1 CIRAD, UMR C53 PVBMT, CIRAD-Université de la Réunion, Pôle de Protection des Plantes, Ligne Paradis, 97410 Saint Pierre, Réunion, France
2 Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory 7925, South Africa
3 Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
4 CIRAD, UMR BGPI, CIRAD-INRA, TA 41/K, 34398 Montpellier Cedex 5, France
Correspondence
Jean-Michel Lett
lett{at}cirad.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AJ865337AJ865341.
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INTRODUCTION |
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TYLCV is an important tomato pathogen that, following its emergence from the Mediterranean Basin in recent years (Moriones & Navas-Castillo, 2000), is progressively spreading throughout the world (Cohen & Antignus, 1994
; Czosnek & Laterrot, 1997
; Moriones & Navas-Castillo, 2000
; Pico et al., 1996
; Polston et al., 1999
). In 1997, a severe outbreak of tomato yellow leaf curl disease occurred in Réunion, one of the South West islands of the Indian Ocean (SWIO). Yield losses reached 85 % the first year of the epidemic on the most susceptible cultivars (Reynaud et al., 2003
) and the disease has become the primary factor limiting both open field and protected greenhouse tomato production on the island. No begomoviruses had been detected in Réunion tomatoes prior to 1997 and it has now been determined that two strains of an exotic virus, the Israel and the Mild strains of TYLCV, are the causal agents of the disease (Peterschmitt et al., 1999
; Delatte et al., 2005a
). There is precedent for human spread of TYLCV into new habitats, i.e. the Carribean and Florida (Polston et al., 1999
), and the finding that whiteflies can acquire the virus from fruits demonstrates yet another route of potential dissemination (Delatte et al., 2003
). The influx of exotic viruses into SWIO is also not restricted to tomato begomoviruses. Other begomovirus of cassava such as the African cassava mosaic virus (ACMV) (Fauquet & Fargette, 1990
), East African cassava mosaic virus (EACMV) (Swanson & Harrison, 1994
) and South African cassava mosaic virus (SACMV) (Berrie et al., 2001
) have also been detected in Madagascar (Ranomenjanahary et al., 2002
).
In 2001, a tomato virus symptom survey on the islands of Madagascar and Mayotte identified both the association of the begomovirus vector species, Bemisia tabaci, with tomato plants and the presence of plants displaying leaf curling and plant stunting symptoms characteristic of begomoviruses. Analysis of partial viral genome fragments isolated from leaf samples collected during this survey indicated the presence of two potentially new Begomovirus species (Delatte et al., 2002; Lett et al., 2004
).
In this study, we report the construction of agro-infectious viral clones, symptom evaluation, whitefly transmission tests and analysis of the full-length DNA sequences of TYLCV-Mld[RE] and two isolates from two new monopartite begomovirus species. The new species, tentatively named Tomato leaf curl Madagascar virus (ToLCMGV) and Tomato leaf curl Mayotte virus (ToLCYTV), belong to the African begomoviruses but represent a distinctly unique monophyletic group that we refer to as the SWIO group. We report that the SWIO isolates appear to have been actively recombining amongst themselves.
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METHODS |
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Sampling and DNA extraction.
Tomato leaves presenting leaf-curling symptoms were collected from individual plants in Saint Pierre (Réunion), Morondova and Toliary (Madagascar), and Dembeni and Kahani (Mayotte). The leaves were preserved by dehydration with CaCl2 (Bos, 1977). Total DNA was extracted from dried samples using the DNeasy Plant miniprep kit (Qiagen) according to the manufacturer's instructions.
PCR detection.
PCR was used to amplify two fragments from the extracted DNA of all samples using two degenerate primer sets: MP16MP82 (Umaharan et al., 1998) and AV494AC1048 (Wyatt & Brown, 1996
). A less degenerate primer set was designed from previously obtained SWIO begomovirus sequences and used to amplify 904 nt of the core region of the coat protein (CP) gene (VD360CD1266; Table 1
). PCR reactions were carried out in 25 µl volumes with the following programme: a cycle of 5 min at 94 °C, then 30 cycles at 95 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and a final cycle at 72 °C for 5 min. The presence/absence of a DNA B genome component was also determined for each of the isolates using the PCR primers: PBL1v2040 and PCRc1 (Rojas et al., 1993
; Table 1
). The presence/absence of DNA
molecules was determined for each of the isolates using the primers Beta 1 and Beta 2 (Briddon et al., 2002
; Table 1
).
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Agro-inoculation.
