disease in Oman
Sohail Akhtar1, Akhtar J. Khan,1 Achuit S. Singh2, Rob W. Briddon3
1
Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod
123, Oman
2
School of Life Sciences, Central University of Gujrat, Gandhinagar, India
3
National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan
Corresponding author
Akhtar J. Khan
Email: [email protected]
Tel: +968 2414 1217 Fax: +968 2441 3418
Running title: Begomovirus-satellite complex infecting okra in Oman
Abstract: Okra leaf curl disease (OLCD) is an important viral disease of okra in tropical and
subtropical areas. The disease is caused by begomovirus-satellite complexes. A begomovirus and
associated betasatellite and alphasatellite were identified in symptomatic okra plants from Barka,
in the Al-Batinah region of Oman. Analysis of the begomovirus sequences showed them to
represent a new begomovirus most closely related to cotton leaf curl Gezira virus (CLCuGeV), a
begomovirus of African origin. The sequences showed less than 85% nucleotide sequence
identity to CLCuGeV isolates. The name okra leaf curl Oman virus (OLCOMV) is proposed for
the new virus. Further analysis revealed that the OLCOMV is a recombinant begomovirus that
evolved by the recombination of CLCuGeV isolates with tomato yellow leaf curl virus-Oman
(TYLCV-OM). An alpha- and a betasatellite were also identified from the same plant sample,
which were also unique when compared to sequences available in the databases. However,
although the betasatellite appeared to be of African origin, the alphasatellite was most closely
related to alphasatellites originating from South Asia. This is the first report of a begomovirussatellite complex infecting okra in Oman.
2
The family Geminiviridae consists of phytopathogenic viruses with circular, single-stranded
(ss)DNA genomes that are encapsidated in twinned icosahedral particles [5]. The family is
divided into seven genera – Mastrevirus, Curtovirus, Begomovirus, Topocuvirus, Becurtovirus,
Turncurtovirus and Eragrovirus – based on genome organization, type of insect vector, and host
range [1]. Most of the economically important geminiviruses belong to the genus Begomovirus,
are transmitted exclusively by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), and infect
dicotyledonous plants [27, 42]. Most begomoviruses originating from the New World (NW) and
a few from the Old World (OW) have bipartite genomes (having two genomic components
known as DNA A and DNA B), while the majority of OW begomoviruses are monopartite
(having a single genomic component homologous to the DNA A of bipartite begomoviruses) [9].
Recently, the first native NW monopartite begomovirus was identified [26, 34]. The genomes of
monopartite begomoviruses have six genes: two in the virion sense (encoding the V2 protein and
the coat protein [CP]) and four in the complementary sense (the replication-associated protein
[Rep], transcriptional activator protein [TrAP], replication enhancer protein [REn] and the C4
protein) [32]. Most monopartite begomoviruses are associated with ssDNA satellites known as
betasatellites (previously called DNA-β) and satellite-like molecules known as alphasatellites
(previously called DNA-1) [3].
Okra (Abelmoschus esculentus, family Malvaceae) is a widely grown vegetable crop that
is believed to have its origin in western Africa. In Oman, okra is mostly cultivated in the AlBatinah region during the winter season to meet the high demand in the domestic market. Okra
leaf curl disease (OLCD) is an important limiting factor for okra production. The disease is
characterized by stunted growth, leaf curling, distortion, mottling and a yellow mosaic. Several
begomoviruses and begomovirus-satellite complexes causing OLCD have been identified around
3
the world. Cotton leaf curl Gezira virus (CLCuGeV), okra yellow crinkle virus (OYCrV) and
okra leaf curl Cameroon virus (OLCuCMV) along with satellite molecules have been shown to
the causal agents of OLCD in Africa [11, 20, 22, 41], while in South Asia (Pakistan and India),
diseases of okra are caused by various begomovirus-betasatellite complexes [24, 38, 43-46].
Here, we describe for the first time the identification and molecular characterization of a distinct
begomovirus, a new betasatellite, and an alphasatellite isolated from okra grown in the AlBatinah region of Oman.
