Genetic diversity of a natural population of Cucurbit yellow stunting disorder virus

C. F. Marco1 and M. A. Aranda2

1 Estación Experimental ‘La Mayora’, Consejo Superior de Investigaciones Científicas, 29750 Algarrobo-Costa, Málaga, Spain
2 Centro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Científicas, Campus Universitario de Espinardo, Apdo Correos 164, 30100 Espinardo, Murcia, Spain

Correspondence
M. A. Aranda
m.aranda{at}cebas.csic.es


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
An analysis of nucleotide sequences in five coding and one non-coding genomic regions of 35 Cucurbit yellow stunting disorder virus (CYSDV) isolates collected on a local scale over an 8 year period is reported here. In total, 2277 nt were sequenced for each isolate, representing about 13 % of the complete virus genome. Mean nucleotide diversity for the whole population in synonymous positions in the coding regions was 0·00068, whilst in the 5' untranslated region (5' UTR) of genomic RNA2, it was 0·00074; both of these values are very small, compared with estimates of nucleotide diversity for populations of other plant viruses. Nucleotide diversity was also determined independently for each of the ORFs and for the 5' UTR of RNA2; the data showed that variability is not distributed evenly among the different regions of the viral genome, with the coat protein gene showing more diversity than the other four coding regions that were analysed. However, the low variability found precluded any inference of selection differences among gene regions. On the other hand, no evidence of selection associated with host adaptation was found. In contrast, at least a single amino acid change in the coat protein appears to have been selected with time.

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY580884–AY580985 and AY583997–AY584064.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
RNA viruses are known to have a high variation potential, because of their high mutation and recombination rates (Drake & Holland, 1999; Bruyere et al., 2000; Malpica et al., 2002). However, this mutability does not necessarily result in a high degree of genetic variability of their populations, as the genetic composition of virus populations will depend on evolutionary forces, such as selection and random genetic drift. In fact, for most of the cases analysed, plant virus populations have been shown to be genetically stable (reviewed by García-Arenal et al., 2001). To explain this stability, population bottlenecks associated with ecological factors, such as colonization of new hosts or new geographical areas, are often invoked (Fraile et al., 1996, 1997a; Sánchez-Campos et al., 2002). Also, selection pressures, including the maintenance of structural features of the virus or interaction with host and vector factors, may contribute to genetic stability (e.g. Moreno et al., 2004) (reviewed by García-Arenal et al., 2001). The relative contribution of these evolutionary forces can be studied by analysing the genetic structure of plant virus populations. Analysis through temporal series might be particularly informative, although studies of this type are scarce (Fraile et al., 1997b; Sánchez-Campos et al., 2002).