While the infectivity of the cloned DNA components of isolates from Réunion and Madagascar were tested using full head-to-tail DNA dimers (constructed at BamHI restriction sites), partial DNA head-to-tail dimers were constructed (at HindIII restriction sites) for the viruses from Mayotte. Both full and partial dimers were inserted into the binary vector, pCAMBIA 2300 (Cambia). Recombinant plasmids were mobilized from Escherichia coli JM-109 cells (Promega) into Agrobacterium tumefaciens (strain C58) by triparental mating using E. coli HMB101 containing the plasmid helper pRK 2013 (Ditta et al., 1980). The identity of all clones was verified by restriction endonuclease analysis. Ten day old susceptible tomato seedlings were agro-inoculated with the five constructs using a needle (Paximadis & Rey, 2001
), and symptoms of infection were evaluated between 15 and 20 days post-inoculation. All the agro-inoculated plants showing symptoms were tested for the presence of viral DNA using either specific degenerate primers designed to amplify the isolates from the two new species (V360CD1266; Table 1
) or a specific non-degenerate primer designed to amplify TYLCV DNA (V196C1000; Table 1
).
Transmission tests.
B. tabaci transmissibility of the viruses was evaluated by determining whether whiteflies could successfully transmit the viruses from symptomatic PCR-positive agro-inoculated plants to healthy tomato plants. B. tabaci adults used for the transmission tests were from a cabbage reared B biotype population initiated from whiteflies initially collected from cabbage in Réunion (Delatte et al., 2005b). In each transmission test, 15 whiteflies were permitted a 3 day acquisition access period on PCR-positive symptomatic agro-inoculated tomato plants. These insects were then transferred onto healthy tomato plants and allowed an inoculation access period (IAP) of 3 days. Twenty-one days following the IAP, symptoms were evaluated and symptomatic plants tested for the presence of DNA by PCR, using the specific primers described above (V360CD1266; V196C1000; Table 1
).
Sequence analysis.
The full DNA sequences of the five isolates were arranged so that the first nucleotide in the sequence corresponds to the first base (A) of virion strand replication (Laufs et al., 1995). Potential open reading frames in each of the isolate sequences were identified using DNAMAN (version 5.2.2, Lynnon Biosoft). Full DNA A-like and A sequences of related viruses used in phylogenetic analyses were obtained from public sequence databases using BLASTN. Two outgroup sequences were used during phylogenetic analyses, an isolate from Australia of the monopartite species Tomato leaf curl virus isolate (GenBank accession no. S53251; Stonor et al., 2003
) and an isolate from Florida of the bipartite species Tomato mottle virus isolate (NC_001938; Polston et al., 1993
). Multiple sequence alignments were performed using the optimal alignment method of DNAMAN. Phylogenetic trees were generated using the neighbour-joining method of PHYLIP (Felsenstein, 1989
) or the JukesCantor corrected distances, 2000 bootstrap replicates were performed.
Detection of potential recombinant sequences, identification of likely parental sequences and localization of possible recombination breakpoints in multiple sequence alignments were carried out using the RDP (Martin & Rybicki, 2000), GENECONV (Padidam et al., 1999
), MAXIMUM
2 (Smith, 1992
), CHIMAERA (Martin et al., 2005a
), RECSCAN (Martin et al., 2005a
) and SISTER SCAN (Gibbs et al., 2000
) methods as implemented in RDP2 (Martin et al., 2005b
). The analysis was performed with default settings for the different detection methods and a Bonferroni corrected P-value cut-off of 0·05.
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RESULTS |
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We detected three anomalies in the nucleotide sequence of ToLCMGV-[Tol] that might explain lack of infectivity of its clone. The first, and potentially most serious, is a single nucleotide frame-shift mutation near the beginning of the V2 ORF. The other anomalies were two unusual termination codons in the C4 ORF. For purposes of comparing the putative ToLCMGV-[Tol] V2 and C4 amino acid sequences with those of other viruses (Tables 2 and 3), we corrected the sequence by inserting a T nucleotide at position 282, and changing an A at position 2163 and an A at position 2376 to a G and a C, respectively.
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The highest nucleotide identity of DNA detected between isolates of ToLCMGV and ToLCYTV, was 86 % when comparing ToLCMGV-[Mor] and ToLCYTV-[Dem]. The greatest degree of genome-wide sequence identity shared by ToLCMGV and ToLCYTV isolates with other currently described full-length sequences was 82 % for ToLCMGV-[Mor] with TYLCV-Mld and 83 % for ToLCYTV-[Dem] with SACMV (Fig. 2). We therefore propose that, according to the ICTV criteria for begomovirus species demarcation using DNA complete sequence (Fauquet et al., 2003
; Fauquet & Stanley, 2003
), both ToLCMGV and ToLCYTV be considered new species as their nucleotide identities with other begomovirus are below 89 %.