Leaf samples were collected from two symptomatic plants of okra grown in fields in the
Barka area (Al-Batinah, North Oman). One okra field was found affected with OLCD, showing
>30% disease incidence. The plants from which samples were collected showed symptoms such
as leaf curling, inter-veinal yellowing, vein thickening, and reduced leaf size, which are
reminiscent of begomovirus infection. Total nucleic acids were extracted from leaf samples by
the method described by Porebski et al. [31], and circular DNA molecules in genomic DNA were
amplified by rolling-circle amplification (RCA; [13]) using an IllustraTM TempliPhi 100
Amplification Kit (GE Healthcare, Sweden) following the manufacturer’s instructions. RCA
products were digested with selected restriction endonucleases to identify enzymes giving fulllength begomovirus (~2.8 kb) and satellite (~1.4 kb) fragments. Putative begomovirus genomes,
obtained by BamHI digestion of the concatameric RCA product, and putative satellites, obtained
by separate digestion of the RCA product with BamHI and SacI, were cloned in the plasmid
vector pUC19. Two clones of ~2.8 kb (OK-2 and OK-3) and two clones of ~1.4 kb (OKB-1 and
OKB-2) were obtained and sequenced in both directions using a primer walking strategy. The
evolutionary history was inferred using the neighbor-joining method [33]. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test (1000
4
replicates) is shown next to the branches [7]. The tree is drawn to scale, with branch lengths in
the same units as those of the evolutionary distances used to infer the phylogenetic tree. The
evolutionary distances were computed using the p-distance method [29] and are in units of the
number of base substitutions per site. Evolutionary analyses were conducted in MEGA5 [40].
The sequences of the clones OK-2 and OK-3 were determined to be 2788 and 2787 nt in
length, respectively, and they were submitted to nucleotide sequence databases under the
accession numbers given in Table 1. Analysis of the sequences showed them to encode all of the
genes that are typical of the genomes of monopartite begomoviruses. The positions and coding
capacities of the six genes are indicated in Table 1. In the intergenic region (IR), there lies a
putative stem-loop structure and the nonanucleotide sequence (TAATATTAC) in the loop,
which forms part of the origin of virion-strand DNA replication [30]. The predicted Rep-binding
site (iteron) was identified as the directly repeated sequence GGTGT (coordinates 2653-2657,
2680-2684 for OK-2 and 2652-2656, 2679-2683 for OK-3) located 5’ of the TATA box of the
putative Rep gene promoter.
The isolates OK-2 and OK-3 shared 99.5% nucleotide sequence identity, indicating that
they belong to a single begomovirus species (89% being the species demarcation threshold for
begomoviruses [6]). An initial comparison to sequences available in the databases using BlastN
showed the clones from okra to have the highest levels of sequence identity to isolates of
CLCuGeV. Subsequent sequence alignment using the ClustalV algorithm of MegAlign
(DNAStar, Madison, WI, USA) showed them to have 80.7 to 84.7% nucleotide sequence identity
to all CLCuGeV isolates available in the databases (42 of which are available at this time). To
hollyhock leaf crumple virus (HoLCrV), another Malvaceae-infecting begomovirus of African
origin, the levels of identity were between 74.8 and 78.9%, followed by tomato yellow leaf curl
5
virus-Oman (TYLCV-OM; 73-75.6 % identity). To all other begomoviruses available in the
databases, the levels of sequence identity were less than 74% (Table 1). These results indicate
that the begomovirus identified in association with OLCD is a member of a distinct begomovirus
species. The name Okra leaf curl Oman virus (OLCOMV) is proposed for this newly identified
species.
A neighbor-joining phylogenetic tree based upon an alignment of the complete sequences
of begomoviruses causing OLCD, including the OLCOMV isolates identified here and selected
other begomovirus sequences from GenBank database, is shown in Fig. 1A. This shows
OLCOMV to be basal to the CLCuGeV sequences, whereas the other begomoviruses involved in
OLCD in Africa (OLCuCMV and OYCrV) grouped separately. These results confirm the
identification of OLCOMV isolates as belonging to a distinct begomovirus species. Furthermore,
the close grouping with CLCuGeV sequences indicate this virus to be the closest relative.
