Genetic diversity and distribution of a distinct strain of Chili leaf curl virus and associated betasatellite infecting tomato and pepper in Oman

Virus Research 177 (2013) 87–97

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Genetic diversity and distribution of a distinct strain of Chili leaf curl virus and associated betasatellite infecting tomato and pepper in Oman Akhtar J. Khan a,∗ , Sohail Akhtar a , Amal M. Al-Zaidi a , Achuit K. Singh b , Rob W. Briddon c a

Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box-34, Al-Khod 123, Oman School of Life Sciences, Central University of Gujarat, Gandhinagar, India c National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan b

a r t i c l e

i n f o

Article history: Received 2 June 2013 Received in revised form 12 July 2013 Accepted 20 July 2013 Available online 30 July 2013 Keywords: Begomovirus Chili leaf curl virus Genetic diversity Oman Pepper Tomato

a b s t r a c t Tomato and pepper are widely grown in Oman for local consumption. A countrywide survey was conducted during 2010–2011 to collect samples and assess the diversity of begomoviruses associated with leaf curl disease of tomato and pepper. A virus previously only identified on the Indian subcontinent, chili leaf curl virus (ChLCV), was found associated with tomato and pepper diseases in all vegetable grown areas of Oman. Some of the infected plant samples were also found to contain a betasatellite. A total of 19 potentially full-length begomovirus and eight betasatellite clones were sequenced. The begomovirus clones showed >96% nucleotide sequence identity, showing them to represent a single species. Comparisons to sequences available in the databases showed the highest levels of nucleotide sequence identity (88.0–91.1%) to isolates of the “Pakistan” strain of ChLCV (ChLCV-PK), indicating the virus from Oman to be a distinct strain, for which the name Oman strain (ChLCV-OM) is proposed. An analysis for recombination showed ChLCV-OM likely to have originated by recombination between ChLCV-PK (the major parent), pepper leaf curl Lahore virus and a third strain of ChLCV. The betasatellite sequences obtained were shown to have high levels of identity to isolates of tomato leaf curl betasatellite (ToLCB) previous shown to be present in Oman. For the disease in tomato Koch’s postulates were satisfied by Agrobacterium-mediated inoculation of virus and betasatellites clones. This showed the symptoms induced by the virus in the presence of the betasatellite to be enhanced, although viral DNA levels were not affected. ChLCV-OM is the fourth begomovirus identified in tomato in Oman and the first in Capsicum. The significance of these findings is discussed. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The family Geminiviridae consists of plant-infecting viruses with circular, single-stranded (ss)DNA genomes encapsidated in small twinned icosahedral virions (Brown et al., 2012). Geminiviruses are among the smallest known viruses transmitted by insect vectors. The family Geminiviridae is classified into seven genera, Begomovirus, Mastrevirus, Curtovirus, Topocuvirus, Becurtovirus, Eragrovirus and Turncurtovirus based on genome structure, host specificity and the type of insect vector (Fauquet et al., 2008; Brown et al., 2012; Adams et al., 2013). The genus Begomovirus constitutes the largest genus of the family encompassing viruses that infect a wide range of economically important dicotyledonous host plants in both the New World (NW) and Old World (OW) that are transmitted by the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) (Moffat,

∗ Corresponding author. Tel.: +968 2414 1217; fax: +968 2441 3418. E-mail address: [email protected] (A.J. Khan). 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.07.018

1999; Varma and Malathi, 2003). The majority of begomoviruses native to the NW are bipartite, having genomes consisting of two components (known as DNA A and DNA B) that share a region of ∼200 nucleotides within the intergenic region called the common region (CR). The CR contains a conserved stem-loop with the nonanucleotide sequence, TAATATTAC, forming part of the loop. Recently the first native NW monopartite begomovirus has been identified (Melgarejo et al., 2013; Sánchez-Campos et al., 2013). Although in the OW a few bipartite begomoviruses have been identified (Padidam et al., 1995a,b; Patil et al., 2004), the majority are monopartite with a single component homologous to the DNA A component of the bipartite begomoviruses (Brown et al., 2012). Tomato (Lycopersicum esculentum) and pepper (Capsicum sp.) are widely grown vegetables. Leaf curl disease of chili and tomato is the major constraint in the production of these crops across the OW and is caused by a number of both monopartite and bipartite begomoviruses. Chili leaf curl disease was first recorded in 1960s in India (Mishra et al., 1963; Dhanraj and Seth, 1968). Later a begomovirus, now known as ChLCV, was shown to be associated