Analysis of recombination
We analysed a 62-sequence alignment of full-length SWIO (Table 2), African and Mediterranean begomovirus DNA sequences for evidence that the SWIO isolates had undergone recombination. We initially screened the alignment looking for all evidence of recombination involving the SWIO isolates either as potential recombinants (i.e. as acceptors of sequence) or as parental donors of sequence in non-SWIO recombinants. Six different detection methods identified an enormous amount of evidence for recombination involving the SWIO sequences as either donors or acceptors of sequences (at least 130 unique events identified by RDP2). We analysed each of the identified events individually and used a phylogenetic approach to verify the parental/donor identifications made by RDP2. This involved construction and comparison of bootstrapped neighbour-joining trees from the two portions of the alignment corresponding to regions of potential recombinants originating from different parental sequences. Wherever there was good phylogenetic evidence that an inferred recombination event involved an SWIO isolate as a donor sequence (i.e. there was little or no evidence that the SWIO isolate was the recombinant), we marked the recombinant region in the non-SWIO recombinant sequence for later removal. Having examined all events with associated P-values <1·0x106 (i.e. the most obvious events), we removed all the identified evidence of non-SWIO isolate recombination from the alignment. This was carried out by treating the identified recombinant region in the recombinant sequence as missing data in subsequent analyses. We scanned the four SWIO isolates in pairs (i.e. six pairs in total) against the rest of the sequences in the alignment. Following identification of the more obvious recombination events (events identified with multiple comparison corrected P-values <1·0x105) that involved SWIO isolates as acceptors of sequence (determined phylogenetically as described above) and removal of the identified recombinant regions from the alignment (also as described above), the six SWIO isolate pairs were screened one last time against the rest of the alignment for the least obvious detectable events.
It was apparent from this analysis that all of the SWIO isolates together bear detectable evidence of at least 15 past recombination events (Fig. 4). In all isolates other than ToLCMGV-[Tol] we detected a complex mosaic of sequences in an
350 nt region spanning sequences encoding the N-terminal portion of Rep. Whereas there is statistically significant evidence that this region of the ToLCYTV-[Dem] sequence has three distinct origins, it has at least four distinct origins in both ToLCYTV-[Kah] and ToLCMGV-[Mor]. Importantly, in all cases the parental sequences identified were one of the SWIO isolates and a sequence only distantly related to previously characterized mainland African begomovirus isolates (either listed as unknown or with a
prefix in Fig. 4
).
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DISCUSSION |
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We have demonstrated the infectivity and whitefly transmissibility of cloned DNA sequences for three of the four SWIO isolates. Our inability to detect either DNA B or DNA in source leaf material, and the induction of leaf curling and stunting symptoms in agro-inoculated tomato plants similar to those observed in the field in the absence of these other genome components, indicates that the SWIO viruses are most likely monopartite.
Recently, a new biotype (Ms) of B. tabaci has been identified on Madagascar and other SWIO (Delatte et al., 2005b). Although biotype Ms is genetically closely related to the B. tabaci B and Q biotypes, it has been estimated that biotype Ms diverged from biotype B and Q as long as 3 (±0·3) million years ago. It is possible that the SWIO viruses have evolved in relative isolation for a similar period and it will be interesting to determine whether the SWIO isolates have any transmission advantage relative to mainland African and Mediterranean isolates in biotype Ms.
The results of our recombination analysis support the fact that the SWIO viruses may have been evolving in relative isolation for a prolonged period. Had there been substantial influx of mainland begomovirus isolates onto the islands it would be expected that genetic exchange between mainland and island isolates would be detectable. Such exchanges are, for example, easily detectable both amongst and between divergent African and Mediterranean isolates (Padidam et al., 1999). None of the sequences within the recombinant regions identified in the SWIO isolates closely resembled that of any known non-SWIO begomovirus, indicating that genetic exchange in these viruses has most likely been limited to that occurring between relatively unique island isolates. It is important to note, however, that the recombination analysis does not preclude the possibility of genetic exchange between viruses on different islands. In fact, there is highly significant evidence that, firstly, an 856 bp fragment of the Madagascar isolate, ToLCMGV-[Tol], originated from a virus closely resembling the Mayotte isolate, ToLCYTV-[Dem] (P-value=2·3x108) (Fig. 4
), and, secondly, that a 122 bp fragment of ToLCYTV-[Dem] originated from a virus closely resembling the Madagascar isolate, ToLCMGV-[Mor] (P-value=1·7x1010). When and where these potential recombination events occurred is an open question but it cannot be discounted that both ToLCYTV and ToLCMGV isolates might occur on both islands.