We report here a local-scale analysis of the genetic diversity of a natural population of Cucurbit yellow stunting disorder virus (CYSDV), a whitefly-transmitted closterovirus that affects cucurbit crops extensively in many warm and temperate areas of production (Hassan & Duffus, 1991; Célix et al., 1996; Wisler et al., 1998; Abou-Jawdah et al., 2000; Desbiez et al., 2000; Kao et al., 2000; Louro et al., 2000). Whitefly-transmitted closteroviruses are responsible for emergent diseases worldwide (Wisler et al., 1998; Karasev, 2000). In Spain, CYSDV was first detected in 1992 (Célix et al., 1996) and, since then, it has become the prevalent virus in protected cucurbit crops of south-eastern regions, where up to 100 % of plants are frequently infected. CYSDV induces a yellowing syndrome that can result in important yield reductions in infected plants, with corresponding economic consequences. CYSDV is a member of the genus Crinivirus (family Closteroviridae) (Martelli et al., 2000, 2002). It has a narrow host range that is limited to species of the family Cucurbitaceae, in which it is confined to phloem-associated cells (Célix et al., 1996; Marco et al., 2003). Its viral particles are flexible rods with lengths of 750–800 nm (Liu et al., 2000) that encapsidate two molecules of single-stranded RNA of positive polarity, designated RNAs 1 and 2 (Aguilar et al., 2003). The variability of two genes in CYSDV RNA2 has been characterized for a collection of isolates from different areas of the world (Rubio et al., 1999, 2001b). Here, we report a study that complements this previous work and includes an analysis of nucleotide sequences in five genomic regions of the CYSDV genomic RNAs 1 and 2 of 35 CYSDV isolates. These isolates were collected in a restricted geographical area over an 8 year period, from the initial outbreaks of the CYSDV epidemic in Spain.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus isolates and RNA extraction.
CYSDV isolates were obtained from cucumber (Cucumis sativus), melon (Cucumis melo) and watermelon (Citrullus lanatus) plants cultivated in plastic houses of the El Ejido area, Almería province, south-eastern Spain. This is the most important area in Spain for protected horticulture, where more than 16 000 hectares of cucurbits are cultivated per annum. Greenhouses here are almost all unheated, symmetrical and covered with polyethylene on a small roof slope. Cultivation is mostly (90 %) in artificial sandy soil. In this area, CYSDV has affected cucurbit crops extensively since the early 1990s; often, 100 % of plants in affected greenhouses show the characteristic yellowing symptoms that are induced by this virus (Célix et al., 1996). To obtain a collection of CYSDV isolates, surveys were conducted in commercial greenhouses within this area during an 8 year period from 1994 to 2001 (Table 1). Surveys could not be carried out during 1995, 1998 or 2000. Within the area surveyed, the two most distant sampling points were 50 km apart. Isolates corresponding to the years 1994, 1996 and 1997 were collected and kindly provided by E. Rodriguez-Cerezo (CNB-CSIC, Madrid, Spain). Melon and watermelon crops were surveyed during late spring, whereas cucumber crops were surveyed in autumn, all during the eighth to twelfth weeks post-transplantation. From each plastic house visited, at least three samples were collected from plants located in separated points of the crop. Each sample consisted of a leaf with clear yellowing symptoms. From this material, total RNA extracts were prepared as described by Díaz et al. (2004) and the presence of CYSDV was determined by dot-blot hybridization (Marco et al., 2003). For the analysis presented in this paper, we took 35 CYSDV isolates at random from this collection (Table 1). Total RNA extracts corresponding to these isolates were used in RT-PCR as described below.


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Table 1. CYSDV isolates analysed in this study, grouped by host and year of collection

 
Nucleotide sequence analysis.
Throughout this paper, numbers for CYSDV nucleotide sequences are as decribed by Aguilar et al. (2003). Primers 5'-GATTGTCACTGAATCACC-3' (MA155) and 5'-TCACATCATCAATCCAAAAG-3' (MA129), complementary to nt 8090–8108 and 8753–8773 of CYSDV RNA1, respectively, and 5'-CAACTTGCCCAATCACAGC-3' (MA249), 5'-CCTCCCAAAAACGAATCACC-3' (MA137) and 5'-CCAAAACTGCGGTAATACCC-3' (MA176), complementary to nt 504–522, 1890–1910 and 5524–5544 of CYSDV RNA2, respectively, were used to synthesize first-strand cDNAs in independent, standard reverse-transcription reactions (Sambrook & Russell, 2000) for each of the isolates. cDNA amplification was carried out in standard PCRs, in which the primers for second-strand synthesis were 5'-GACTTGCAATAATCATAGCC-3' (MA143) and 5'-GAAGAATTCCAGGCAAGG-3' (MA156), identical to nt 7573–7593 and 8191–8209 of CYSDV RNA1, respectively, and 5'-GGTAAATCCATTGGGATACGG-3' (MA227), 5'-GGTGGGTAGGTGTTGACAG-3' (MA208) and 5'-GGATCGTTCTCATTATCGG-3' (MA217), identical to nt 11–31, 1442–1461 and 4843–4862 of CYSDV RNA2, respectively. In every case, the RT-PCR DNA fragments that were obtained had the expected size and in no case were DNA products obtained from RNA extracts prepared from non-infected control plants. RT-PCR products were fractionated by electrophoresis in agarose gels and, after elution, both strands were sequenced in an automatic sequencer (ABI Prism 3700 DNA analyser; Applied Biosystems) by priming the sequencing reactions with the same oligonucleotides as were used for the cDNA synthesis. Whenever a discrepancy was found, internal primers were used to resequence the corresponding region. Sequence alignments were obtained by using the CLUSTAL_X program (Thompson et al., 1997). Genetic distances were estimated from sequence data as described by Pamilo & Bianchi (1993) and Li (1993) (PBL method) and also by means of the Kimura two-parameter method (Kimura, 1980). Confidence estimates for dNS and dS values were calculated by using the bootstrap method (Nei & Kumar, 2000) with 500 replicates. The program used was MEGA2 (Kumar et al., 2001), available at http://www.megasoftware.net. Within-population and between-population nucleotide diversities were estimated from genetic distances as described by Nei (1987).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
DNA fragments corresponding to five regions (Fig. 1) of the genomic RNAs of each CYSDV isolate (Table 1) were generated by RT-PCR and subsequently sequenced. According to the numbering of the CYSDV nucleotide sequence described by Aguilar et al. (2003), the genomic regions sequenced were: region I, nt 7584–8066 (RNA1) of ORFs 2 and 3; region II, nt 8324–8720 (RNA1) of ORF 4; region III, nt 23–480 (RNA2) of the 5' untranslated region (UTR); region IV, nt 1455–1864 (RNA2) of the ORF that encodes the heat-shock protein 70 homologue (HSP70h); region V, nt 4926–5454 (RNA2) of the coat protein (CP) ORF (Fig. 1). Regions I, II and III were chosen randomly, whereas regions IV and V were chosen because information on CYSDV variability pre-existed for them (Rubio et al., 2001b). In this way, we sequenced a total of 2277 nt for each isolate, representing about 13 % of the complete virus genome.