Detection of potential recombinant sequences and localization of recombination
breakpoints were carried out with the Simplot program, version 3.2 [23], and the Recombination
Detection Program (RDP version 3; [25]). Default RDP settings were used throughout, with a pvalue cutoff of 0.01 and the standard Bonferroni correction. For the bootscan analysis, 200
replicates with a 95% cutoff were taken, and for the GENECONV analysis [37], the g-scale
parameter was set to 1. The two isolates of OLCOMV showed identical recombination patterns,
and the analysis of one isolate, OK-3 (HF536716), is shown (Fig. 2). Bootscan and RDP
analyses indicated that OLCOMV evolved by recombination between CLCuGeV and TYLCVOM. The RDP analysis showed the major parent of OLCOMV to be most closely related to
CLCuGeV from Egypt (AF155064), with two inserted fragments, the first being a fragment in
the middle of the coat protein gene (coordinates 457 to 863) originating from an isolate of
6
CLCuGeV from Burkina Faso (FN554520) and the second covering most of the Rep gene and
part of IR (coordinates 1755 to 16), derived from TYLCV-OM (DQ644565) (Fig. 2). This
conclusion was supported by all of the methods implemented in the RDP package, with low pvalues for the detected recombination events (Supplementary Table 1). It is interesting to note
that CLCuGeV has so far only been identified as a defective molecule (not the full genome) in
Oman [15]. The results here suggest that the diversity of CLCuGeV in Oman is greater than
previously believed or that OLCOMV was introduced into Oman. Little is known about the
diversity of begomoviruses causing diseases of okra on the Arabian Peninsula; OLCD occurs in
Saudi Arabia but the causative agent has yet to be characterized at the sequence level [8].
Digestion of the RCA product with BamHI also resulted in a subgenomic molecule of
approximately 1.4 kb. The fragment was cloned and sequenced. The sequence of the clone OKB1 was determined to be 1351 nt and is available in the databases under accession number
KF267444. The sequence contains the features typical of betasatellites: a single open reading
frame in complementary-sense orientation, known as βC1 (coordinates 243-596), encoding a
predicted protein of 118 amino acids; a region of sequence highly conserved between all
betasatellites, known as the satellite conserved region (SCR; coordinates 1232-22); and a region
of sequence rich in adenine residues, known as the A-rich region (coordinates 750-953,
containing 55% A). The SCR contains a nonanucleotide sequence (TAATATTAC) that is
identical to that of begomoviruses. An initial comparison of the betasatellite sequence using
BlastN search showed highest levels of sequence identity to isolates of cotton leaf curl Gezira
betasatellite (CLCuGeB). Subsequent sequence alignment using ClustalV (MegAlign) showed
the betasatellite from okra in Oman to have highest levels of sequence identity (between 68.1 and
76.7%) to the isolates of CLCuGeB, followed by the isolates of tomato leaf curl Yemen
7
betasatellite (ToLCYB; 43.5 and 44 %). To all other betasatellites, the levels of identity were
below 40%. These results indicate that the betasatellite identified in okra in Oman is a previously
unidentified betasatellite (the demarcation threshold for betasatellites being 78% [4]), for which
the name okra leaf curl Oman betasatellite (OLCOMB) is proposed.
A phylogenetic tree was constructed from an alignment of the nucleotide sequences of
the betasatellite from Oman with selected betasatellite sequences available in the databases (Fig.
1B). The sequence of OKB-1 formed a distinct branch basal to the CLCuGeB isolates,
confirming its identification as a distinct betasatellite most closely related to CLCuGeB, as
suggested by the sequence comparisons. The tree also shows the phylogeographic segregation of
the betasatellites from Africa and Asia, which suggests that OLCOMB has its origins in Africa.
Bootscan and RDP analysis of the sequence of OLCOMB revealed a single recombinant
fragment between nt coordinates 870 and 1233 derived from an isolate of CLCuGeB (JX649952)
but with the major parent being unknown (Supplementary Table 1). OLCOMB is only the
second betasatellite to have been identified in Oman. The other, tomato leaf curl betasatellite
(ToLCB), originates from South Asia and is associated with diseases of tomato and pepper [14,
16].
