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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY580884AY580985 and AY583997AY584064.
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INTRODUCTION |
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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 750800 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.
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METHODS |
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RESULTS |
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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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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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DISCUSSION |
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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 19942001 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 19971998 (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
).
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ACKNOWLEDGEMENTS |
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Received 3 September 2004;
accepted 15 November 2004.
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