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Fig. 1. Genomic structure of CYSDV and regions sequenced in this work. Genomic RNAs 1 and 2 are each represented by a line, with the ORFs indicated by boxes. ORFs are shown above, below or in the middle of the line, depending on the frame in which they are found. The start of ORF 1b is drawn, assuming a +1 ribosomal translational frameshifting. Domains predicted from deduced amino acid sequences are indicated inside the boxes: L-Pro, putative papain-like leader proteinase, for which an arrow and a dotted line indicate the predicted autocatalytic cleavage site; MTR, methyltransferase domain; HEL, helicase domain; RdRp, RNA-dependent RNA polymerase domain; Hsp70h, heat-shock protein 70 homologue; CP, coat protein; CPm, minor coat protein. Approximate molecular mass is shown in parentheses if no function could be predicted for potential peptides coded by ORFs. Black segments represent directly sequenced RT-PCR products.

 
Genetic diversity in coding regions of the virus genome
Nucleotide sequences corresponding to putative CYSDV coding regions were aligned and used to estimate genetic distances between each pair of isolates by using the PBL method. Values of genetic distances at synonymous positions between pairs of isolates for all coding regions ranged from 0 to 0·00672. The maximum value was found between pairs of isolates sampled during different years, but also between isolates sampled during the same year. The mean nucleotide diversity at synonymous positions for the whole population was 0·00068, a very small figure compared with nucleotide diversity estimates of populations of other plant viruses (see Discussion). Values of nucleotide diversity were also estimated independently for each of the ORFs included in this analysis (Table 2). For ORF 2, all isolates were identical and, thus, genetic distances were null. Highest diversity values were found for ORF 3 and the CP gene (Table 2).


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Table 2. Nucleotide diversities for different coding regions of the CYSDV genome

Nucleotide diversity is defined here as the mean number of nucleotide substitutions per site. Nucleotide diversities were computed separately for non-synonymous (dNS) and synonymous (dS) positions by using the PBL method (see Methods). Standard errors (SE) were calculated by using the bootstrap method (Nei & Kumar, 2000) with 500 replicates.

 
The direction and degree of the selective constraints operating in a coding region can be estimated by the ratio between nucleotide diversity values in non-synonymous and synonymous positions (dNS/dS ratio) (Nei & Gojobori, 1986; Nei, 1987; Yang & Bielawski, 2000). For the CYSDV population analysed here, this ratio was below unity for ORF 3. In contrast, the dNS/dS ratio was >1 for the CP and HSP70h genes and ORF 4, as nucleotide changes were only detected in non-synonymous positions in ORF 4 (Table 2). However, the calculated standard errors (Nei & Kumar, 2000) were of the same order of magnitude as dNS and dS values (Table 2) and, therefore, these data are inconclusive for inferring selection differences among gene regions.

