Abstract
Turnip curly top virus (TCTV) is the only member of the newly established genus Turncurtovirus (family Geminiviridae). As part of an ongoing study to identify additional plant hosts and the diversity of turncurtoviruses, between 2012 and 2014, we sampled symptomatic turnip plants and other crops in the provinces Fars and Khorasan Razavi (southern and northeastern Iran, respectively). Infection by turncurtoviruses was tested by PCR and/or rolling-circle amplification (RCA) coupled with restriction enzyme digests. Turncurtoviruses were identified in turnip as well as seven other field crops, including eggplant, basil, radish, lettuce, sugar beet, red beet and spinach. Full turncurtovirus genomes were recovered from 25 of these samples, leading to the identification of TCTV and a new putative turncurtovirus, turnip leaf roll virus (TLRV; 13 isolates), which shares <80 % genome-wide pairwise identity with TCTV. Agroinoculation of plants with an infectious clone of TLRV demonstrated that this virus could infect several plant hosts under greenhouse conditions and could be transmitted by the leafhopper Circulifer haematoceps (Mulsant and Rey, 1855) from agroinoculated to healthy plants.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Geminiviridae is a large family of plant-infecting viruses, and its members are associated with substantial crop losses of economically important food and fiber crops worldwide [40]. This family is divided into seven genera: Mastrevirus, Curtovirus, Topocuvirus, Begomovirus, Becurtovirus, Eragrovirus and Turncurtovirus [41]. Furthermore, over the last few years, several novel geminiviruses have been discovered infecting alfalfa, Euphorbia caput-medusae, faba bean, and grapevine as well as apple, citrus and mulberry trees [3, 24–26, 37]. Geminiviruses have twinned quasi-isometric particles with a diameter of approximately 20 nm and a length of 30 nm [6, 44]. Their genomes consist of one or two circular single-stranded DNA molecules with a length of ~2.5-3.0 kb [8].
Over the last decade, the use of rolling-circle amplification (RCA) coupled with restriction enzyme digests has enabled the identification of a large number of diverse circular DNA viruses, especially geminiviruses [4, 7, 18, 20, 24, 25, 27, 38, 42]. Using these methods, our research group has identified turnip curly top virus (TCTV) infecting turnip (Brassica rapa L. var. rapa), radish (Raphanus sativus L.) and five weeds species [7, 12, 35]. TCTV is the type member of the genus Turncurtovirus of family Geminiviridae, and it is vectored by the leafhopper Circulifer haematoceps [7, 35, 36, 41].
The 20 TCTV genome sequences that have been characterized previously [7, 36] share >87 % pairwise identity, and they can be assigned to four strains: TCTV-A (n = 7), -B (n = 8), -C (n = 4) and -D (n = 1). Except for an isolate of TCTV-B that was found in radish, all isolates have been identified only in turnip.
To complement the dataset of 20 full-length genomes of TCTV from previous studies [7, 35, 36] for more in-depth evolutionary analysis with a greater diversity of turncurtoviruses, we have continued to sample plants of turnip and other crops displaying symptoms similar to those induced by TCTV. In this study, we report the identification of a new turncurtovirus (13 isolates) and also demonstrate that the leafhopper C. haematoceps is able to transmit this virus from agroinoculated plants.
