Estación Experimental La Mayora, Consejo Superior de Investigaciones Científicas, 29750 Algarrobo-Costa, Málaga, Spain1
Istituto di Fitovirologia Applicata, CNR, Strada delle Cacce 73, 10135 Torino, Italy2
Direcçâo Geral de Protecçâo das Culturas, Quinta do Marquês, 2780 Oeiras, Portugal3
Author for correspondence: Enrique Moriones. Fax +34 952552677. e-mail moriones{at}eelm.csic.es
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Abstract |
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TYLCV has been known in the Iberian Peninsula since 1992, when the species TYLCV-Sar was reported as the causal agent of yellow leaf curl epidemics in tomatoes in southern Spain (Noris et al., 1994 ). The TYLCV-Is species was also found associated with epidemics in tomato in 1995 in southern Portugal and 1997 in southern Spain, either alone or in mixed infections with TYLCV-Sar (Louro et al., 1996
; Navas-Castillo et al., 1997
). In this paper, we report the genetic characterization of infectious clones from TYLCV-Is isolates collected in Spain and Portugal and we describe evidence of natural recombination between TYLCV-Is and the begomovirus Tomato leaf curl virus (ToLCV).
Infectious clones that produced Bemisia tabaci-transmissible progeny were obtained as described in Navas-Castillo et al. (1999 ) from the TYLCV-Is-infected tomato samples SP72/97, collected in Spain in June 1997, and Port2/95, collected in Portugal in September 1995. The complete genome sequences of these clones were determined from both strands of the full-length inserts cloned in pSP72/97 and pPort2 [GenBank accession numbers AF071228 (2791 nt) and AF105975 (2793 nt)] by automated sequencing with pUC/M13 universal primers and specific primers based on partial sequences. The DNA sequences of SP72/97 and Port2/95 were compared with those of TYLCV-Sar isolate Sardinia (GenBank accession no. X61153) and TYLCV-Is isolates for which complete genome information was available: Japan Shizuoka (Japan-S), GenBank accession no. AB014346; Japan Aichi (Japan-A), AB014347; Israel, X15656; Israel/Mild, X76319; Cuba, AJ223505; Dominican Republic, AF024715; and Iran, AJ132711. Isolates SP72/97 and Port2/95 appeared to be closely related variants of TYLCV-Is and their genomes strongly resembled those of the Japan-A and Japan-S isolates (Kato et al., 1998
) (Table 1
). In addition, the isolates from the Iberian Peninsula and Japan were closely related to the Israel/Mild isolate and more distantly related to the Israel, Cuba, Dominican Republic and Iran isolates. Sequence identities among the IRs or complete genomes followed a similar pattern. Nucleotide sequence comparisons of the different ORFs provided further support for the observed relationships between the TYLCV-Is isolates; interestingly, ORFs C1 and C4 were less conserved than V1, V2, C2 and C3 (Table 1
). The presence of such closely related variants in geographical regions so distantly separated can only be explained by movement of infected plant material or viruliferous B. tabaci individuals via plant trade or human movement. In fact, this was argued as the cause of the primary spread of Middle East TYLCV-Is to the Caribbean and then to USA (Polston & Anderson, 1997
; Polston et al., 1999
).
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Phylogenetic relationships of SP72/97 and Port2/95 to other TYLCV-Is isolates (listed above) were studied on the basis of the nucleotide sequences of the IRs and ORFs V1, V2 and C1 to C4. As changes in the topological position of certain isolates occurred depending on which part of the genome was compared (not shown), a more detailed comparison throughout the genome was done with the aid of PLOTSIMILARITY diagrams (Wisconsin GCG software package; Devereux et al., 1984 ). In these comparisons, the nucleotide sequences of the SP72/97, Port2/95, Japan-A and Japan-S isolates on the one hand and the Israel, Cuba and Dominican Republic isolates on the other proved to be almost identical throughout the genome (not shown). Therefore, in Fig. 1
, the first group of isolates is represented by SP72/97 while the second is represented by Israel. Comparisons revealed that a large portion of the genome, hereafter referred to as region I (see below), was quite well conserved among all TYLCV-Is isolates (Fig. 1
). The nucleotide sequence of the Israel isolate differed from that of SP72/97 in the 5'-proximal two-thirds of the C1 ORF and the 5'-half of the IR (regions II and III; Fig. 1A
). The Israel and Iran sequences also differed in the same part of the genome (Fig. 1B
). However, when the sequences of SP72/97 and Iran were compared, differences were only observed in region III (Fig. 1C
). The Israel/Mild sequence was highly similar to that of SP72/97 except for the central part of the IR (region IV; Fig. 1D
). Nucleotide sequences in regions II to IV of TYLCV-Is isolates were then compared with other available geminivirus sequences, the limits of each region established by visual inspection of sequence alignments (indicated in Fig. 1
). The sequence in region III of the Iran isolate was more closely related (91% identity) to the sequence of the equivalent region of the Bangalore-4 isolate of ToLCV (GenBank accession no. AF165098) than to that of any other TYLCV-Is isolate (below 75% identity). Sequences in regions II and III of the Israel, Cuba and Dominican Republic isolates were 97100% identical, their closest relationships (between 73 and 83% identity) being to sequences in the equivalent regions of the Bangalore-2 (GenBank accession no. U38239) and Bangalore-4 isolates of ToLCV.
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The retention of the 5'-proximal part of the IR with the cognate 5' region of ORF C1 might be due to functional constraints. In fact, ORF C1 (rep gene) encodes the replication-associated protein (Rep) and it is known that the N-terminal region of Rep interacts specifically with the cognate 5' region of the IR for DNA replication and autoregulation of the rep gene (Fontes et al., 1994 ; Eagle et al., 1994
; Orozco et al., 1997
). The 3' limit of the putative ToLCV recombined fragments occurred in a highly variable region of the IR in both the Iran and Israel isolate genomes (not shown), so it was difficult to define the precise putative 3' cross-over site. However, in both cases, it occurred close to the conserved stemloop structure of the IR, in which ori occurs (Fig. 1
), and ori has been suggested as a hot-spot for recombination in the geminiviruses (Stanley, 1995
; Sanz et al., 1999
). The precise putative 5' cross-over site could not be defined by comparison of the Iran and Israel genomes with their closest related ancestors because stretches of identical nucleotides occurred in the region involved.
The existence of additional recombinations cannot be excluded. For example, in the genome of the Israel isolate, a short stretch of 113 nt was found within the central part of ToLCV-related region III that was almost identical (>96% identity) to the equivalent sequence of related TYLCV-Is isolates (see Fig. 1A for comparison with SP72/97).
Recombination is not a rare phenomenon among begomoviruses (Zhou et al., 1997 ; Harrison & Robinson, 1999
; Padidam et al., 1999
; Sanz et al., 1999
), providing additional sources of variation with unpredictable effects on virus pathogenicity. Knowledge of sequence variants of TYLCV-Is present in a given geographical region is essential for the correct establishment of effective control measures, particularly because breeding is the best way to control this virus.
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References |
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Received 12 May 2000;
accepted 28 July 2000.