In order to analyse whether host adaptation could constitute a selection factor, nucleotide diversity values were estimated between and within CYSDV subpopulations, considering a subpopulation as the group of isolates that were originally collected from a given host species (melon, watermelon or cucumber). Between-subpopulation diversity values were, when greater than zero, similar in their order of magnitude to those corresponding to within-subpopulation diversity values (i.e. differences among subpopulations are smaller than or similar to differences within subpopulations) (Table 3). This indicates that there is no differentiation of the population according to the host species from which the isolates were taken. In addition, the dNS/dS ratios determined here did not seem to follow any pattern that could be associated with the host. For example, the dNS/dS ratios were >1 when isolates from melon or watermelon were compared, and <1 when isolates from cucumber were compared with those from melon or watermelon (Table 3). Therefore, host adaptation does not seem to play a role in the variability of the CYSDV population analysed here.


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Table 3. Within- and between-population nucleotide diversities in coding regions for CYSDV subpopulations by host species

A subpopulation was considered here as the set of isolates from a given host species (melon, watermelon or cucumber). Nucleotide diversities were computed separately for non-synonymous (dNS) and synonymous (dS) positions in coding regions by using the PBL method (see Methods). Between-population diversity values correspond to net nucleotide substitutions as given on p. 276 in Nei (1987).

 
Genetic diversity in a non-coding region of the virus genome
Nucleotide sequences corresponding to the 5' UTR of RNA2 were aligned and used to estimate genetic distances between each pair of isolates by using the Kimura two-parameter method (Kimura, 1980). Values ranged between 0 and 0·0044. The maximum value was found between two isolates sampled during 1996 and 2001. The mean nucleotide diversity for this region of the CYSDV genome was 0·00074, a value similar in its order of magnitude to that found for the coding regions.

It has been shown that maintenance of a functional secondary structure may constitute a constriction to the genetic variability of non-coding RNAs (e.g. Aranda et al., 1997). In order to study whether this could be the case for the non-coding region considered in this study, the nucleotide substitutions identified for this region were compared with a secondary-structure model that was predicted by using the Mfold program (Zuker, 1989). Interestingly, all of the nucleotide substitutions that were identified are located in the loops and therefore would not affect the stability of the predicted structure (data not shown). However, only a small number of substitutions were found, which precluded a proper statistical analysis to confirm or reject this hypothesis.

As for the coding regions, between- and within-subpopulation nucleotide diversity values were estimated, considering as a subpopulation the group of isolates that were sampled from the same host species. All intrapopulational diversity values were small, although the diversity found for the watermelon subpopulation was around eight to nine times higher than the diversities found for the subpopulations from the other hosts (Table 4). In contrast, interpopulational diversity values were null or insignificant (Table 4), indicating again that the population cannot be considered to be subdivided according to the host species from which the isolates were taken.


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Table 4. Within- and between-population nucleotide diversities in the 5' non-coding region of RNA2 for CYSDV subpopulations by host species

A subpopulation was considered here as the set of isolates from a given host species (melon, watermelon or cucumber). Nucleotide diversities in the 5' non-coding region of RNA2 were computed by using the Kimura two-parameter method (see Methods). Between-population diversity values correspond to net nucleotide substitutions as given on p. 276 in Nei (1987).