Materials and methods
Sampling and recovery of turncurtovirus genomes
Symptomatic leaves (n = 220) were sampled as part of ongoing field surveys between 2012 and 2014 in turnip-growing areas and vegetable farms around turnip plantations in the provinces Fars and Khorasan Razavi, which are located in southern and northeastern Iran, respectively. Total DNA of all 220 samples was extracted from leaves using the CTAB method as described by Zhang et al. [45]. DNA from the samples was enriched for circular molecules by RCA using Phi29 DNA polymerase (TempliPhi, GE Healthcare, USA) as described in Shepherd et al. [38]. The high-molecular-weight RCA amplicons from each sample were digested with different restriction endonucleases, including PstI, KpnI, HindIII, BglII or EcoRI, to yield unit-size molecules. In addition to this, a specific back-to-back primer pair (TCTV-F: 5’-AGG TTT GTC TGC CAC TCC TTT-3’/TCTV-R 5’-GCA GAC AAA CCT CAA ATA CGG-3’) was used to recover complete genomes using the RCA DNA as template with KAPA HiFi DNA polymerase (Kapa Biosystems, USA). The following thermal cycling protocol was used: initial denaturation at 95 °C for 3 min, followed by 25 cycles of 98 °C for 20 s, 60 °C for 15 s, and 72 °C for 3 min, and then a final extension at 72 °C for 3 min. According to restriction enzyme digests, BglII or EcoRI restriction enzymes were used to clone the full-length genome of IR:Zaf:Z11-Bgl:Tur:12, IR:Zaf:Z11-4:Tur:12 and IR:Lap:L14:Tur:12 isolates. The ~3-kb fragments amplified by RCA reaction or PCR for 25 symptomatic leaves from various hosts in Homayejan (n = 5; lat.: 30° 12′ 35.9″N, long.: 52° 03′ 59.1″O), Zafar-Abad (n = 18; lat.: 29° 24′ 34.8″N, long.: 52° 35′ 39″O), Lapouei (n = 1; lat.: 29° 48′ 44.5″N, long.: 52° 37′ 40.1″O) and Neyshabour (n = 1; lat.: 36° 14′ 41.4″N, long.: 58° 46′ 50″O) were ligated into pTZ57R or pJET1.2 vectors (Thermo Fisher Scientific, USA) followed by transformation of Escherichia coli strain XL1blue or DH5α. One clone per isolate was sequenced from both directions by primer walking at either Bioneer or Macrogen Inc. (South Korea).
Construction of an infectious clone of a divergent turncurtovirus
Since a subset of divergent turncurtoviruses was identified in this study (n = 13), an infectious clone of isolate IR:Zaf:Z11:12 (GenBank accession no. KT388087) was constructed as described previously [20] in order to study biological properties of this isolate and compare it to TCTV-[IR:Zaf:B11:06] (GenBank accession no. GU456685) from our previous study [36]. In brief, the genome of Z11 was cloned as a dimer into EcoRI-digested pGreen0029 plasmid [19]. The resulting recombinant pGreen0029 plasmid together with a pSoup helper plasmid was used to transform Agrobacterium tumefaciens strain C58.
Three to four leaf seedlings of turnip (B. rapa cv. Purple top white globe), cabbage (Brassica oleracea L. cvs Capitata and Kohlrabi white vienna), sugar beet (Beta vulgaris L. cv. Universe), red beet (Beta vulgaris subsp. esculenta (L.) Arcang. cv. Conditiva), spinach (Spinacia oleracea L. cv. Winter giant), pepper (Capsicum annuum L. cv. California Wonder), radish (R. sativus cv. Sparkler), okra (Abelmoschus esculentus (L.) Moench cv. Clemson Spineless), cowpea (Vigna unguiculata (L.) Walp. cv. Mashhad), eggplant (Solanum melongena L. cv. Black Beauty), lettuce (Lactuca sativa L. cv. Attraction), tomato (Solanum lycopersicum L. cv. Moneymaker), canola (Brassica napus L. cv. Mendel), thale cress (Arabidopsis thaliana (L.) Heynh. ecotype Columbia) and Nicotiana benthamiana L. were agroinoculated with the infectious clone of the turncurtovirus IR:Zaf:Z11:12 isolate according to the method described by Grimsley et al. [16]. Similarly, turnip and radish seedlings were agroinoculated with A. tumefaciens cultures bearing pGreen plasmid without the turncurtovirus genome and used as negative controls. Agroinoculated plants were kept in a greenhouse at ~25 °C and monitored for the appearance of curly top symptoms. Total DNA from symptomatic and non-symptomatic plants (top non-inoculated leaves) was extracted 4 weeks post-agroinoculation, and PCR assays were used to confirm turncurtovirus infection.
Leafhopper transmission of the turncurtovirus IR:Zaf:Z11:12 isolate
Symptomatic turnip plants agroinoculated with the infectious clone of the IR:Zaf:Z11:12 isolate were used for leafhopper transmission using C. haematoceps under insect-proof conditions in a greenhouse at ~25 °C as described previously [20]. Non-viruliferous leafhoppers (nymph stage and/or adults) were caged with agroinoculated infected turnip plants showing typical curly top symptoms for one week to allow for acquisition of the virus. The constructed cage contained a plastic cylinder sealed at one end with cotton mesh. Following acquisition, leafhoppers were transferred to healthy turnip or radish seedlings (one leafhopper per seedling), and the inoculated plants were checked four weeks later for appearance of curly top symptoms. In addition, the agroinoculated plants were tested by PCR using the specific primer pair TCTV11-1676-F-GCGTAAATCCCTCACCACAGC/TCTV11-2699-R-CAGCTGCAGAACCTCGCCTGT, which direct the amplification of a ~1060-bp fragment of the genome of the IR:Zaf:Z11:12 isolate to confirm turncurtovirus infection. PCR conditions consisted of initial denaturation at 95 °C for 3 min and 35 cycles of 94 °C for 1 min, 57 °C for 1 min and 72 °C for 1.5 min, followed by one cycle of 72 °C for 10 min.