This study highlights the need for further sampling and monitoring of begomovirus diversity in both tomato and non-tomato hosts on the SWIO such as Madagascar, Mayotte and the Comoros archipelago. Such activities would almost certainly lead to the identification of more novel species and provide early warning of the presence of newly imported and potentially dangerous exotic begomoviruses. Many of the SWIO are small enough that repetitive and reasonably exhaustive begomovirus surveys on them are feasible. Isolated begomovirus populations on the smaller, remote SWIO such as Mayotte could provide one of the last and best remaining opportunities to non-destructively test begomovirus evolutionary hypotheses and population genetic models. Continuous maintenance of sampling projects on these islands might also provide opportunities for testing begomovirus epidemiological models whenever importation of exotic viruses to these islands does occur.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bos, L. (1977). Persistance of infectivity of three viruses in plant material dried over CaCl2 and stored under different conditions. Neth J Plant Pathol 83, 217220.
Briddon, R. W., Bull, S. E., Mansoor, S., Amin, I. & Markham, P. G. (2002). Universal primers for the PCR-mediated amplification of DNA : a molecule associated with some monopartite begomoviruses. Mol Biotechnol 20, 315318.[CrossRef][Medline]
Cohen, S. & Antignus, Y. (1994). Tomato yellow leaf curl virus (TYLCV), a whitefly-borne geminivirus of tomatoes. Adv Dis Vector Res 10, 259288.
Czosnek, H. & Laterrot, H. (1997). A worldwide survey of tomato yellow leaf curl viruses. Arch Virol 142, 13911406.[CrossRef][Medline]
Delatte, H., Reynaud, B., Lett, J. M., Peterschmitt, M., Granier, M., Ravololonandrianina, J. & Goldbach, W. R. (2002). First molecular identification of a begomovirus isolated from tomato in Madagascar. Plant Dis 86, 1404.
Delatte, H., Dalmon, A., Rist, D., Soustrade, I., Wuster, G., Lett, J. M., Goldbach, R. W., Peterschmitt, M. & Reynaud, B. (2003). Tomato yellow leaf curl virus can be acquired and transmitted by Bemisia tabaci (Gennadius) from tomato fruit. Plant Dis 87, 12971300.
Delatte, H., Holota, H., Naze, F., Peterschmitt, M., Reynaud, B. & Lett, J. M. (2005a). The presence of both recombinant and non recombinant strains of Tomato yellow leaf curl virus on tomato in Réunion Island. Plant Pathol 54, 000000.
Delatte, H., Reynaud, B., Granier, M., Thornary, L., Lett, J. M., Goldbach, R. & Peterschmitt, M. (2005b). A new silverleaf-inducing biotype Ms of Bemisia tabaci (Hemiptera: Aleyrodidae) indigenous of the islands of the south-west Indian Ocean. Bull Entomol Res 95, 2935.[CrossRef][Medline]
Ditta, G., Stanfield, S., Corbin, D. & Helsinki, D. R. (1980). Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A 77, 73477351.[Abstract]
Fauquet, C. & Fargette, D. (1990). African cassava mosaic virus: etiology, epidemiology and control. Plant Dis 74, 404411.
Fauquet, C. M. & Stanley, J. (2003). Geminivirus classification and nomenclature: progress and problems. Ann Appl Biol 142, 165189.
Fauquet, C. M., Bisaro, D. M., Briddon, R. W., Brown, J. K., Harrison, B. D., Rybicki, E. P., Stenger, D. C. & Stanley, J. (2003). Revision of taxonomic criteria for species demarcation in the family Geminiviridae, and an updated list of begomovirus species. Arch Virol 148, 405421.[CrossRef][Medline]
Felsenstein, J. (1989). PHYLIP - Phylogeny inference package (version 3.2). Cladistics 5, 164166.
Gibbs, M. J., Armstrong, J. S. & Gibbs, A. J. (2000). Sister-scanning: a Monte Carlo procedure for assessing signals in recombinant sequences. Bioinformatics 16, 573582.[Abstract]
Laufs, J., Traut, W., Heyraud, F., Matzeit, V., Rogers, S. G., Schell, J. & Gronenborn, B. (1995). In vitro cleavage and joining at the viral origin of replication by the replication protein of the tomato yellow leaf curl virus. Proc Natl Acad Sci U S A 92, 38793883.