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with leaf curl disease of pepper in Pakistan, India and Oman (Shih et al., 2003; Khan et al., 2006; Senanayake et al., 2006). In common with all monopartite begomoviruses, ChLCV encodes six genes; two in the virion-sense and four in the complementary-sense. The virion-sense genes encode the coat protein (CP), involved in encapsidation, virus movement in plants and interactions with whitefly vector for transmission, and the V2 protein, possibly involved in virus movement that is also involved in overcoming RNA-based host defenses (Rojas et al., 2005; Luna et al., 2012). Genes in the complementary-sense encode the replication associated protein (Rep), required for rolling circle replication of viral DNA (HanleyBowdoin et al., 1999, 2004), the transcriptional activator protein (TrAP) involved in up-regulating late virus genes and overcoming RNA-based host defenses (Bisaro, 2006), the replication enhancer protein (REn), that enhances virus replication by interacting with Rep and host factors (Settlage et al., 2005), and the C4 protein, involved in symptom determination and overcoming RNA-based host defenses (Luna et al., 2012). Most monopartite begomoviruses are associated with small symptom modulating circular ssDNA molecules known as betasatellites (earlier referred to as DNA ␤; Briddon and Stanley, 2006; Briddon et al., 2008). These satellites are half of the size of their helper viruses (∼1.4 kb) and depend entirely on the helper virus for their replication, movement in plants and transmission between plants. They lack significant nucleotide sequence homology to the helper virus with the exception of a predicted hairpin structure encompassing, within the loop, a nonanucletide motif with similarities to origin of virion-sense DNA replication of geminiviruses (Briddon et al., 2012). Although betasatellite sequences are very diverse, they have a highly conserved structure comprising of a region of sequence rich in adenine (A-rich), a sequence highly conserved between distinct betasatellites (known as the satellite conserved region [SCR]) and a single gene in complementary-sense orientation known as ␤C1 (Briddon et al., 2004). The ␤C1 gene encodes a protein of ∼118 amino acids which is DNA binding, a pathogenicity determination, a suppressor of gene silencing and may be involved in virus movement in plants (Saunders et al., 2004; Kon et al., 2007; Qazi et al., 2007; Saeed et al., 2007; Amin et al., 2011a). Previously studies have shown three begomoviruses that infect tomato in Oman; tomato yellow leaf curl virus (TYLCV; Khan et al., 2008), tomato leaf curl Oman virus (ToLCOMV; Idris et al., 2011) and tomato leaf curl Sudan virus (ToLCSDV; Khan et al., 2013a). TYLCV and ToLCOMV, but not ToLCSDV, infections of tomato were shown to be associated with the tomato leaf curl betasatellite (ToLCB), a satellite previously only identified in Pakistan and India (Briddon et al., 2003, 2008). The study conducted here shows that a fourth begomovirus species, with a wide geographic distribution, occurs in Oman and also infects peppers. 2. Materials and methods

Fig. 1. Map of Oman showing origins of virus isolates. Indicated are the northern and southern agricultural regions. The numbers of ChLCV clones from tomato and pepper, as well as the numbers of betasatellite clones are given in brackets (tomato/pepper/betasatellite) for each location.

2013b) for begomovirus genomes (or DNA A components) and primers beta01/beta02 (Briddon et al., 2002) for begomovirusassociated betasatellites, was carried out as described previously (Khan et al., 2013a). Potentially full-length begomovirus genomes (or genomic components) and satellites were amplified by rolling circle amplification (RCA; Haible et al., 2006) using an Illustra TempliPhi 100 Amplification kit (Amersham Biosciences, Piscataway, NJ, USA) following the manufacturer’s instructions. The resulting high molecular weight products were digested with a range of restriction endonucleases to identify enzymes yielding unit length (∼2.8 kb for begomovirus components or ∼1.4 kb for satellites), which were then cloned into the plasmid vector pUC19 and sequenced commercially in both orientations using the primer walk strategy (Macrogen Inc., Seoul, Korea).

2.1. Virus source and DNA extraction Leaf samples of tomato and pepper plants showing typical leaf curl disease symptoms were collected from widely separated areas of Oman in 2010–2011. The origins of these samples are shown in Fig. 1 and listed in Table 1. Total nucleic acids were extracted from leaf samples by the method described by Porebski et al. (1997). Total nucleic acid extracts were resuspended in sterile distilled water and stored at −20 ◦ C. 2.2. Viral DNA amplification, cloning and sequencing Diagnostic PCR for the identification of samples harboring begomoviruses, using primers FDCP-382/RDCP-1038 (Khan et al.,

2.3. Sequence assembly and analysis DNA sequences were assembled and analyzed using SeqMan, part of the Lasergene suite of sequence analysis programs (DNAStar Inc., Madison, WI, USA). The ORF Finder program (http://www.ncbi.nlm.nih.gov/projects/gorf/) was used to identify putative genes. Sequence similarity searches were performed by comparing sequence to all sequences available in the GenBank database using BlastN (Altschul et al., 1990). Multiple sequence alignments were performed using MegAlign (Lasergene, DNASTAR Inc., Madison, WI, USA), using the ClustalV algorithm. Phylogenetic relationships were inferred using the NeighborJoining (distance) algorithm (Saitou and Nei, 1987) with

Table 1 Origins and features of begomovirus and betasatellite clones obtained from Oman. Isolate descriptor

Origin

Host

Begomovirus

Betasatellite

Accession number

Size (nt)