Turncurtovirus sequence analysis
The genomes of turncurtoviruses available in GenBank (n = 20) together with the ones determined in this study (n = 25) were aligned using MUSCLE [11]. The aligned genome sequences were used to construct a neighbor-joining tree using the Jukes-Cantor model with 1000 bootstrap replicates. The tree was rooted with the genome sequences of eragroviruses [42]. Branches with less than 60 % support were collapsed.
The aligned turncurtovirus dataset was also used for analysis of evidence of recombination in the genomes using RDP4 [28, 29], which implements the methods RDP [31], GENECONV [33], Bootscan [30], Maxchi [39], Chimaera [34], Siscan [14] and 3Seq [5]. Recombination events detected by RDP4 were deemed credible if there was strong phylogenetic support for the recombination event and the event was detected by a minimum of three methods with p-values of <10−3.
A maximum-likelihood phylogenetic tree with recombinant regions removed from the genomes was constructed using PHYML 3.0 [17], using the T92+G nucleotide substitution model inferred using jModelTest [9] and 1000 bootstrap replicates. Branches with less than 60 % support were collapsed.
Maximum-likelihood phylogenetic trees of the amino acid sequences of replication-associated protein (Rep) and coat protein (CP) were constructed using PHYML 3.0 [17] with the WAG+G substitution model, determined as the best model using Prottest [1], and with an approximate-likelihood ratio test (aLRT) for branch support. Branches with aLRT branch support <80 % were collapsed.
The pairwise identities of the genome nucleotide sequences as well as amino acid sequences of CP and Rep were determined using SDT v1.2 [32].
Results
Turncurtovirus host range and symptoms
The surveys and molecular detection of turncurtoviruses revealed that in addition to turnip and radish [7, 36], a number of crops in vicinity of turnip-growing farms are hosts to turncurtoviruses (25 out of 220 samples). With the exception of turnip, turncurtoviruses were identified in eggplant showing mild yellowing (Fig. 1A), basil (Ocimum basilicum L.) with symptoms of dwarfing and severe yellowing (Fig. 1B), radish showing inward rolling of the leaf margins, brittle leaves and swelling of veins on the lower leaf surfaces (Fig. 1C), lettuce showing dark green vein banding (Fig. 1D), and sugar beet, red beet and spinach with typical curly top symptoms (Fig. 1E, F and G, respectively).
Turncurtoviruses were identified in Beta vulgaris (n = 4), B. vulgaris subsp. esculenta (n = 1), Brassica rapa var. rapa (n = 13), Lactuca sativa (n = 4), Ocimum basilicum (n = 1) and Raphanus sativus (n = 2). Taking into account previously identified turncurtoviruses, the amongst collected samples, the dominant host is B. rapa (n = 32; n = 13 from this study and n = 19 from previous studies) (Table 1).
Diversity of turncurtoviruses
Twenty-five turncurtovirus genomes were recovered, and their sequences were analyzed together with 20 turncurtovirus genomes from previous studies. A pairwise identity analysis revealed that 12 of the turncurtovirus genomes from this study grouped with TCTV isolates, sharing >85 % pairwise identity. In addition, a group of 13 turncurtoviruses recovered from B. rapa var. rapa, L. sativa, B. vulgaris subsp. esculenta and B. vulgaris shared <80 % pairwise identity with TCTV isolates and had among them a diversity of ~17 % (TCTV isolates showed 15 % diversity; Fig. 2). We analyzed 991 pairwise comparisons of these turncurtovirus isolates, and the distribution of these identities shows a trough between 81-82 % and 95 % (Fig. 2). We recommend that turncurtoviruses sharing less than 80 % pairwise identity to known turncurtoviruses be considered members of tentative new species. The diverse group was well supported in the neighbor-joining phylogenetic tree (Fig. 3). Based on the phylogenetic support coupled with the distribution of pairwise identities and rough recommendations set out by Varsani et al. [41], these diverse isolates would be classified as members of a different species, and we propose the name turnip leaf roll virus (TLRV) for this virus.
