2049 Ariana, Université de Carthage, Tunisie 2 IRD, UMR CBGP (INRA/IRD/Cirad/Montpellier SupAgro), Campus international de Baillarguet,

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1 Journal of Plant Pathology (2014), 96 (1), Edizioni ETS Pisa, 2014 Mnari-Hattab et al. 195 Short Communication NATURAL OCCURRENCE OF BEGOMOVIRUS RECOMBINANTS ASSOCIATED WITH TOMATO YELLOW LEAF CURL DISEASE CO-EXISTING WITH PARENTAL VIRUSES IN TOMATO CROPS AND WEEDS IN TUNISIA M. Mnari-Hattab 1, S. Zammouri 1, F. Pellegrin 2 and N. Gauthier 2 1 Laboratoire de Biotechnologie appliquée à l agriculture, Institut National de la Recherche Agronomique de Tunisie, 2049 Ariana, Université de Carthage, Tunisie 2 IRD, UMR CBGP (INRA/IRD/Cirad/Montpellier SupAgro), Campus international de Baillarguet, CS 30016, F Montferrier-sur-Lez cedex, France SUMMARY Tomato yellow leaf curl virus disease (TYLCVD) is the main limiting factor for tomato production in the Mediterranean Basin, and particularly in Tunisia where climatic and crop production conditions, as well as the occurrence of many weeds, provide suitable conditions for the presence and spread of TYLCVD all year round. In Tunisia, epidemics in tomato-growing regions have been associated with two begomoviruses: Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) but the presence of recombinants between them has never been investigated. A large-scale survey was conducted on leaves sampled from late-season tomato crops exhibiting severe curling symptoms, from beans, and from nine nearby weed species in the South and Sahel regions of Tunisia to investigate: (i) the natural occurrence of the TYLCV and TYLCSV species; (ii) the presence of the two recombinant types, RecA and RecB, derived from these species. Identification of TYLCV and TYLCSV was based on a multiplex PCR with primers generating a typical pattern on gels (750 bp and 366 bp fragments, respectively). Recombinants were identified by simplex PCR, which amplified the intergenic region, the most common recombination site described for begomoviruses, and sequencing. The occurrence of TYLCSV and TYLCV was confirmed in both Tunisian regions. Their co-existence was shown on tomato and in new weed species. This is the first time that recombinants between TYLCV and TYLCSV species showing 93% to nucleotide identity with tomato yellow leaf curl recombinant viruses, are reported in Tunisia in tomato and black nightshade. Key words: Tomato, Begomovirus, TYLCV, TYLCSV, recombinant virus. Corresponding author: M. Mnari Hattab Fax: hattab.monia@iresa.agrinet.tn; mmnari@gmail.com Tomato yellow leaf curl disease (TYLCVD) is one of the most devastating disorders of tomato crops worldwide (Czosnek and Laterrot, 1997) which can also affect many other crops and weeds (Brown et al., 2011). It causes serious damage and severely constrains production of susceptible crops, particularly in some Mediterranean areas where suitable climatic conditions in addition to complementary agricultural structures allow year-round crop production. There, TYLCVD is more likely to entrench and spread. In Tunisia, production of ornamentals and vegetables, particularly tomato (Solanum lycopersicum L.), is particularly constrained by TYLCD which causes significant yield losses in the main tomato-growing regions (Cherif and Russo, 1983). The tomato crop is of major economic importance with a total production of about 26,000 ha/ year (i.e. 17% of the land used for cultivating vegetables) (Anonymous, 2010). From November to June, tomatoes are mainly produced in geothermally heated greenhouses in the south of the country and in unheated tunnels in the Sahel. During the rest of the year, tomatoes are mainly grown in open fields in Cap Bon and in the central region. The causal agents of this whitefly-transmitted virus disease are members of a virus complex belonging to the genus Begomovirus, family Geminiviridae, which are also found on a wide range of host plant species worldwide. Until recently, TYLCVD epidemics in Mediterranean countries were mainly associated with two viruses: Tomato yellow leaf curl virus (TYLCV) first described in Israel, and Tomato yellow leaf curl Sardinia virus (TY- LCSV) (Kheyr-Pour et al., 1991). Distinct strains of the same virus species and/or different virus species can be associated with TYLCVD either as single or recombinant forms (Brown et al., 2011). The begomoviruses associated with TYLCVD are characterized by small circular single-stranded (ss) DNA genomes which replicate in their host cell via double stranded intermediates by a rolling-circle mechanism analogous to that used by ssdna phages (Saunders et al., 1991; Stanley, 1995) and by recombination-dependent replication (RDR) as confirmed