Salalah

Pepper

JN604489

2747

OM:Sal2:pep:10

Salalah

Pepper

JN604490

2747

OM:Sh1:pep:10

Shinas

Pepper

JN604491

2758

OM:Taq1:tom:10

Taqah

Tomato

JN604492

2759

OM:Taq2:tom:10

Taqah

Tomato

JN604493

2758

OM:Taq3:tom:10

Taqah

Tomato

JN604494

2759

OM:Taq4:tom:10

Taqah

Tomato

JN604495

2759

OM:Thm1:tom:10

Thumrait

Tomato

JN604496

2759

OM:Thm2:tom:10

Thumrait

Tomato

JN604497

2759

OM:Thm3:tom:10

Thumrait

Tomato

JN604498

2759

OM:Thm4:tom:10

Thumrait

Tomato

JN604499

2759

OM:KW1:tom:10

Kamil Wafi

Tomato

JN604500

2768

OM:Mct1:tom:10

SQU, Muscat

Tomato

HE819238

2758

OM:Swq1:pep:11

Suwaiq

Pepper

HE806431

2748

OM:Sal1:tom:11

Salalah

Tomato

HE806432

2763

OM:Sal2:tom:11

Salalah

Tomato

HE806433

2760

OM:Thm5:tom:10

Thumrait

Tomato

HE806434

2759

OM:Thm6:tom:10

Thumrait

Tomato

HE806435

2759

OM:Thm7:tom:10

Thumrait

Tomato

HE806436

2759

Accession number

CP

V2

Rep

TrAP

REn

C4

310–1071/252 [28] 310–1071/252 [28] 309–1082/256 [28] 310–1083/256 [28] 309–1082/256 [28] 310–1083/256 [28] 310–1083/256 [28] 310–1083/256 [28] 310–1083/256 [28] 310–1083/256 [28] 310–1083/256 [28] 310–1092/259 [28] 309–1079/256 [28] 311–1069/252 [28] 311–1081/256 [28] 311–1081/256 [28] 310–1080/256 [28] 310–1080/256 [28] 310–1080/256 [28]

150–515/121 [13] 150–515/121 [13] 149–514/121 [13] 150–515/121 [13] 149–505/118 [13] 150–515/121 [13] 150–515/121 [13] 150–515/121 [13] 150–515/121 [13] 150–515/121 [13] 150–515/121 [13] 150–515/121 [13] 149–514/121 [13] 151–516/121 [13] 151–516/121 [13] 151–516/121 [13] 150–515/121 [13] 150–515/121 [13] 150–515/121 [13]

1517–2602/361 [40] 1517–2602/361 [40] 1528–2613/361 [40] 1529–2614/361 [40] 1528–2613/361 [40] 1529–2614/361 [40] 1529–2614/361 [40] 1529–2614/361 [40] 1529–2614/361 [40] 1529–2614/361 [40] 1529–2614/361 [40] 1538–2623/361 [40] 1528–2613/361 [40] 1518–2603/361 [40] 1533–2618/361 [40] 1530–2615/361 [40] 1529–2614/361 [40] 1529–2614/361 [40] 1535–2614/359 [39]

1210–614/134 [15] 1210–1614/134 [15] 1221–1625/134 [15] 1222–1626/134 [15] 1221–1625/134 [15] 1222–1626/134 [15] 1222–1626/134 [15] 1222–1626/134 [15] 1222–1626/134 [15] 1222–1626/134 [15] 1222–1626/134 [15] 1231–1635/134 [15] 1221–1625/134 [15] 1211–1615/134 [15] 1223–1630/135 [15] 1223–1627/134 [15] 1222–1626/134 [15] 1222–1626/134 [15] 1222–1632/136 [15]

1065–1469/134 [15] 1065–1469/134 [15] 1076–1480/134 [15] 1077–1481/134 [15] 1076–1480/134 [15] 1077–1481/134 [15] 1077–1481/134 [15] 1077–1481/134 [15] 1077–1481/134 [15] 1077–1481/134 [15] 1077–1481/134 [15] 1086–1490/134 [15] 1076–1480/134 [15] 1066–1470/134 [15] 1078–1485/135 [15] 1078–1482/134 [15] 1077–1481/134 [15] 1077–1481/134 [15] 1077–1487/136 [15]

2152–2445/97 [11] 2152–2445/97 [11] 2163–2456/97 [11] 2164–2457/97 [11] 2163–2456/97 [11] 2164–2457/97 [11] 2164–2457/97 [11] 2164–2457/97 [11] 2164–2457/97 [11] 2164–2457/97 [11] 2164–2457/97 [11] 2173–2466/97 [11] 2163–2456/97 [11] 2153–2446/97 [11] 2168–2461/97 [11] 2165–2458/97 [11] 2164–2457/97 [11] 2164–2457/97 [11] 2170–2457/95 [10]

– HE800541

Size (nt)

– 1373

Position of ␤C1 gene (coordinates/no. of amino acids/coding capacity [kDa])

–

–

–

201–557/118 [13] –

–

–

–

–

–

–

–

–

–

–

–

–

HE800544

1375

–

–

201–557/118 [13] 201–557/118 [13] –

HE800545

1374

–

–

–

HE800546 –

1379 –

HE800542

1381

HE800547

1370

201–557/118 [13] –

–

–

201–557/118 [13] 201–557/118 [13] –

–

–

–

HE800548

1376

HE800549

1374

A.J. Khan et al. / Virus Research 177 (2013) 87–97

OM:Sal1:pep:10

Position of genes (coordinates/no. of amino acids/coding capacity [kDa])

201–557/118 [13] 201–557/118 [13]