Further, the 12 TCTV sequences from this study can be grouped, as outlined by Razavinejad et al. [36] and Varsani et al. [41], into the existing strains TCTV-B (n = 3), TCTV-C (n = 3), TCTV-D (n = 2) and the new strain TCTV-E (n = 4). Similarly, the 13 TLRV sequences can be grouped into the four strains TLRV-A (n = 4), TLRV-B (n = 1), TLRV-C (n = 1) and TLRV-D (n = 7).
Overall, based on the 45 genomes of turncurtoviruses, TCTV-A (n = 7), TCTV-C (n = 7), TCTV-D (n = 3), TLRV-A (n = 4) and TLRV-B (n = 1) have only been recovered from B. rapa (Table 1).
Inter- and intraspecies recombination in turncurtoviruses
Amongst the turncurtoviruses, evidence of recombination was found for almost all isolates, with recombinant regions accounting for 5 to 40 % of the genome. In total, nine well-supported detectable recombination events were detected (summarized in Fig. 4), with up to three recombinant regions in a genome. The genomes of TLRV-A and TLRV-C isolates were found to have a ~400-nt recombinant region derived from TCTV.
Nonetheless, taking recombination into account, it is clear from the recombination-free maximum-likelihood phylogenetic tree that TCTV and TLRV are members of two distinct species and that the bulk of the diversity between these two viruses arose primarily by recombination (Fig. 4).
Analysis of CP and Rep sequences revealed that the CPs of both TCTV and TLRV are more conserved than Rep (Fig. 5). The Rep sequences of TCTV and TLRV isolates share >87 % and >74 % pairwise amino acid sequence identity, respectively, whereas the CP sequences of TCTV and TLRV isolates share >88 % and >95 % pairwise amino acid sequence identity, respectively. Overall, the CP sequences of turncurtoviruses show 30 % diversity, whereas the Rep sequences have a diversity of 38 %.
Agroinfection studies with the infectious clone of TLRV-A[IR:Zaf:Z11:12]
Agroinoculation of all of the hosts with the infectious clone of TLRV-A[IR:Zaf:Z11:12] resulted in systemic infection only in turnip (7/10), radish (3/16), rapeseed (5/10), thale cress (3/13) and N. benthamiana (10/17). Infected plants of turnip and radish showed inward rolling of the leaf margin and swelling of veins on the lower leaf surfaces (Fig. 6A and B). For N. benthamiana, thale cress and rapeseed, virus could be detected by PCR in systemic top leaves, but no symptoms were observed on the plants. Agroinoculated cowpea seedlings (5/15) showed formation of local lesions on the leaves (Fig. 6C), and virus was not detected in top non-inoculated leaves. Viral infection was not detected in cabbage, sugar beet, red beet, spinach, pepper, okra, eggplant, lettuce, tomato and canola.
Leafhopper transmission of TLRV-A[IR:Zaf:Z11:12]
Nymphs and/or adults of C. haematoceps successfully transmitted TLRV-A[IR:Zaf:Z11:12] from infected turnip plants to 83.3 % (30/36) and 72.2 % (13/18) of the turnip and radish seedlings, respectively. Leafhopper-inoculated plants showed typical turncurtovirus symptoms similar to those of agroinoculated plants. The turncurtovirus infection was confirmed by PCR using back-to-back primers.
Discussion
Amongst geminiviruses, host range varies from wide to restricted [8]. While beet curly top virus (BCTV) has a broad host range, horseradish curly top virus infects only cruciferous species [10]. Prior to this study, it was believed that in contrast to other reported curly top viruses from Iran, i.e., BCTV and beet curly top Iran virus (BCTIV), TCTV would have a limited natural host range [2, 13, 35]. Based on the results of this study, in addition to the main host (turnip), TCTV and/or TLRV were detected in radish, sugar beet, red beet, lettuce, eggplant, spinach and basil. However, most of these plants, i.e., sugar beet, red beet, lettuce, eggplant and spinach, did not become infected after agroinoculation with TLRV-A [IR:Zaf:Z11:12] in the greenhouse. Furthermore, our earlier studies have shown that, besides turnip, radish and sugar beet, the infectious clone of TCTV-A[IR:Zaf:B11:06] could not establish an infection in other experimental test plants, and only 6.7 % of the agroinoculated sugar beet seedlings became infected [36]. The reason may be due to different cultivars used in the field as compared to the greenhouse studies. In addition, the reaction of these plants was tested against only one TLRV genotype (TLRV-A[IR:Zaf:Z11:12] isolate) using agroinoculation. Therefore, due to the genetic variability of turncurtovirus isolates, the infectivity of agroinoculated clones may differ between TCTV and TLRV genotypes.