The sequence of OKB-2 was determined to be 1368 nt and is available in the databases
under accession number KF267445. The sequence contains a single predicted gene (coordinates
71-1018) in the virion sense, encoding a predicted protein of 315 amino acids, an A-rich region
(coordinates 1044-1248, containing 54% A), and a predicted hairpin structure containing, within
the loop, the nonanucleotide sequence TAGTATTAC, typical of alphasatellites [2]. By BlastN
search, the sequence of OKB-2 showed the highest levels of identity to isolates of Gossypium
darwinii symptomless alphasatellite (GDarSLA). Subsequent sequence alignment using ClustalV
8
showed the OKB-2 sequence to have 77.7 to 81.2% sequence identity to isolates of GDarSLA.
To all other sequences available in the databases, the sequence of OKB-2 showed less than 73%
identity. These results indicate that OKB-2 represents a distinct, previously unidentified
alphasatellite (the demarcation threshold for distinct alphasatellites being 83% [28]), for which
the name okra leaf curl Oman alphasatellite (OLCOMA) is proposed. A phylogenetic analysis
based upon an alignment of the complete nucleotide sequences of selected alphasatellites with
the sequences of OKB-2 is shown in Fig. 1C. This shows the sequence of OKB-2 to fall basal to
the GDarSLA sequences and to form a distinct clade with GDarSLA and okra leaf curl
alphasatellite (OLCA). The tree also suggests that OLCA originates from South Asia rather than
Africa; the alphasatellites from Africa have been shown to be distinct from those occurring in
Eurasia [35]. OLCOMA is also only the second alphasatellite identified on the Arabian
Peninsula, the other, ageratum yellow vein Singapore alphasatellite, only having been identified
in Oman and Singapore [12, 36]. A single recombinant fragment between nt coordinates 630 and
833, which was derived from an isolate of CLCuMuA (FR873571) with GDarSLA (FR772090)
being the major parent, was detected by Bootscan and RDP analysis (Supplementary Table 1).
With the possible exception of watermelon chlorotic stunt virus [17], which is endemic
across the Middle East and North Africa, the geminiviruses and associated satellites present in
Oman have either been introduced or have evolved from introduced viruses by recombination
[12, 14, 16, 18, 19]. This has led to new disease complexes consisting of viruses and satellites of
differing origins – for example a virus of Middle Eastern origin (TYLCV-OM) interacting with a
betasatellite originating from South Asia (ToLCB) and an alphasatellite possibly from Southeast
Asia associated with tomato leaf curl disease in tomato [12, 14]. Here, this unusual convergence
of disparate components in Oman has been shown to also occur in okra, with a virus and
9
betasatellite likely originating from Africa and an alphasatellite that clearly has its origins in
South Asia. With Oman being a major hub for trade and travel by air and sea, the likely means
by which the viruses were introduced, there is the concern that the new viruses and associated
components may spread out of Oman.
Acknowledgements
This study was supported by research grant number ORG/EBR/09/03, funded by The Research
Council, Oman to AJK. RWB is supported by the Higher Education Commission, Government
of Pakistan, under the ‘‘Foreign Faculty Hiring Program.’’
10
References
1.
Adams MJ, King AMQ, Carstens EB (2013) Ratification vote on taxonomic proposals to the International
Committee on Taxonomy of Viruses (2013). Arch Virol, DOI 10.1007/s00705-013-1688-5
2.
Briddon RW, Bull SE, Amin I, Mansoor S, Bedford ID, Rishi N, Siwatch SS, Zafar Y, Abdel-Salam AM,
Markham PG (2004) Diversity of DNA 1: a satellite-like molecule associated with monopartite
begomovirus-DNA β complexes. Virology 324: 462 - 474
3.
Briddon RW, Stanley J (2006) Sub-viral agents associated with plant-infecting single-stranded DNA
viruses. Virology 344:198-210
4.
Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X, Fauquet CM (2008)
Recommendations for the classification and nomenclature of the DNA-β satellites of begomoviruses. Arch
Virol 153:763-781
5.
Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, Navas-Castillo J (2012) Geminiviridae. In
Virus Taxonomy, ed. by King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ. Ninth Report of the
International Committee on Taxonomy of Viruses. London, UK: Elsevier Publication.351-373
6.
Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X (2008) Geminivirus
strain demarcation and nomenclature. Arch Virol 153:783-821
7.
Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783791
8.
Ghanem GAM (2003) Okra leaf curl virus: A monopartite begomovirus infecting okra crop in Saudi
Arabia. Arab J Biotechnol 6:139-152
9.
Hanley-Bowdoin L, Settlage SB, Orozco BM, Nagar S, Robertson D (1999) Geminviruses: models for
plant DNA replication, transcription, and cell cycle regulation. Crit Rev Plant Sci 18:71-106
10. Hou Y-M, Gilbertson RL (1996) Increased pathogenicity in a pseudorecombinant bipartite geminivirus
correlates with intermolecular recombination. J Virol 70:5430-5436
11. Idris AM, Hussein MH, Abdel-Salam AM, Brown JK (2002) Phylogenetic relationships for okra leaf curland hollyhock leaf crumple-associated begomoviruses and first report of associated satellite DNAs. Arab J
Biotech 5:67-82
11
12. Idris AM, Shahid MS, Briddon RW, Khan AJ, Zhu JK, Brown JK (2011) An unusual alphasatellite
associated with monopartite begomoviruses attenuates symptoms and reduces betasatellite accumulation. J
Gen Virol 92:706-717
13. Inoue-Nagata AK, Albuquerque LC, Rocha WB, Nagata T (2004) A simple method for cloning the
complete begomovirus genome using the bacteriophage phi29 DNA polymerase. J Virol Methods 116:209211
14. Khan AJ, Idris AM, Al-Saady NA, Al-Mahruki MS, Al-Subhi AM, Brown JK (2008) A divergent isolate of
tomato yellow leaf curl virus from Oman with an associated DNAβ satellite: an evolutionary link between
Asian and the Middle Eastern virus-satellite complexes. Virus Genes 36:169-176
15. Khan AJ, Akhtar S, Al-Shihi AA, Al-Hinai FM, Briddon RW (2012) Identification of Cotton leaf curl
Gezira virus in papaya in Oman. Plant Dis 96:1704
16. Khan AJ, Akhtar S, Al-Zaidi AM, Singh AK, Briddon RW (2013) Genetic diversity and distribution of a
distinct strain of Chili leaf curl virus and associated betasatellite infecting tomato and pepper in Oman.
Virus Res in press (DOI 10.1016/j.virusres.2013.07.018)
17. Khan AJ, Akhtar S, Briddon RW, Ammara U, Al-Matrushi AM, Mansoor S (2012) Complete nucleotide
sequence of Watermelon chlorotic stunt virus originating from Oman. Viruses 4:1169-1181
18. Khan AJ, Akhtar S, Singh AK, Briddon RW (2013) A distinct strain of Tomato leaf curl Sudan virus causes
tomato leaf curl disease in Oman. Plant Dis, in press (DOI 10.1094/PDIS-02-433 13-0210-RE)
19. Khan AJ, Akhtar S, Singh AK, Al-Shehi AA, Al-Matrushi AM, Ammara U, Briddon RW (2012) Recent
evolution of a novel begomovirus species causing tomato leaf curl disease in the Al-Batinah region of
Oman. Arch Virol in press
20. Kon T, Rojas MR, Abdourhamane IK, Gilbertson RL (2009) Roles and interactions of begomoviruses and
satellite DNAs associated with okra leaf curl disease in Mali, West Africa. J Gen Virol 90:1001-1013
21. Lefeuvre P, Martin DP, Hoareau M, Naze F, Delatte H, Thierry M, Varsani A, Becker N, Reynaud B, Lett
JM (2007) Begomovirus 'melting pot' in the south-west Indian Ocean islands: molecular diversity and
evolution through recombination. J Gen Virol 88:3458-3468
12
22. Leke WN, Sattar MN, Ngane EB, Ngeve JM, Kvarnheden A, Brown JK (2013) Molecular characterization
of begomoviruses and DNA satellites associated with okra leaf curl disease in Cameroon. Virus Res
174:116-125
23. Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW,
Ray SC (1999) Full-length human immunodeficiency virus type1 genomes from subtype C-infected
seroconverters in India, with evidence of intersubtype recombination. J Virol 73:152-160
24. Mansoor S, Amin I, Hussain M, Zafar Y, Bull S, Briddon RW, Markham PG (2001) Association of a
disease complex involving a begomovirus, DNA 1 and a distinct DNA beta with leaf curl disease of okra in