2 196 Begomovirus recombinants in tomato Journal of Plant Pathology (2014), 96 (1), Table 1. Results of the survey conducted in the late tomato crop season in the Sahel and southern regions of Tunisia, in 2009, with TYLCV species and detection of recombinants. Region Plant species TYLSCV (366 bp) Single infection by Mixed infection Recombinant TYLCV (750 bp) TYLSCV and TYLCV (366 bp and 750 bp) TYLSCV/TYLCV (570bp) GenBank accession Nos. Vegetables Tomato (Solanum lycopersicum) 1/63 8/63 46/63 8/63 a reca GU Faba bean (Vicia faba) 0/4 0/4 3/4 Pepper (Capsicum annuum) 0/5 0/5 5/5 Sahel Weeds Solanum nigrum 0/19 6/19 12/19 0/19 Lantana camara 0/1 0/1 1/1 Malva parviflora 0/3 0/3 3/3 Sisymbrium irio 0/2 0/2 2/2 Emex spinosa 0/4 0/4 3/4 Melilotus officinalis 0/1 0/1 1/1 Sonchus arvensis 0/1 0/1 0/1 Amaranthus lividus 0/1 0/1 1/1 Vegetables Tomato (Solanum lycopersicum) 2/8 0/8 4/8 0/8 Faba bean (Vicia faba) 1/1 0/1 0/1 South Weeds Solanum nigrum 1/4 0/4 3/4 Solanum nigrum 5/6 0/6 1/6 1 reca GU Chenopodum album 0/1 0/1 1/1 Total number of positive samples observed/tested 10/124 14/124 86/124 9 reca a rec = recombinant by Jeske et al. (2001). Their genome is also characterized by the presence of an intergenic region (IR) of about 300 nucleotides including a stem-loop and a conserved nonanucleotide sequence (TAATATTAC) placed in this loop, where the breaking and joining site for rolling-circle replication occurs (Hanley-Bowdoin et al., 2000). This IR is a well-known recombination site in geminiviruses (Stanley, 1995; Navas-Castillo et al., 2000; Monci et al., 2002; García-Andrés et al., 2006), particularly in the recombinant progeny of TYLCV and TYLCSV (Garcia-Andrés et al., 2007; Davino et al., 2009). In Tunisia, TYLCVD was first reported in 1983 (Cherif and Russo, 1983). The associated virus was first observed by electron microscopy and the cytological changes observed were similar to those previously observed in diseased tomato plants suffering from tomato yellow leaf curl disease in Israel (Cherif and Russo, 1983). The presence of TYLCSV species was then identified through molecular characterisation from symptomatic tomato plants collected in 2001 and 2002 (Fekih-Hassen et al., 2003) in the Sahel and the southern areas of Tunisia. The Tomato yellow leaf curl virus-israel species (TYLCV-[IL]) was molecularly identified in tomato, pepper (Capsicum annuum) and faba bean (Vicia faba) in the 2004 and 2006 growing seasons in different locations of Sahel (Gharsallah-Chouchane et al., 2007). Thus, in Tunisia, TYLCVD may be associated with distinct species. Recently, the simultaneous presence of TYLCV and TYLCSV has been reported in mixed infections in the same host plant (Pellegrin et al., 2008), but the occurrence of recombinants between these two viruses had not been looked for. Therefore, in this study we investigated: (i) the occurrence of both viruses (i.e. TYLCSV and TYLCV), alone or together, in Tunisian tomato, faba bean and pepper crops and in nearby weed species; (ii) the presence of potential recombinants between these two parental TYLCV species. Our research has focused on the presence of two recombinant types (namely, RecA and RecB) of TYLCV and TYLCSV, commonly found in nearby Mediterranean countries, particularly in Spain (Garcia- Andrés et al., 2007) and Italy (Davino et al., 2009). To this aim, a large survey was conducted sampling leaves from late field tomato crops exhibiting severe begomovirus-like curling and yellowing symptoms, but also from faba bean and pepper crops and nine weed species showing different degrees of leaf yellowing and curling in the Sahel and southern Tunisian areas in 2009 (Table 1). Leaves from 124 plants sampled were stored separately in a freezer at 20 C until molecular characterisation. Genomic DNA was extracted and purified according to Carling (2004) with slight modifications. About 50 mg of leaf per sample were ground in liquid nitrogen, extracted in 500 µl freshly prepared buffer (Carling, 2004) and incubated for 30 min in a waterbath at 65 C with shaking