89

90

A.J. Khan et al. / Virus Research 177 (2013) 87–97

bootstrapping (Felsenstein, 1985). Evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000) using MEGA5 (Tamura et al., 2011). Detection of potential recombinant sequences and localization of recombination breakpoints were carried out with the Simplot program version 3.2 (Lole et al., 1999) and the Recombination Detection Program (RDP version 3; Martin and Rybicki, 2000). Default RDP settings were used throughout with a p-value cutoff of 0.01 and the standard Bonferroni correction. For bootscan analysis, 200 replicates with a 95% cut-off were taken, and for GENECONV (Sawyer, 1989) analysis, the g-scale parameter was set to 1. 2.4. Agrobacterium-mediated inoculation A partial direct repeat construct of begomovirus clone Mct1 (HE819238) was produced for Agrobacterium-mediated inoculation. An ∼580 bp BamHI and SacI fragment of Mct1 was ligated in the binary vector pGreen0029 (Hellens et al., 2000). This partial clone was then linearized with BamHI and the full-length insert of Mct1, released with BamHI, ligated in this. Similarly, for the betasatellite clone P-23, a construct was produced by digestion with BamHI and XbaI to release a fragment of ∼593 bp which was cloned in binary vector pCAMBIA-1301. This partial clone was then linearized with XbaI and ligated with the full-length betasatellite component, released with XbaI, to yield a partial direct repeat construct. The binary vector constructs were electroporated into competent Agrobacterium tumefaciens strain LBA4404 cells. Inocula were prepared by growing A. tumefaciens cultures in LB medium supplemented with rifampicin (25 ␮g/ml), kanamycin (50 ␮g/ml) and tetracycline (10 ␮g/ml) at 28 ◦ C for 48 h to an optical density (OD600 ) of 0.6–1.0. The cells were harvested by centrifugation (4400 rpm for 15 min at 22 ◦ C) and resuspended in 10 mM MgCl2 containing 100 ␮M acetosyringone. Cells were incubated for 3 h and then infiltrated into the undersides of the young leaves of 3–4-week old Nicotiana benthamiana, tomato var. Indam and pepper var. Lido plants using a 5 ml syringe as described previously (Amin et al., 2011b). Plants were maintained in an insect-free glasshouse at 26 ◦ C with supplementary lighting to give a 16 h day length. The presence of virus and betasatellite in inoculated plants was confirmed by PCR using primers FDCP-382/RDCP-1038 and beta01/beta02, respectively. 2.5. Detection of viral DNA in plants by Southern blot hybridization DNA extracted from plants was electrophoresed on 1% agarose gels run in 0.5X TAE buffer. Nucleic acids were transferred to nylon membranes (Roche GmbH, Germany) by capillary transfer. Blots were probed with an ∼970 bp XbaI and SacI fragment of clone Mct1, or an ∼780 bp XbaI and BamHI fragment of clone P-23, labeled with Digoxigenin (DIG) using a DIG High Prime DNA Labeling and Detection Starter Kit I (Roche GmbH, Germany). 3. Results 3.1. Cloning and sequencing of begomoviruses infecting tomato and pepper in Oman Leaf samples of tomato and pepper plants showing leaf curl symptoms were collected from the major agricultural areas of Oman in 2010–2011. A summary of all plants examined and the viruses identified are given in Supplementary Table 1. All collected samples were subjected to PCR for the detection of putative begomoviruses using genomic DNA as template. A pair of degenerate

primers (Khan et al., 2013b) designed on the CP region of begomoviruses was used which gave an amplification product of 650 bp (results not shown). PCR positive samples were used to amplify fulllength circular genome using 29 DNA polymerase. The resulting high molecular weight concatameric molecule was digested with PstI and BamHI restriction enzymes and resulting DNA fragments of ∼2.8 kb (unit length of begomoviruses) were cloned in the pUC19 vector and sequenced in both directions. A total of 19 clones were sequenced in their entirety and the sequences are available in nucleotide sequence databases under the accession numbers given in Table 1. The geographic origins of the samples from which clones were obtained are indicated on the map given in Fig. 1. Of the 19 samples from which begomovirus clones were obtained, eight samples of tomato and pepper were PCR positive for the presence of betasatellites. RCA amplified betasatellites (∼1.4 kb) were cloned and sequenced. The eight sequences are available in the sequence databases under the accession numbers listed in Table 1.

3.2. Analysis of begomovirus sequences The sequences of the 19 clones were determined to be between 2747 and 2768 nt in length, typical of the genomes (or DNA A components) of begomoviruses. All sequences were shown to contain six genes with an arrangement typical of monopartite (or DNA A component of bipartite) begomoviruses originating from the Old World (OW); two overlapping genes in virion-sense (the V2 and coat protein [CP] genes) and four in the complementary-sense (the replication associated protein [Rep], the transcriptional-activator protein [TrAP], the replication enhancer protein [REn] and the C4 genes). For each clone, the coordinates of genes and their predicted coding capacities are given in Table 1. Between the 5 ends of the V2 and Rep genes, there lies a non-coding, intergenic region with a predicted hairpin structure, encompassing the sequence ‘TAATATTAC’ in the loop, known as nonanucleotide sequence, which forms part of the origin of virion-strand DNA replication (Orozco and Hanley-Bowdoin, 1996). Sequence alignments using the ClustalV algorithm implemented in MegAlign (Lasergene) showed the begomovirus clones from Oman to share 96.9–100% nucleotide sequence identity, indicating that they are isolates of a single species, based on presently applicable species demarcation criteria for begomoviruses (89%; Fauquet et al., 2008). An initial comparison of the sequences using NCBI database was performed by BlastN that showed the clones from Oman to have highest levels of sequence identity to isolates of ChLCV. Subsequent alignments of the ChLCV isolates from Oman with all ChLCV isolates available in the databases (23 available at this time) showed these to have 82.7–91.1% nucleotide sequence identity (Table 2). To all other begomovirus sequences in the database the ChLCV isolates from Oman showed less than 85% identity with the highest levels (84.6%) to an isolate of tomato leaf curl Joydebpur virus (JQ654463). These results indicate that the begomovirus clones characterized here are isolates of ChLCV. At this time four distinct strains of ChLCV are recognized; the “India”, “Pakistan” and “Khanewal” strains, as well as a strain, which has yet to be named, which will here be referred to as the “Guntur” strain (for the isolates within each strain, and their accession numbers, see Fig. 2). Comparisons with the ChLCV sequences from Oman showed them to have the highest levels of nucleotide sequence identity to isolates of the “Pakistan” strain (88.0–91.1%) followed by the “Guntur strain” (88.2–89.1%; Table 2). This indicates that the isolates from Oman represent a distinct strain within the species (the demarcation threshold for strains of begomoviruses being 93%; Fauquet et al., 2008). The name “Oman” strain is proposed for this newly identified strain.