 
Temporal analysis of the genetic diversity of the CYSDV population
CYSDV isolates were collected from 1994 to 2001, thus allowing an analysis of the evolution of the CYSDV population during a temporal lapse of 8 years. To do this, we studied the CYSDV population, considering as subpopulations those isolates that were collected during a given growing season. Diversity values were calculated between and within subpopulations, both for coding regions (Table 5) and for the 5' UTR (Table 6). In the coding regions, intrapopulational diversity values in non-synonymous positions increased slightly with time (except for years 1994 and 1999, when diversity values were zero), the 2001 subpopulation having the greatest diversity. As regards diversity in synonymous positions, the highest value was found for the 1997 subpopulation. When interpopulational diversity values were computed, in many cases, null values were obtained for dS and, thus, the dNS/dS ratio provided little information. However, variability was found frequently at non-synonymous positions (i.e. dNS>0), suggesting that different peptide types were being selected with time (Table 5). Given that the overall number of nucleotide substitutions is low, these results should be verifiable through visual inspection of the alignments of amino acid sequences deduced from the nucleotide sequences. In fact, one case was particularly clear: the amino acid at position 1348 of the CP [amino acid numbering as deduced for the complete CP nucleotide sequence described by Aguilar et al. (2003)] changed during the period 1994–2001, from aspartic acid for the oldest isolates to asparagine for the more recent isolates (Fig. 2).


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Table 5. Within- and between-population nucleotide diversities in coding regions for CYSDV subpopulations by year

A subpopulation was considered here as the set of isolates collected during a growing season (years 1994, 1996, 1997, 1999 and 2001). Nucleotide diversities were computed separately for non-synonymous (dNS) and synonymous (dS) positions in coding regions by using the PBL method (see Methods). Between-population diversity values correspond to net nucleotide substitutions as given on p. 276 in Nei (1987).

 

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Table 6. Within- and between-population nucleotide diversities in the 5' non-coding region of RNA2 for CYSDV subpopulations by year

A subpopulation was considered here as the set of isolates collected during a growing season (years 1994, 1996, 1997, 1999, 2001). Nucleotide diversities in the 5' non-coding region of RNA2 were computed by using the Kimura two-parameter method (see Methods). Between-population diversity values correspond to net nucleotide substitutions as given on p. 276 in Nei (1987).

 


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Fig. 2. Alignment of the amino acid sequence of a fragment of the coat protein (CP) of the CYSDV isolates analysed here. Amino acid numbering is shown as deduced from the complete CP gene nucleotide sequence described by Aguilar et al. (2003). Identical amino acids in all isolates are indicated by dots. Note that the amino acid at position 1348 of the CP changed during the period 1994–2001 from aspartic acid (D) for the oldest isolates to asparagine (N) for the more recent isolates.

 
In the 5' UTR of RNA2, within-population nucleotide diversity was also low. Excluding the 1997 value, the diversity for each subpopulation seemed to increase steadily with time. However, between-population diversity values were null in most cases, indicating that the population as a whole cannot be considered to be made up of yearly subpopulations (Table 6).


   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this work, we have analysed on a local scale the genetic diversity of a CYSDV population during an 8 year period. The population characterized here was sampled in the geographical area where CYSDV was first detected in Spain, from the beginning of the CYSDV epidemic. It is important to mention that, for this virus, genetic diversity studies were only available for populations that were sampled on a regional or global scale and included isolates that were separated by a maximum of 4 years (Rubio et al., 1999, 2001b). In the same studies, only two coding regions of the CYSDV genome were considered (Rubio et al., 1999, 2001b), whereas we have studied new regions of the CYSDV genome, including a non-coding region. The most interesting observation to arise from our study is that the CYSDV population is extremely uniform. Thus, the value of nucleotide diversity at synonymous positions in coding regions of the CYSDV genome was 30 times smaller than an equivalent estimate for a population of Tobacco mild green mosaic virus (TMGMV), a tobamovirus that is typically considered to be very stable (Fraile et al., 1996). The same can be concluded when comparing the results presented here with those obtained for viruses in the family Closteroviridae (Rubio et al., 1999, 2001a, b). In fact, the nucleotide diversity value in synonymous positions that was estimated for the CYSDV population is smaller than all examples cited in a review (García-Arenal et al., 2001). Importantly, the genetic uniformity of the CYSDV population was maintained during an 8 year period and, therefore, the case analysed here appears to constitute an example of significant genetic stability over time.