Iran is a part of the Old World with a long history of agriculture coupled with hot and dry climatic conditions. The climate is influenced by Iran’s location between the subtropical aridity of the Arabian Desert areas and the subtropical humidity of the eastern Mediterranean area. This is favorable for crop cultivation, but also for the leafhopper C. haematoceps [23, 43], the vector of several curtoviruses, becurtoviruses and turncurtoviruses [21, 22, 35]. Four geminiviruses causing curly top symptoms, i.e., BCTV, BCTIV, TCTV and TRLV, have been reported from sugar beet and turnip of farms in Iran [4, 7, 15], and all can be vectored by C. haematoceps (Fig 7). Adaptation of vector and virus to a system with long-term agricultural activities provides suitable conditions for variability and emergence of distinct viruses, and it appears that the Middle East region is a diversity hotspot for the emergence of new curly top viruses where recombination is playing a significant role in generating diversity.
References
Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21:2104–2105
Bennett CW (1971) The curly top disease of sugarbeet and other plants. Monograph 7. American Phytopathological Society, St Paul
Bernardo P, Golden M, Akram M, Naimuddin Nadarajan N, Fernandez E, Granier M, Rebelo AG, Peterschmitt M, Martin DP, Roumagnac P (2013) Identification and characterisation of a highly divergent geminivirus: evolutionary and taxonomic implications. Virus Res 177:35–45
Bolok Yazdi HR, Heydarnejad J, Massumi H (2008) Genome characterization and genetic diversity of beet curly top Iran virus: a geminivirus with a novel nonanucleotide. Virus Genes 36:539–545
Boni MF, Posada D, Feldman MW (2007) An exact nonparametric method for inferring mosaic structure in sequence triplets. Genetics 176:1035–1047
Böttcher B, Unseld S, Ceulemans H, Russell RB, Jeske H (2004) Geminate structures of African cassava mosaic virus. J Virol 78:6758–6765
Briddon RW, Heydarnejad J, Khosrowfar F, Massumi H, Martin D, Varsani A (2010) Turnip curly top virus, a highly divergent geminivirus infecting turnip in Iran. Virus Res 152:169–175
Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, Navas-Castillo J (2012) Geminiviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy. Ninth report of the ICTV. Elsevier/Academic Press, London, pp 351–373
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772
Duffus JE, Milbrath GM, Perry R (1982) Unique type of curly top virus and its relationship with horseradish brittle root. Plant Dis 66:650–652
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797
Farzadfar Sh, Pourrahim R (2013) Wild radish (Raphanus raphanistrum) a new host for Turnip curly top virus. In: 7th international geminivirus symposium and 5th international ssDNA comparative virology workshop, Hangzhou. http://www.geminivirus.org/doc/Program_and_Abstracts.pdf (Accessed 7 Jan 2014)
Gharouni Kardani S, Heydarnejad J, Zakiaghl M, Mehrvar M, Kraberger S, Varsani A (2013) Diversity of Beet curly top Iran virus isolated from different hosts in Iran. Virus Genes 46:571–575
Gibbs MJ, Armstrong JS, Gibbs AJ (2000) Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16:573–582
Gibson KE (1967) Possible incidence of curly top in Iran a new record. Plant Dis 51:976–977
Grimsley N, Hohn B, Hohn T, Walden R (1986) Agroinfection, an alternative route for viral-infection of plants by using the Ti plasmid. Proc Natl Acad Sci USA 83:3282–3286
Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321
Haible D, Kober S, Jeske H (2006) Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses. J Virol Methods 135:9–16
Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832
Heydarnejad H, Keyvani N, Razavinejad S, Massumi H, Varsani A (2013) Fulfilling the Koch’s postulate for beet curly top Iran virus and proposal for consideration of new genus in the family Geminiviridae. Arch Virol 158:435–443
Hosseini Abhari E, Heydarnejad J, Massumi H, Hosseini Pour A, Izadpanah K (2005) Natural hosts, vector and molecular detection of Beet curly top virus (BCTV) in Southeast of Iran. In: The second Asian conference on plant pathology, Singapore, p 62
Kheyri M, Alimoradi I, Davatchi A (1969) The leafhoppers of sugar beet in Iran and their role in curly top virus disease. Sugar beet Seed Inst, Agric College, Karaj