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Table 2 Percentage nucleotide sequence identities for the full-length genome and ORFs of Chili leaf curl virus-Oman (ChLCV-OM) and selected monopartite begomoviruses. Virus species (-straina )

Genomec

Percentage amino acid sequence identityc CP

ChLCV-PK (6)b ChLCV-IN (14)b ChLCV-Kha (1)b ChLCV-Gun (2)b PepLCLV (3)b PepLCBDV (3)

88.0–91.1 82.7–86.8 85.7–87.1 88.2–89.1 79.5–81.5 79.7–80.8

93.0–97.3 92.5–94.8 91.0–95.3 93.0–97.3 93.4–96.9 92.5–93.7

V2 88.1–89.0 87.3–91.7 84.7–85.6 90.0–90.9 90.9–91.5 90.7–91.5

Rep 91.7–95.1 88.1–91.1 91.4–95.0 91.4–95.0 71.2–74.6 74.5–77.8

C2 91.8–95.5 82.8–94.8 81.3–91.8 85.1–95.5 88.1–96.3 85.1–92.5

REn 73.1–90.3 74.6–84.3 75.4–86.6 78.4–88.1 74.6–84.3 73.1–86.5

C4 83.2–91.8 73.7–85.6 84.2–92.8 84.2–94.8 34.1–40.0 34.1–38.8

a

Chili leaf curl virus (ChLCV) strains are indicated as India (IN), Khanewal (Kha), Guntur (Gun) and Pakistan (PK). The figures in brackets indicate the numbers of sequences available in the databases for comparison. c The values highlighted in gray indicate the highest nucleotide sequence identities, for full length genomes, or highest amino acid sequence identities, for predicted gene products, with ChLCV-OM. b

3.3. Phylogenetic analysis of begomovirus sequences A phylogenetic analysis, based upon alignments of the complete sequences of all available ChLCV isolates, including the ChLCV-OM isolates identified here, and selected other begomovirus sequences is shown in Fig. 2. This shows the ChLCV isolates to form separate groupings according to the aforementioned strains of the species. The ChLCV-OM isolates clearly form a distinct group, confirming their identification as a distinct strain, that are most closely related to ChLCV-Gun isolates which are basal to the Oman strain isolates. One ChLCV-OM isolate, Sh1 (JN604491), is basal to all others in the in the tree, suggesting that it is distinct. It is also interesting to note that there is little sequence variation in ChLCV-OM,

short branch lengths, in comparison to isolates of the “India” and “Pakistan” strains (for the “Guntur” and “Khanewal” strains too few sequences are available to judge the levels of sequence variability at this time). It is interesting to note that a set of deletions/insertions in the IR regions of ChLCV isolates distinguishes the India, Khanawal and Guntur/Pakistan/Oman (these three strains having near identical deletions/insertion) strains (Supplementary Fig. 1). 3.4. Analysis of ChLCV-OM isolates for recombination Bootscan and RDP analyses of the sequence of ChLCV-OM isolate Sal2 (JN604490) are shown in Fig. 3. Both analyses provide

Fig. 2. Phylogenetic dendrograms based upon the alignments of the sequences of ChLCV-OM with all sequences of ChLCV available in the nucleotide sequence databases with selected other begomovirus sequences (panel A) and the sequences of ToLCB isolates identified in this study with all other ToLCB isolates available in the databases with selected other betasatellite sequences (panel B). In each case, the isolate descriptor and database accession number is given. The numbers at nodes represent percent bootstrap confidence score (1000 replicates). The begomovirus tree was arbitrarily rooted on the DNA A component of pepper golden mosaic virus (PepGMV), a distantly related begomovirus, as outgroup whereas the betasatellite tree was arbitrarily rooted on ageratum yellow vein Singapore alphasatellite (AYVSGA), a distantly related sequence of a similar size, as outgroup. The virus acronyms used are as defined in Fauquet et al. (2008) and the betasatellite acronyms are as in Briddon et al. (2008). For the virus tree the positions of the ChLCV strains are indicated on the right. For both trees the sequences produced as part of this study are in gray.

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Fig. 3. Analysis of ChLCV-OM isolate Sal2 (JN604490) for recombination. (A) Boot scanning analysis of ChLCV-OM and other begomoviruses was performed using the SimPlot program with a sliding window of 200 nucleotides moving in 20-nucleotide steps. The graphs were built by using the neighbor-joining algorithm, the Kimura 2-parameter distance model, and 100 pseudo-replicates. The dotted line shows an arbitrary 70% reliability threshold, and the solid line shows the 89% species demarcation threshold for begomoviruses. (B) Schematic representation of the linearized genome of ChLCV-OM isolate Sh1 showing the origin of recombinant fragments, recombination breakpoints, p-values determined by the RDP method (Table 3) and putative parental viruses. 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 chili leaf curl virus (ChLCV; Pakistan [PK] and Guntur [Gun] strains), pepper leaf curl Lahore virus (PepLCLV), tomato yellow leaf curl virus (TYLCV). The database accession numbers are given in each case.

evidence of recombination suggesting that recombination of the progenitor virus of ChLCV-OM has occurred with pepper leaf curl Lahore virus (PepLCLV) and with a Guntur strain isolate of ChLCV. The RDP analysis identified the breakpoints for the recombinant fragment originating from PepLCLV at coordinates 276 and 1011 and for the fragment originating from ChLCV-Gun coordinates 1531–2060 (Table 3 and Fig. 3). The RDP analysis also suggested that the major parent of ChLCV-OM was a ChLCV-PK. The results of an RDP analysis for all ChLCV-OM isolates is shown in Supplementary Fig. 2. This shows the 19 ChLCV-OM isolates to contain the same recombinant fragments except for isolate Sh1 (JN604491) for which the PepLCLV is smaller which may explain why this isolate is basal to all other ChLCV-OM isolates in the phylogenetic tree.