Lett, J. M., Delatte, H., Naze, F., Reynaud, B., Abdoul-Karime, A. L. & Peterschmitt, M. (2004). A new tomato begomovirus from Mayotte. Plant Dis 88, 681.
Martin, D. & Rybicki, E. (2000). RDP: detection of recombination amongst aligned sequences. Bioinformatics 16, 562563.[Abstract]
Martin, D. P., Posada, D., Crandall, K. A. & Williamson, C. (2005a). A modified bootscan algorithm for automated identification of recombinant sequences and recombination breakpoints. AIDS Res Hum Retroviruses 21, 98102.[CrossRef][Medline]
Martin, D. P., Williamson, C. & Posada, D. (2005b). RDP2: recombination detection and analysis from sequence alignments. Bioinformatics 21, 260262.
Moriones, E. & Navas-Castillo, J. (2000). Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Res 71, 123134.[CrossRef][Medline]
Navot, N., Pichersky, E., Zeidan, M., Zamir, D. & Czosnek, H. (1991). Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology 185, 151161.[CrossRef][Medline]
Padidam, M., Sawyer, S. & Fauquet, C. M. (1999). Possible emergence of new geminiviruses by frequent recombination. Virology 265, 218225.[CrossRef][Medline]
Patel, V. P., Rojas, M. R., Paplomatas, E. J. & Gilbertson, R. L. (1993). Cloning biologically active geminivirus DNA using PCR and overlapping primers. Nucleic Acids Res 21, 13251326.[Medline]
Paximadis, M. & Rey, M. E. C. (2001). Genome organization of Tobacco leaf curl Zimbabwe virus, a new distinct monopartite begomovirus associated with subgenomic defective DNA molecules. J Gen Virol 82, 30913097.
Peterschmitt, M., Granier, M., Mekoud, R., Dalmon, A., Gambin, O., Vayssières, J. F. & Reynaud, B. (1999). First report of tomato yellow leaf curl virus in Réunion Island. Plant Dis 83, 303.
Pico, B., Diez, M.-J. & Nuez, F. (1996). Viral diseases causing the greatest economic losses to the tomato crop. II. The tomato yellow leaf curl virus - a review. Sci Hortic 67, 151196.[CrossRef]
Polston, J. E., Hiebert, E., McGovern, R. J., Stansly, P. A. & Schuster, D. J. (1993). Host range of tomato mottle virus, a new geminivirus infecting tomato in Florida. Plant Dis 77, 11811184.
Polston, J. E., McGovern, R. J. & Brown, L. G. (1999). Introduction of tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato. Plant Dis 83, 984988.
Ranomenjanahary, S., Rabindran, R. & Robinson, D. J. (2002). Occurrence of three distinct begomoviruses in cassava in Madagascar. Ann Appl Biol 140, 315318.
Reynaud, B., Wuster, G., Delatte, H., Soustrade, I., Lett, J. M., Gambin, O. & Peterschmitt, M. (2003). Les maladies à bégomovirus chez la tomate dans les départements français d'Outre-Mer. Le Tomato yellow leaf curl virus (TYLCV) à la Réunion. Phytoma 562, 1317.
Rojas, M. R., Gilbertson, R. L., Russel, D. R. & Maxwell, D. P. (1993). Use of degenerate primers in the polymerase chain reaction to detect whitefly-transmitted geminivirus. Plant Dis 77, 340347.
Smith, J. M. (1992). Analyzing the mosaic structure of genes. J Mol Evol 34, 126129.[Medline]
Stonor, J., Hart, P., Gunther, M., DeBarro, P. & Rezaian, M. A. (2003). Tomato leaf curl geminivirus in Australia: occurrence, detection, sequence diversity and host range. Plant Pathol 52, 379388.[CrossRef]
Swanson, M. M. & Harrison, B. D. (1994). Properties, relationships and distribution of cassava mosaic geminiviruses. Trop Sci 34, 1525.
Umaharan, P., Padidam, M., Phelps, R. H., Beachy, R. N. & Fauquet, C. M. (1998). Distribution and diversity of geminiviruses in Trinidad and Tobago. Phytopathology 88, 12621268.
Wyatt, S. D. & Brown, J. K. (1996). Detection of subgroup III geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction. Phytopathology 86, 12881293.
Received 3 December 2004;
accepted 21 January 2005.
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