Klein M (1992) Role of Circulifer/Neoaliturus in the transmission of plant pathogens. In: Harris KF (ed) Advances in disease vector research, vol 9. Springer, New York, pp 151–193
Krenz B, Thompson JR, Fuchs M, Perry KL (2012) Complete genome sequence of a new circular DNA virus from grapevine. J Virol 86:7715
Liang P, Navarro B, Zhang Z, Wang H, Lu M, Xiao H, Wu Q, Zhou X, Di Serio F, Li S (2015) Identification and characterization of a novel geminivirus with monopartite genome infecting apple trees. J Gen Virol. doi:10.1099/vir.0.000173
Loconsole G, Saldarelli P, Doddapaneni H, Savino V, Martelli GP, Saponari M (2012) Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae. Virology 432:162–172
Ma Y, Navarro B, Zhang Z, Lu M, Zhou X, Chi S, Di Serio F, Li S (2015) Identification and molecular characterization of a novel monopartite geminivirus associated with mulberry mosaic dwarf disease. J Gen Virol. doi:10.1099/vir.0.000175
Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P (2010) RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26:2462–2463
Martin DP, Murrell B, Golden M, Khoosal A, Muhire B (2015) RDP4: detection and analysis of recombination patterns in virus genomes. Virus Evol 1:1–5. doi:10.1093/ve/vev003
Martin DP, Posada D, Crandall KA, Williamson C (2005) A modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints. AIDS Res Hum Retroviruses 21:98–102
Martin D, Rybicki E (2000) RDP: detection of recombination amongst aligned sequences. Bioinformatics 16:562–563
Muhire BM, Varsani A, Martin DP (2014) SDT: A virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One 9:e108277
Padidam M, Sawyer S, Fauquet CM (1999) Possible emergence of new geminiviruses by frequent recombination. Virology 265:218–225
Posada D, Crandall KA (2001) Evaluation of methods for detecting recombination from DNA sequences: computer simulations. Proc Natl Acad Sci USA 98:13757–13762
Razavinejad S, Heydarnejad J (2013) Transmission and natural hosts of Turnip curly top virus. Iran J Plant Pathol 49:83–91
Razavinejad S, Heydarnejad J, Kamali M, Massumi H, Kraberger S, Varzani A (2013) Genetic diversity and host range studies of turnip curly top virus. Virus Genes 46:345–353
Roumagnac P, Granier M, Bernardo P, Deshoux M, Ferdinand R, Galzi S, Fernandez E, Julian C, Abt I, Filloux D, Mesléard F, Varsani A, Blanc S, Martin DP, Peterschmitt M (2015) Alfalfa leaf curl virus: an aphid transmitted geminivirus. J Virol 89:9683–9688
Shepherd DN, Martin DP, Lefeuvre P, Monjane AL, Owor BE, Rybicki EP, Varsani A (2008) A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. J Virol Methods 149:97–102
Smith JM (1992) Analyzing the mosaic structure of genes. J Mol Evol 34:126–129
Varma A, Malathi VG (2003) Emerging geminivirus problems: a serious threat to crop production. Ann Appl Biol 142:145–164
Varsani A, Navaz-Castillo J, Moriones E, Hernández-Zepeda C, Idris A, Brown JK, Zerbini FM, Martin DP (2014) Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Arch Virol 159:2193–2203
Varsani A, Shepherd DN, Dent K, Monjane AL, Rybicki EP, Martin DP (2009) A highly divergent South African geminivirus species illuminates the ancient evolutionary history of this family. Virol J 6:36
Young DA, Frazier NW (1954) A study of the leafhoppers genus Circulifer Zakhvatkin (Homoptera: Cicadellidae). Hilgardia 23:25–52
Zhang W, Olson NH, Baker TS, Faulkner L, Agbandje-McKenna M, Boulton MI, Davies JW, McKenna R (2001) Structure of the Maize streak virus geminate particle. Virology 279:471–477
Zhang YP, Uyemoto JK, Kirkpatrick BC (1998) A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay. J Virol Methods 71:45–50
Acknowledgments
This study was supported by a grant from Shahid Bahonar University of Kerman, Kerman, Iran, and a block grant from the University of Cape Town, South Africa, awarded to Arvind Varsani. The authors would like to thank Zerina Beslija for excellent technical assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kamali, M., Heydarnejad, J., Massumi, H. et al. Molecular diversity of turncurtoviruses in Iran. Arch Virol 161, 551–561 (2016). https://doi.org/10.1007/s00705-015-2686-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00705-015-2686-6