3.5. Association of tomato leaf curl betasatellite (ToLCB) with ChLCV-OM The screening of field-collected samples indicated that some, but not all, ChLCV-OM virus isolates are associated with a betasatellite (Table 1). The complete sequences of eight potentially full-length betasatellite clones were obtained and sequenced. The sequences are between 1370 and 1381 nt in length and are available in the sequence databases under the accession numbers listed in Table 1. The sequences have the typical arrangement of betasatellites, consisting of a single gene (known as ␤C1; Table 1), in complementary-sense orientation, encoding a predicted 118 aa product, a region of sequence rich in adenine and a sequence that is conserved between distinct betasatellites (known as the satellite

2.99 × 10−12 3.66 × 10−3 3.60 × 10−7 2.35 × 10−5

Siscan

6.54 × 10−9 1.93 × 10−3

Chimera Maxchi

1.32 × 10−8 1.05 × 10−3 6.91 × 10−17 3.98 × 10−15 5.72 × 10−13 1.73 × 10−3

Bootscan GENECONV RDP

ChLCV-PK (AF336806) ChLCV-PK (AF336806) PepLCLV (AM691745) ChLCV-Gun (HM007100) 1011 2060

3

5

276 1531

Detection methods Major parent Minor parent

 

Breakpoint positions

Table 3 Results of a recombination analysis of ChLCV-OM (isolate Sal2 [JN604490]) using RDP.

3.66 × 10−7 5.81 × 10−6

3 Seq

A.J. Khan et al. / Virus Research 177 (2013) 87–97

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conserved region [SCR]), that encompasses a predicted hairpin structure containing the sequence “TAATATTAC” within the loop, with similarity to the origin of virion-strand DNA replication of geminiviruses (Briddon et al., 2003). The betasatellites characterized as part of this study share 91–95% nucleotide sequence identity with the only betasatellite previously reported from Oman (tomato leaf curl betasatellite [ToLCB]; 2 sequences available in the databases; Khan et al., 2008; Idris et al., 2011), and 85–90% with ToLCB isolates originating from the Indian sub-continent (7 sequences available in the databases). A phylogenetic analysis based upon an alignment of the complete sequences of betasatellite isolates from Oman with selected betasatellites available in the databases is shown in Fig. 2B. This shows the betasatellites isolated from tomato and pepper plants infected with ChLCV to be most closely related to the previous isolates of ToLCB obtained from Oman, which were isolated from tomato (Khan et al., 2008; Idris et al., 2011). Basal to these sequences are the sequences of ToLCB originating from India and Pakistan from which the Oman isolates are distinct. What is not evident is any phylogeographic segregation (no distinction between ToLCB isolates occurring in the north and south of the country) nor any evidence of segregation according to host (either tomato or pepper). The tree also shows that ToLCB is distinct from a number of other betasatellites, which are commonly identified in solanaceous hosts that were included in the analysis.

3.6. Infectivity of ChLCV-OM and ToLCB to N. benthamiana and tomato The infectivity of ChLCV-OM isolate Mct1 in the presence and absence of ToLCB isolate P-23 was assessed by Agrobacteriummediated inoculation to N. benthamiana and tomato var. Indam. To N. benthamiana ChLCV-OM was highly infectious in both the presence and absence of ToLCB, with all plants becoming infected (Table 4). For N. benthamiana inoculated with the virus alone or with the virus and betasatellite the first symptoms of infection were visible at 12 days post-inoculation (dpi). Symptoms for plants inoculated with only ChLCV-OM consisted of upward curling of the edges of the youngest leaves with downward rolling of the tips, mild downward curling (cupping) of the edges of older leaves, leaf crumpling, vein thickening, mild chlorosis and stunting of plants (plants ceased to grow at approx. 3 weeks post inoculation) (Fig. 4H). N. benthamiana plants inoculated with ChLCV and ToLCB developed slightly more severe symptoms consisting of deep downward cupping of leaves, severe leaf crumpling, interveinal chlorosis, vein thickening and stunting (Fig. 4I). ChLCV induced slightly more severe symptoms in tomato in the presence of ToLCB than alone, although the levels of infectivity were similar in each case. Tomato plants inoculated with ChLCV-OM or with ChLCV-OM and ToLCB developed the first symptoms of infection at approx. 17 dpi. For tomato plants inoculated with ChLCV alone, the symptoms consisted of severe stunting with reduced internodes and a greatly reduced leaflet size (with a normal leaflet shape) giving the plants a bunchy appearance at the growing points (Fig. 4E). Leaves showed some crumpling and mild interveinal chlorosis, particularly at the edges (Fig. 4B). For tomato plants inoculated with ChLCV and ToLCB leaflets were greatly reduced in size with downward cupping and plants had a reduced internode size (Fig. 4F). Leaflets also showed severe interveinal chlorosis with necrosis (Fig. 4C). All attempts to infect pepper var. Lido with ChLCV-OM in the presence and absence of ToLCB (three experiments involving a total of 30 plants) were unsuccessful, with no evidence of the presence of the virus in tissues distal to the inoculation site as determined by PCR (results not shown).