3 Journal of Plant Pathology (2014), 96 (1), Mnari-Hattab et al. 197 at 5 min intervals. An equal volume of phenol-chloroform isoamyl alcohol ( ) was then added and centrifuged at 13,000 rpm for 5 min. From this stage on, the protocol used followed that described by Carling (2004). Extracted DNA was quantified using 1% agarose gel and diluted when necessary before amplification. Detection of TYLCSV and TYLCV in our plant sampling was based on multiplex PCR using primers TY209, TY575, TY613, TY1363 (Pellegrin et al., 2008) which generate a typical amplified fragment size pattern (750 bp for TYLCV, 366 bp for TYLCSV) on gels. Detection of potential recombinants between TYLCV and TYLCSV in samples previously shown to be infected by one or both parental viral species (Pellegrin et al., 2008) was based on separate PCRs using primers designed to target the IR (Davino et al., 2008). The following viral DNAs were amplified (primer pairs in brackets): recombinant type A, namely RecA (TY2463+/TY247-) of ca. 570 bp with the sequence from TYLCV to the left and TYLCSV to the right of the recombination site in the IR; recombinant type B, namely RecB (TY2222+/TY255-) of ca. 800 bp with the sequence from TYLCSV to the left and TYLCV to the right of the recombination site, as detailed in Davino et al. (2008). DNA amplicons from samples infected by parental viral forms were compared with the amplicon reference that corresponded to a 570 bp recombinant isolate of TYLCAxV-Sic1-[IT: Sic2/2:04) (GenBank accession No. NC_011024) provided by Dr. G.P. Accotto (Fig. 1). Some amplified products (570 bp) from our samples were either directly sequenced using the primer set (TY2463+/TY247- ) or cloned before sequencing. These amplicons were purified (Wizard PCR DNA purification system, Promega, USA), and ligated into the pgem-t Easy vector (Promega, USA) following the manufacturer s recommendations. Briefly, Escherichia coli competent cells (Promega strain JM 109) were transformed with the resultant recombinant plasmids and cultured onto solid Luria-Bertani medium for blue/white colony selection. Recombinant plasmids were tested by EcoRI digestion, which gives the linear plasmid form and an approximately 600 bp insert fragment, then purified with Qiaprep spin miniprep kit (Qiagen, USA) before sequencing. For each insert, two independent clones were sequenced on both directions using T7 and Sp6 primers. All sequencing was done with an automated ABI3730 DNA sequencer (Applied Biosystems, USA) and the resulting consensus sequences were aligned before being deposited in GenBank and analysed. They were aligned with Clustal W (Thompson et al., 1994) and compared among themselves and with sequences from GenBank database using BLASTn ( Multiplex PCR reactions tested on the 124 leaves collected revealed that 110 samples (including all host plants collected except for Sonchus arvensis) were positive, that is, were infected with TYLCV and/or TYLCSV (Table 1). Plants infected by a single virus species (14 by only 500 bp 250 bp M Fig. 1. Agarose gel electrophoresis of the PCR products obtained after amplification with the primer pair TY247 and TY2463. M: GeneRuler 50 bp DNA ladder from Fermentas (Lithuania). Lane 1, reference positive TYLCAxV-Sic1-[IT: SIC2 / 2:04]; lane 2, negative control; lanes 3 to 8, tomato samples (lane 6: negative sample, other lanes: positive samples); lane 9, blank (bi-distilled sterile water). TYLCV, 10 by only TYLCSV) were more rarely observed than plants infected by both species (86/110 infected). In south Tunisia and Sahel 50 and 73% of symptomatic tomato plants, respectively, were found to be infected by both TYLCV and TYLCSV. Such mixed infections were also observed in the other vegetable crops (faba bean, pepper) but above all, and with a very high occurrence, in all weed species (i.e. except one) collected (Table 1) regardless of the severity of the symptoms shown. Out of the 110 samples infected with TYLCV and/or TYLCSV and tested for a potential recombinant type virus, eight tomato samples and one S. nigrum sample yielded a 570 bp amplicon identical in size to the 570 bp IR amplified fragment of the reference recombinant A (Fig. 1). The amplified product derived from S. nigrum was directly sequenced (accession No. GU322874). Four out of the eight amplified fragments derived from tomato samples were cloned and sequenced (GU GU322873). When aligned and compared to TYLCV and TYLCSV nucleotide sequences retrieved from GenBank (EF and DQ317784, respectively), the five sequences obtained representing recombinant virus sequences RecA respectively, were shown to match the TYLCV sequence to the left of the IR and the TYLCSV sequence to the right of this region characterised by the conserved TAATATTAC nona-nucleotide sequences and the nicking site for initiation of virus DNA replication indicated by an open box in Fig. 2. Significant nucleotide identity (nid) between these five sequences (GU GU322874) and more sequences from GenBank were also found (Table 2). They