Pakistan. Plant Dis 85:922
25. Martin DP, Rybicki EP (2000) RDP: detection of recombination amongst aligned sequences.
Bioinformatics 16:562-563
26. Melgarejo TA, Kon T, Rojas MR, Paz-Carrasco L, Zerbini FM, Gilbertson RL (2013) Characterization of a
New World monopartite begomovirus causing leaf curl disease of tomato in Ecuador and Peru reveals a
new direction in geminivirus evolution. J Virol 87:5397-5413
27. Moffat AS (1999) Geminiviruses emerge as serious crop threat. Science 286:1835
28. Mubin M, Briddon RW, Mansoor S (2009) Complete nucleotide sequence of chili leaf curl virus and its
associated satellites naturally infecting potato in Pakistan. Arch Virol 154:365-368
29. Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics. Oxford University Press, New York
30. Orozco BM, Hanley-Bowdoin L (1996) A DNA structure is required for geminivirus replication origin
function. J Virol 7:148-158
31. Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants
containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15:8-15
32. Rojas MR, Hagen C, Lucas WJ, Gilbertson RL (2005) Exploiting chinks in the plant's armor: Evolution
and emergence of geminiviruses. Ann Rev Phytopathol 43:361-394
33. Saitou N, Nei M (1987). The neighbor-joining method: A new method for reconstructing phylogenetic
trees. Mol Biol Evol 4:406-425
13
34. Sánchez-Campos S, Martínez-Ayala A, Márquez-Martín B, Aragón-Caballero L, Navas-Castillo J,
Moriones E (2013) Fulfilling Koch's postulates confirms the monopartite nature of tomato leaf deformation
virus, a begomovirus native to the New World. Virus Res 173:286-293
35. Sattar MN, Kvarnheden A, Saeed M, Briddon RW (2013) Cotton leaf curl disease – an emerging threat to
cotton production worldwide. J Gen Virol 94:695-710
36. Saunders K, Bedford ID, Stanley J (2002) Adaptation from whitefly to leafhopper transmission of an
autonomously-replicating nanovirus-like DNA component associated with ageratum yellow vein disease. J
Gen Virol 83:909-915
37. Sawyer S (1989) Statistical tests for detecting gene conversion. Mol Biol Evol 6:526-538
38. Sohrab SS, Mirza Z, Karim S, Rana D, Abuzenadah AM, Chaudhary AG, Bhattacharya PS (2013)
Detection of begomovirus associated with okra leaf curl disease. Arch Phytopath Plant Prot 46:1047-1053
39. Tahir MN, Amin I, Briddon RW, Mansoor S (2011) The merging of two dynasties--identification of an
African cotton leaf curl disease-associated begomovirus with cotton in Pakistan. PLoS One 6:e20366
40. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: Molecular Evolutionary
Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods.
Mol Biol Evol 28: 2731-2739
41. Tiendrébéogo F, Lefeuvre P, Hoareau M, Villemot J, Konaté G, Traoré AS, Barro N, Traoré VS, Reynaud
B, Traoré O (2010) Molecular diversity of Cotton leaf curl Gezira virus isolates and their satellite DNAs
associated with okra leaf curl disease in Burkina Faso. Virol J 7:48
42. Varma A, Malathi VG (2003) Emerging geminivirus problems: A serious threat to crop production. Ann
App Biol 142:145-164
43. Venkataravanappa V, Lakshminarayana Reddy C, Jalali S, Krishna Reddy M (2012) Molecular
characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India.