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Table 4 Infectivity of ChLCV-OM in the presence and absence of ToLCB to N. benthamiana and tomato. Inoculum

Infectivity (No. of plants infected/No. of plants inoculated) N. benthamiana

ChLCV-OM ChLCV-OM + ToLCB

Tomato

Expt. 1

Expt. 2

Expt. 3

Expt. 1

Expt. 2

Expt. 3

6/6 5/5

6/6 5/5

5/5 5/5

5/8 6/8

6/10 7/10

8/10 8/10

Southern blot analysis of DNA extracted from symptomatic N. benthamiana and tomato plants inoculated with ChLCV-OM, and ChLCV-OM/ToLCB showed the presence of single stranded (ss) and double-stranded (ds) viral DNA forms typical of geminivirus replication (Fig. 5). The results show that in both N. benthamiana and tomato ChLCV-OM is able to maintain ToLCB. However, there is no evidence that the betasatellite leads to an increase in the titer of the virus. 4. Discussion Previous studies of the begomoviruses causing leaf curl disease of tomato in Oman have identified three species; TYLCV, ToLCSDV and a begomovirus species so far only identified in Oman, ToLCOMV (Khan et al., 2008, 2013a,b; Idris et al., 2011). The results presented

here show that a fourth species of begomovirus, ChLCV, also causes the disease in Oman. ChLCV has previously only been reported in Pakistan and India (Senanayake et al., 2006, 2012; Shih et al., 2003; Mubin et al., 2009). With the possible exception of watermelon chlorotic stunt virus (Khan et al., 2012a,b), a bipartite begomovirus, which is endemic across the Middle East and North Africa, all the begomoviruses reported so far in Oman have their origin on either the Indian subcontinent, the Middle East/Mediterranean or Africa (Khan et al., 2012a,b, 2013a,b). ToLCOMV is only known in Oman and is believed to have emerged through recombination between two begomoviruses introduced into Oman (Idris et al., 2011). The evidence suggests that ChLCV-OM has similarly been introduced, likely from the subcontinent, by way of the extensive trade links between Oman (and/or other countries on the Arabian Peninsula). Vegetable

Fig. 4. Symptoms induced by ChLCV-OM (panels E and H) and ChLCV-OM with ToLCB (panels F and I) by Agrobacterium-mediated inoculation of tomato (panels E and F) and N. benthamiana (panels H and I). For comparison healthy, non-inoculated tomato (panel D) and N. benthamiana (panel G) plants are shown. Field infected tomato plants are shown in panel (A) and close-up photographs of the plants in panels E and F are shown in panels B and C, respectively.

A.J. Khan et al. / Virus Research 177 (2013) 87–97

Fig. 5. Southern blot detection of ChLCV-OM (A) and ToLCB (B) in agroinoculated N. benthamiana and tomato plants. The samples loaded on the gel in panel A were total DNA extracted from a mock inoculated tomato plant (lane 1) and symptomatic young leaves of N. benthamiana plants inoculated with ChLCV-OM (lane 2) or ChLCVOM and ToLCB (lane 3) and tomato plants inoculated with ChLCV-OM (lane 4) or ChLCV-OM and ToLCB (lane 5). The samples loaded on the gel in panel B were total DNA extracted from a mock inoculated tomato plant (lane 1) and symptomatic young leaves of N. benthamiana plants (lane 2 and 3) and tomato plants (lanes 4 and 5) inoculated with ChLCV-OM and ToLCB. For both blots the samples in lanes 6 and 7 were extracted from field collected tomato plants from which both ChLCV-OM and ToLCB were cloned. Approximately equal amounts of total DNA (10 ␮g) were loaded in each case (the ethidium bromide-stained genomic DNA on the agarose gel is shown above the Southern blot in each case). The positions viral circular (oc), linear (lin), super coiled (sc) and single stranded (ss) DNA forms are indicted.

production in Oman only meets about 20–25% of the demand in the country (Ministry of Agriculture and Fisheries, personal communication), meaning that substantial amounts of produce are imported from surrounding countries, including Pakistan and India, providing a possible route for the introduction of ChLCV. Recombination is a major mechanism of evolution of begomoviruses and has been shown to have played a part in the evolution of the distinct strains of TYLCV and ToLCSDV, as well as the distinct species ToLCOMV, in Oman (Idris et al., 2011; Khan et al.,