The high genetic stability found for the CYSDV population could be attributed to negative or purifying selection to maintain the functional integrity of the viral genome (e.g. Moreno et al., 2004) (reviewed by García-Arenal et al., 2001). The degree of negative selection in genes, or the degree of functional constraint for the maintenance of the encoded protein sequence, can be estimated by the ratio between the nucleotide diversities in non-synonymous and synonymous positions (dNS/dS ratio) (Nei & Gojobori, 1986; Nei, 1987; Yang & Bielawski, 2000). For most coding genes, the dNS/dS ratio is below unity, which is consistent with negative selection against protein change (Nei, 1987). In contrast, a dNS/dS ratio above unity may be an indication that adaptive or positive selection is driving gene divergence (Nei & Gojobori, 1986). In this work, we observed both situations, depending on the gene considered. For ORF 3, a dNS/dS ratio below 1 may suggest that the conservation of the amino acid sequence encoded by this ORF constitutes an impediment to variation. In contrast, dNS values were higher than dS values for ORF 4 and the Hsp70h and CP genes, suggesting positive selection of amino acid sequence variants encoded by these genes. However, the low amount of diversity found resulted in high standard errors for the dNS and dS estimations (Table 2) and, therefore, the CYSDV data presented here are essentially inconclusive for inferring selection differences among gene regions.

Positive selection of better-adapted variants could also be a factor responsible for reduced genetic variability (Fitch et al., 1991; Moya et al., 1993; Ina & Gojobori, 1994; Fraile et al., 1996). A possible advantage conferred by amino acid changes could be associated with host adaptation. In fact, there are examples in the literature showing that this type of selection may act during serial host-passage experiments (e.g. Moriones et al., 1991). However, we did not find any indication that host adaptation may play a role in shaping the CYSDV population characterized here and, thus, other selection factors should be considered in future studies. In this regard, it would be particularly interesting to study possible factors involved in an amino acid change that was identified in the sequences of the CP: the amino acid at position 1348 changed during the 1994–2001 period from aspartic acid to asparagine and, so, variants with asparagine in this position seemed to have been favoured. Given the implication of the CP in transmission, as demonstrated for the related crinivirus Lettuce infectious yellows virus (Tian et al., 1999), and as a change from B to Q biotype prevalence may have occurred in Spanish Bemisia tabaci populations around 1997–1998 (Banks et al., 1998; Moya et al., 2001), it is tempting to speculate that selection of CP variants has arisen through vector adaptation. This hypothesis should be tested experimentally when CYSDV infectious clones become available.

Founder effects may have contributed to shaping the genetic structure of populations of diverse viruses, such as Cucumber mosaic virus (CMV), Citrus tristeza virus, TMGMV and Zucchini yellow mosaic virus (Fraile et al., 1996, 1997a; Ayllón et al., 1999; Desbiez et al., 2002), and could also be responsible for the low genetic variability observed for the CYSDV population analysed here. It can be speculated that the CYSDV epidemic in Almería (Spain) was associated with a marked founder effect, because the virus may not be autochthonous to this geographical area, but imported from distant areas together with contaminated material or B. tabaci individuals. Thus, the establishment of one or a few initial infection foci, from which the virus could have been spread through its abundant vector, may have resulted in a genetically very uniform virus population. However, it is unlikely that population bottlenecks have reoccurred during recent years, taking into account the overlapping of crops that are hosts for this virus and the high B. tabaci population numbers that normally occur throughout the year in the geographical area considered.

Taking all the data obtained here together, it is possible to propose the following evolutive scenario: 12 years ago, when the first CYSDV epidemic outbreak occurred, one or a few genetic types established the initial epidemic foci, but spread very rapidly because of the abundance of B. tabaci and cucurbit crops in the area. The year-round maintenance of vector populations and the overlapping of hosts probably impeded the occurrence of new, i.e. seasonal (as could be the case for CMV; Fraile et al., 1997a), genetic bottlenecks and could also have had an influence on the possible generation and selection of better-adapted variants (Elena et al., 1998).


   ACKNOWLEDGEMENTS
 
We thank E. Rodríguez-Cerezo for providing CYSDV isolates corresponding to years 1994, 1996 and 1997. We also wish to thank P. Moreno, E. Moriones and V. Truniger for critical reviewing of the manuscript, M. V. Martín for technical assistance and P. Thomas for checking the English. Financial support from Fundación Séneca de la Región de Murcia (Spain; grant PB/6/FS/02) is gratefully acknowledged.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 3 September 2004; accepted 15 November 2004.



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