4 198 Begomovirus recombinants in tomato Journal of Plant Pathology (2014), 96 (1), Table 2. Characterization of tomato yellow leaf curl recombinant viruses from infected leaves sampled in Tunisia. Percentages of nucleotide identity (nid) and matching zones between the viral sequences obtained in our study and sequences retrieved from GenBank, either parental virus species (TYLCV and TYLCSV) or previously described recombinants. Nucleotide identity between sequences Accession Nos. (this study) Nucleotide positions Virus species and accession Nos. (from GenBank) Nucleotide positions nid GU GU GU GU GU TYLCAxarV DQ TYLCV/TYLCSV recragusa EU TYLCAxarV DQ TYLCV-Mild AJ TYLCMaV DQ TYLCV/TYLCSV a rec Ragusa EU % 97% 93% 96% 100% 93% 97% 94% 94% 96% a rec = recombinant Fig. 2. Multiple sequence alignment of five recombinant species of TYLCV obtained from this study (GU32270-GU32274) and two sequences (TYLCV and TYLCSV) retrieved from GenBank (EF and DQ317784, respectively). The nucleotide sequences have been aligned by introducing gaps (shown as dashes) to maximize identity. The conserved TAATATTAC nona-nucleotide sequence that contains the nicking site for initiation of virus DNA replication is marked by an open box and the sequence of the conserved stem-loop structure is underlined in red. displayed more than 93% nucleotide identity with different tomato yellow leaf curl recombinant viruses (Table 2). Thus, among the sequences derived from tomato samples, GU and GU shared 93% nid with the Tomato yellow leaf curl Axarquia virus (TYLCAxV; DQ ), GU shared 94% nid with the Tomato yellow leaf curl Malaga virus (TYLCMalV; DQ ) and GU nid with TYLCV/TYLCSV recombinant Ragusa (EU ). The sequence from the infected weed S. nigrum (GU322874) shared 94% nid with the TYLCV/TYLCSV recombinant Ragusa (EU ). Our results confirm that TYLCV and TYLCSV species are widely distributed and established in Tunisia, either in single or mixed infections. They naturally and

5 Journal of Plant Pathology (2014), 96 (1), Mnari-Hattab et al. 199 predominantly co-occur in the same plant, as previously reported by Pellegrin et al. (2008), in the two major Tunisian agricultural areas studied. Mixed infections are widely distributed in tomato, faba bean, pepper and many weed species. The following weed species: Solanum nigrum, Chenopodium album, Lantana camara, Amaranthus lividus, Emex spinosa, Melilotus officinalis and Sisymbrium irio, which are significant components of natural and agricultural ecosystems, are here described for the first time as being infected by both TYLCV and TYLCSV species in Tunisia. Although the strains were sometimes different from those in Tunisia, such mixed infections in crops and weeds have been already widely reported in Mediterranean countries, particularly in Spain (Garcia-Andrés et al., 2007) and Italy (Davino et al., 2009). Co-occurrence of different strains and species is a prerequisite for the emergence of new recombinant viruses associated with TYLCVD. Thus, in the present study and for the first time in Tunisia, natural recombinants between TYLCV and TYLCSV on tomato and S. nigrum, two solanaceous plant species, were observed in the major tomato growing areas in both heated and unheated tunnels. No recombinants have yet been detected in the other weed species analysed, although mixed infections have been widely reported. But it is likely that some identical or distinct TY- LCV recombinants will also be isolated and characterised in some of these plants in the future. Garcia-Andres et al. (2007) provided clear evidence for frequent emergence of recombinant species during mixed begomovirus infection in the same plant. These recombination events might be a major driving force for the evolution of begomoviruses through the generation of new variants, and their ability to emerge in nature and break resistance in crop plants (Monci et al., 2002; Garcia-Arenal and McDonald, 2003). Based on our results, further field surveys will be undertaken to investigate: (i) whether the recombinant species may be more damaging to tomato crops than the parental species; (ii) the importance of weed species, as these might play a critical role in the ecology and epidemiology of viruses as potential reservoir plants. Furthermore, they offer suitable conditions for the presence and spread of TYLCVD all year round, in periods when no tomato crops are available. Further targeted surveys