Virus Genes 44:522-535
44. Venkataravanappa V, Lakshminarayana Reddy CN, Swarnalatha P, Devaraju, Jalali S, Reddy MK (2012)
Molecular evidence for association of Cotton leaf curl Allahabad virus with yellow vein mosaic disease of
okra in North India. Arch Phytopath Plant Prot 45:2095-2113
14
45. Venkataravanappa V, Reddy CNL, Jalali S, Reddy MK (2013) Molecular characterization of a new species
of begomovirus associated with yellow vein mosaic of bhendi (Okra) in Bhubhaneswar, India. Eur J Plant
Pathol 136:811-822
46. Zaffalon V, Mukherjee S, Reddy V, Thompson J, Tepfer M (2011) A survey of geminiviruses and
associated satellite DNAs in the cotton-growing areas of northwestern India. Arch Virol 157:483-495
15
Table 1 Sequence comparisons of the okra leaf curl Oman virus (OLCOMV) isolates with other selected begomoviruses
Begomovirus*
OLCOMV
isolates
CLCuGeV [42]
OLCuCMV [4]
OYCrV [2]
HoLCrV [3]
TYLCV-OM [18]
80.7-84.7
74.2-75.6
63.6-64.9
74.8-78.9
72.6-75.6
Highest and lowest percentage nucleotide sequence identities and predicted amino acid sequences for pairwise
comparisons of the gene sequences of the OLCOMV isolates with selected other begomoviruses**
CP
V2
Rep
TrAP
REn
C4
89.4-90.9
93.5-95.9
77.1-79.1
97.5-100
96.8-99.5
64.6-69.4
(91.1-95.4)
(83.7-92.7)
(81-84)
(94.8-99.3)
(94.8-98.5)
(44.9-48)
67.6-69
65-68.9
76.3-77.8
97.3-99
97.3-98
65-69
(78-78.8)
(56.9-60.7)
(79.3-82.1)
(94.8-97)
(95.5-98.5)
(44.9-50)
66-67.2
68.1-68.4
70.8-72
65.4
62.7-63.4
69.8-74
(74.3-78.4)
(58.1-59)
(76.7-78.9)
(56.3)
(59-59.7)
(47.7-51.2)
84.2-84.4
70.7-71
77.8-80
84-97
69.9-96.5
66.3-70.1
(91.5-91.9)
(65-65.9)
(81-83.5)
(76.3-96.3)
(65.7-94)
(46.9-48)
73.4-74
66.4-69.2
83.5-87.5
63.5-64.9
64.4-66.2
90.6-99.6
(83.2-84.6)
(60.7-64.1)
(80.2-88.2)
(53.3-56.3)
(57.5-60.4)
(78.3-97.8)
* The values in brackets are the numbers of sequences available in the databases and used in the analysis.
** The values in parentheses are highest and lowest percentage amino acid sequence identity values for the predicted product of each gene. The highest values in
each case are underlined.
16
Supplementary Table 1 Recombination breakpoint analysis of Okra leaf curl Oman virus (OLCOMV), okra leaf curl Oman betasatellite (OLCOMB), and okra
leaf curl Oman alphasatellite (OLCOMA) sequences using different methods implemented in the RDP program and their putative parental sequences
Breakpoint
positions
5’
3’
457
863
Recombinant
sequence
Minor
parent
Major
parent
OLCOMV
(HF536716)
CLCuGeV
(FN554520)
CLCuGeV
(AF155064)
1755
16
OLCOMV
(HF536716)
TYLCV-OM
(DQ644565)
870
1233
OLCOMB
(KF267444)
630
883
OLCOMA
(KF267445)
Detection methods
RDP
Genconv
3.59 × 10
-03
CLCuGeV
(AF155064)
2.65 × 10
CLCuGeB
(JX649952)
Unknown
3.65 × 10
CLCuMuA
(FR873571)
GDarSLA
(FR772090)
1.87 × 10-11
Bootscan
3.58 × 10
-04
-69
1.24 × 10
-05
6.77 × 10
Chimera
1.26 × 10
2.59 × 10
-65
2.13 × 10
-66
1.26 × 10
-04
1.20 × 10
-04
3.89 × 10
9.34 × 10-12
17
Maxchi
-02
1.04 × 10-05
-07
Siscan
1.01 × 10
-05
4.59 × 10
-02
-26
1.37 × 10
-28
1.81 × 10
-35
-09
1.52 × 10
-10
4.91 × 10
-10
4.59 × 10-04
2.21 × 10-07
2.77 × 10-08
Figure Legends
Fig. 1 Phylogenetic dendrograms based upon alignments of the complete nucleotide sequences