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2013a). The results presented here suggest that recombination has similarly led to the distinct strain of ChLCV occurring in Oman. The recombination analysis suggests that ChLCV-OM has obtained at least some of its sequence by recombination from PepLCLV, a virus, which has only been reported from India and Pakistan (Tahir et al., 2010; Srivastava et al., 2013) but has not been identified in Oman. This suggests that recombination between the parents of ChLCV-OM either occurred before introduction to Oman or that PepLCV is present in the country but has yet to be identified. Overall the results of the recombination analysis, phylogenetic analysis and the deletions/insertions within the IR suggest that ChLCV-Om evolved from ChLCV-PK (the major parent) with some contribution of PepLCLV and ChLCV-Guntur. The betasatellite identified in association with ChLCV-OM, ToLCB, has previously been reported in association with TYLCV and ToLCOMV in Oman (Khan et al., 2008; Idris et al., 2011) and is so far the only betasatellite identified there. In common with ChLCV, ToLCB has its origins on the Indian sub-continent. ToLCB is an infrequently encountered betasatellite. Despite the large numbers of studies that have analyzed begomovirus infections of tomatoes in India and Pakistan, only 7 isolates of ToLCB have been identified (deposited with the nucleotide sequence databases). Far more frequently encountered in tomato is tomato leaf curl Bangladesh betasatellite (ToLCBDB; Chattopadhyay et al., 2008; Sivalingham et al., 2010). On the sub-continent ChLCV has not been reported in association with ToLCB. In Capsicum spp. numerous other betasatellites are reported to be associated with begomovirus diseases, of which the major one in Pakistan is chili leaf curl betasatellite (Hussain et al., 2009). It is thus somewhat surprising to find ToLCB associated with a disease of tomato and, particularly, chili and also surprising that of the numerous betasatellites associated with diseases of tomato and chili on the Indian sub-continent only one, ToLCB, was introduced into Oman. The reason for this is unclear. The finding also highlights the relaxed specificity for interaction between helper begomoviruses and their dependent betasatellites, as shown experimentally by Saunders et al. (2008). The interaction of betasatellites with begomoviruses can be categorized into two types. The first is an obligate interaction where the begomovirus has an absolute requirement for the betasatellite to infect a particular host and is best exemplified by the viruses and the betasatellite causing cotton leaf curl disease in southern Asia (Briddon et al., 2001; Sattar et al., 2013). The second are begomoviruses that have a more facultative interaction with betasatellites. For these viruses, in the field, some, but not all, virus isolates are associated with a betasatellite. This type of interaction is best exemplified by the interaction of tobacco curly shoot virus with its betasatellite tobacco curly shoot betasatellite (Li et al., 2005). The results presented here indicate that ChLCVOM has a facultative interaction with ToLCB in both pepper and tomato – the betasatellite having been identified in only 2 of 4 pepper plants and only 6 of 15 tomato plants in which the virus was present. This conclusion is supported by the infectivity studies, which indicate that ChLCV-OM can induce symptoms typical of the disease in tomato in the absence of the betasatellite. Koch’s postulates have previously been satisfied only for one ChLCV isolate (a “Pakistan” strain isolate), by inoculation of a cloned virus (Chattopadhyay et al., 2008). This work showed that the associated betasatellite (ToLCBDB) enhances the symptoms induced by the virus, as was the case here. However, the study did not examine whether the betasatellite enhanced virus levels in plants. Chattopadhyay et al. (2008) showed the infectivity of their virus to Capsicum annuum. The virus (and betasatellite) here were not infectious to Capsicum. The reason for this is unclear. It may indicate that the virus (or betasatellite) used are host adapted (infectivity here was assessed with clones obtained from tomato). Subsequent

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inoculations with a set of virus/betasatellite clones isolated from pepper (JN604490 and HE800541) produced the same results; infectivity in tomato but not pepper (results not shown). This would seem to discount the host adaptation hypothesis which is also not supported by the lack of segregation according to host of either the virus or betasatellite in phylogenetic analyses. Alternatively it is possible that the Agrobacterium strain we used is incompatible with Capsicum or that some other factor is required for infectivity in this host. Further studies will be needed to investigate this possibility. The necrotic response of tomato to infection with ChLCV-OM and ToLCB, which was not evident in plants inoculated with only ChLCV-OM, is reminiscent of a hypersensitive response. Since betasatellites encode only a single product, the ␤C1 protein, the results might suggest that ToLCB ␤C1 may be an avirulence determinant in tomato. For two bipartite begomoviruses products encoded by the DNA B component (Garrido-Ramirez et al., 2000; Hussain et al., 2007) and for a monopartite begomovirus the V2 protein (Mubin et al., 2010) have been shown to be avirulence determinants in various hosts. ␤C1 has not previously been implicated as an avirulence determinant. Further studies will be required to confirm this suggestion. The results here show that ChLCV-OM occurs in both the major agricultural regions of Oman. However, this is not the case for the other viruses causing disease of tomato. ToLCSDV has been identified only south of the Al Hajar Mountains, which separate the northern coastal areas from the rest of the country (Khan et al., 2013b). In contrast, TYLCV and ToLCOMV are only found in the north. This suggests that the mountains present a barrier to the movement of insects, and thus also their associated viruses, between the two regions. The finding of ChLCV on both sides of the Al Hajar Mountains may suggest that this virus species has been imported twice (once into each region of Oman), an idea not supported by the close genetic relationship between the virus isolates in the north and south of the country. Alternatively the finding may suggest that ChLCV was introduced much earlier than the other viruses and has had time to spread throughout the country. Intensive agriculture in Oman is a relatively recent innovation and will need to expand considerably to meet present and future demand for produce in the country. The presence of quite a high diversity of begomoviruses, particularly affecting tomato production, is thus worrying since already it is having detrimental effects on productivity. A further worry is that additional viruses may be introduced, aggravating the problem, either directly by causing new diseases or indirectly by interacting (recombining) with viruses already present leading to increased pathogenicity. There is thus a need for more detailed studies of the diversity and extent of spread of begomoviruses in Oman and monitoring to identify any possible future introductions.

Acknowledgements This work was supported by research grant number ORG/EBR/09/03 from The Research Council of Oman, to AJK. RWB is supported by the Higher Education Commission (Government of Pakistan) under the “Foreign Faculty Hiring Program”.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.virusres.2013.07.018.

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