must therefore be undertaken to advance our knowledge of the role of weeds in TYLCVD epidemics and emergence of new tomato yellow leaf curl recombinant viruses. ACKNOWLEDGEMENTS The authors thank G.P. Accotto (Istituto di Virologia Vegetale del CNR, Torino, Italy) for kindly providing the DNA of reference recombinant isolates of TYLCV. REFERENCES Anonymous, Document technique sur la culture de tomate en Tunisie. Agence de la Vulgarisation et de la Formation Agricoles, Tunis, Tunisie Brown J.K., Fauquet C.M., Briddon R.W., Zerbini M., Moriones E., Navas-Castillo J., Family Geminiviridae. In: King A.M.Q, Adams M.J., Carstens E.B., Lefkowitz E.J. (eds). Virus Taxonomy. Ninth Report of the International Committee on Taxonomy of Viruses, pp Elsevier-Academic Press, Amsterdam, The Netherlands. Carling J., Available via DIALOG. com.au/content/dna-preparation.html. Accessed 5 March Cherif C., Russo M., Cytological evidence of the association of a geminivirus with the tomato yellow leaf curl disease in Tunisia. Journal of Phytopathology 108: Czosnek H., Laterrot H., A worldwide survey of tomato yellow leaf curl viruses. Archives of Virology 124: Davino S., Davino M., Accotto G.P., A single-tube PCR assay for detecting viruses and their recombinants that cause tomato yellow leaf curl disease in the Mediterranean basin. Journal of Virological Methods 147: Davino S., Napoli C., Dellacroce C., Miozzi L., Noris E., Davino M., Accotto G.P., Two new natural begomovirus recombinants associated with the tomato yellow leaf curl disease co-exist with parental viruses in tomato epidemics in Italy. Virus Research 143: Fekih-Hassen I., Gorsane F., Djilani F., Fakhfakh H., Makha M., Maxwell D.P., Marrakchi M., Detection of Tomato yellow leaf curl Sardinia virus in Tunisia. Bulletin OEPP/EPPO Bulletin 33: Garcia-Andrés S., Monci F., Navas-Castillo J., Moriones E., Begomovirus genetic diversity in the native plant reservoir Solanum nigrum: evidence for the presence of a new virus species of recombinant nature. Virology 350: García-Andrés S., Miguel-Tomás D., Sánchez-Campos S., Navas-Castillo J., Moriones E., Frequent occurrence of recombinants in mixed infections of tomato yellow leaf curl disease-associated begomoviruses. Virology 365: García-Arenal F., McDonald B.A., An analysis of the durability of resistance to plant viruses. Phytopathology 93: Gharsallah-Chouchane S., Gorsane F., Nakhla M.K., Maxwell D.P., Marrakchi M., Fakhfakh H., First report of Tomato yellow leaf curl virus-israel species infecting tomato, pepper and bean in Tunisia. Journal of Phytopathology 155: Hanley-Bowdoin L., Settlage S.B., Orozco B.M., Nagar S., Robertson D., Geminiviruses - models for plant DNA replication, transcription and cell cycle regulation. Critical Reviews in Biochemistry and Molecular Biology 35: Jeske H., Lütgemeier M., Preiss W., Distinct DNA forms indicate rolling circle and recombination-dependent replication of Abutilon mosaic geminivirus. The EMBO Journal 20: Kheyr-Pour A., Bendahmane M., Matzeit V., Accotto G.P., Crespi S., Gronenborn B., Tomato yellow leaf curl

6 200 Begomovirus recombinants in tomato Journal of Plant Pathology (2014), 96 (1), virus from Sardinia is a whitefly-transmitted monopartite geminivirus. Nucleic Acids Research 19: Monci F., Sanchez-Campos S., Navas-Castillo J., Moriones E., A natural recombinant between the geminiviruses Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus exhibits a novel pathogenic phenotype and is becoming prevalent in Spanish populations. Virology 303: Navas-Castillo J., Sanchez-Campos S., Noris E., Louro D., Accotto G.P., Moriones E., Natural recombination between Tomato yellow leaf curl virus-is and Tomato leaf curl virus. Journal of General Virology 81: Pellegrin F., Mnari-Hattab M., Tahiri A., Dalleau-Clouet C., Peterschmitt M., Bonato O., First report of simultaneous presence of Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl Israel virus infecting crops and weeds in Tunisia. Journal of Plant Pathology 90: 145 Saunders K., Lucy A., Stanley J., DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication. Nucleic Acids Research 19: Stanley J., Analysis of African cassava mosaic virus recombinants suggests strand nicking occurs within the conserved nonanucleotide motif during the initiation of rolling circle DNA replication. Virology 206: Thompson J.D., Higgins D.G., Gibson T.J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionsspecific gap penalties and weight matrix choice. Nucleic Acids Research 22: Received April 18, 2013 Accepted September 9, 2013