of selected begomovirus genomes (A), betasatellites (B), and alphasatellites (C). In each case,
the sequences characterized in this study are highlighted as white text on a black background,
and for each isolate the database accession numbers are given. The values at nodes represent
percentage bootstrap confidence scores (1000 replicates). The begomovirus dendrogram was
arbitrarily rooted on tomato pseudo curly top virus, a distantly related geminivirus, as an
outgroup. Begomovirus acronyms used are chili leaf curl virus (ChLCV), cotton leaf curl Gezira
virus (CLCuGeV), hollyhock leaf crumple virus (HoLCrV), Okra leaf curl Cameroon virus
(OLCuCMV), okra leaf curl Oman virus (OLCOMV), okra yellow crinkle virus (OYCrV),
tomato leaf curl Oman virus (ToLCOMV), tomato leaf curl Sudan virus (ToLCSDV), and
tomato yellow leaf curl virus (TYLCV). The betasatellite acronyms used are bhendi yellow vein
betasatellite (BhYVB), bhendi yellow vein India betasatellite (BhYVIB), chili leaf curl
betasatellite (ChLCB), cotton leaf curl Gezira betasatellite (CLCuGeB), cotton leaf curl Multan
betasatellite (CLCuMB), okra leaf curl betasatellite (OLCB), okra leaf curl Mali betasatellite
(OLCMLB), okra leaf curl Oman betasatellite (OLCOMB), tomato leaf curl betasatellite
(ToLCB), and tomato leaf curl Bangladesh betasatellite (ToLCBDB). The betasatellite tree was
arbitrarily rooted on tomato yellow leaf curl Thailand alphasatellite (TYLCTHA) as an outgroup.
The alphasatellite acronyms used are ageratum yellow vein Singapore alphasatellite (AYVSGA),
bhendi yellow vein mosaic alphasatellite (BhYVMA), cotton leaf curl Gezira alphasatellite
(CLCuGeA), cotton leaf curl Multan alphasatellite (CLCuMuA), Gossypium darwinii
symptomless alphasatellite (GDarSLA), okra leaf curl alphasatellite (OLCA), okra leaf curl
18
Barkina Faso alphasatellite (OLCBFA), okra leaf curl Oman alphasatellite (OLCOMA), and okra
yellow crinkle Cameroon alphasatellite (OYCrCMA).
Fig. 2 Analysis of okra leaf curl Oman virus (OLCOMV) isolate OK-3 (HF536716) for
recombination. (A) Boot scanning analysis of OLCOMV using the SimPlot program with a
sliding window of 200 nucleotides moving in 20-nucleotide steps. The Bootscan graph was built
using the neighbor-joining tree algorithm, the Kimura 2-parameter distance model, and 100
pseudo-replicates. (B) Schematic representation of the results of RDP3 analysis shown as a linear
representation of the OLCOMV genome indicating the origins of the fragments, recombination
breakpoints and putative parental viruses determined by the RDP method. Above each panel, a
linearized version of the virus genome shows the approximate positions of genes (the V2 and
coat protein [CP] genes in the virion sense and replication-associated protein [Rep],
transcriptional-activator protein [TrAP], replication-enhancer protein [REn], and C4 genes in the
complementary sense) and the non-coding intergenic region (IR). The virus sequences used were
from cotton leaf curl Gezira virus (CLCuGeV; FN554520), cotton leaf curl Gezira virus
(CLCuGeV; AF155064), cotton leaf curl Gezira virus (CLCuGeV; FR751146), hollyhock leaf
crumple virus (HoLCrV; AJ542439) and tomato yellow leaf curl virus Oman strain (TYLCVOM; DQ644565).
19
20
21