One Shields Ave, Plant Pathology Dept, University of California, CA 95616 Davis, USA1
Faculty of Agricultural and Food Sciences, American University of Beirut, Lebanon2
Author for correspondence: Bryce Falk. Fax +1 5307525674. e-mail bwfalk{at}ucdavis.edu
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Abstract |
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We studied the genetic variation of the plant virus Cucurbit yellow stunting disorder virus (CYSDV), a member of the genus Crinivirus within the family Closteroviridae (Martelli et al., 2000 ). CYSDV has a bipartite single-strand plus-sense RNA genome encapsidated in long filamentous and flexuous particles. CYSDV infections are phloem-limited and the virus is transmitted in a semipersistent manner by the whiteflies Bemisia tabaci (Gennadius) and B. argentifolii (Bellows & Perring) (Célix et al., 1996
; Martelli et al., 2000
). CYSDV was first identified in the United Arab Emirates in the early 1990s (Hassan & Duffus, 1991
) and since then it has been reported in other Middle East countries and in the Mediterranean basin, where it is recognized as a rapidly emerging and economically important plant virus (Célix et al., 1996
; Wisler et al., 1998
; Rubio et al., 1999
; Abou-Jawdah et al., 2000
). Just recently, CYSDV has been reported from North America (Kao et al., 2000
).
We used single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses of the CYSDV coat protein (CP) gene to estimate the population structure and genetic variation within individual CYSDV isolates, and between CYSDV isolates collected in different years from different areas of the world. Also, we estimated the genetic variation of a region of the CYSDV HSP70 homologue gene (HSP70) from data obtained in a previous report (Rubio et al., 1999 ) and from new data obtained after analysing additional CYSDV isolates.
Seventy-one CYSDV isolates were collected from cucurbit plants (squash, Cucurbita pepo L.; cucumber, Cucumis sativus L.; watermelon, Citrullus lanatus Schard; and melon, Cucumis melo L.) from different curcubit-growing regions included in the Mediterranean basin, Middle East and North America (Table 1). Total RNAs were extracted from curcubit leaf tissue by using Tri Reagent (Molecular Research Center) according to manufacturers instructions, and were used as the template for RTPCR. Specific oligonucleotide primers CYSCPf (5' ATGGCGAGTTCGAGTGAGAATAA 3') and CYSCPr (5' ATTACCACAGCCACCTGGTGCTA 3') corresponding to both ends of the CYSDV CP gene were designed based on the nucleotide sequence published by Livieratos et al. (1999)
. RT was performed in a reaction mixture (20 µl) of 1xAMV buffer, 200 µM of each dNTP, 40 ng of CYSCPr primer, 2 units of Rnasin Ribonuclease Inhibitor (Promega), 0·3 units of AMV reverse transcriptase (Promega). The mixture was incubated at 42 °C for 45 min. PCR was performed in a 20 µl reaction containing 2 µl of the synthesized cDNA, 2 µl 10xbuffer, 5 units Pfu (Stratagene), 1 ng/µl of each primer. The mixture was incubated first at 94 °C for 4 min, followed by 30 cycles at 94 °C for 30 s, 50 °C, 72 °C for 2 min, and by a final cycle at 72 °C for 5 min. When the RTPCR products were electrophoresed in agarose gels, a unique DNA species of 755 nt, the expected size, was detected for each CYSDV isolate. No DNA was obtained from non-CYSDV-infected plants (data not shown).
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The low genetic diversity found in the Western group, distributed in such an extensive area (Europe, Middle East and North America) is surprising for an RNA virus. One possible cause of this low diversity could be the rapid expansion of CYSDV, possibly related with the recent colonization and explosion of the CYSDV whitefly vectors into new areas (Brown et al., 1995 ). For example, in Spain the displacement of another Crinivirus affecting cucurbits, Beet pseudo-yellows virus (BPYV), by CYSDV has been associated with the displacement of the BPYV vector, Trialeurodes vaporariorum, by the CYSDV vector, B. tabaci (Célix et al., 1996
; Berdiales et al., 1999
). However, the low genetic variation found here was not only for CYSDV isolates geographically distant, but also for CYSDV isolates separated temporally over a 3 year period (Table 1
). Negative selection due to functional constraints of virus-encoded proteins can limit the extent of genetic variation in virus populations. Therefore, we estimated the degree and sense of selection by calculating the ratio of nucleotide diversity at nonsynonymous (dN) to synonymous sites (dS) (as described by Pamilo & Bianchi, 1993
; Li, 1993
) of the HSP70 and CP coding regions between CYSDV Western and Eastern subpopulations. The dN/dS ratio was 0·07048 for CP and 0·10452 for HSP70, suggesting that both coding regions are under high negative selective constraints. However, this cannot explain the low variation at the synonymous positions found between isolates within the Western subpopulation. Other constraints, such as those imposed by secondary structure or expression recognition signals might have also limited the genetic variation (Roossinck, 1997
). A model of periodic selection, proposed by Moya et al. (1993)
, consisting of negative selection alternating with rapid expansion of a genome with high fitness (positive selection), could account for the genetic stability observed for CYSDV.
Spatial and temporal genetic stability have also been reported for the tobamoviruses Pepper mild mottle virus (Rodríguez-Cerezo et al., 1989 ) and Tobacco mild green mosaic virus (Fraile et al., 1996
). Within the genus Crinivirus, data concerning the genetic variation are scarce and fragmented. Analysis of partial HSP70 homologue sequences of eight BPYV isolates from Italy, Crete and California showed very low genetic diversity (Rubio et al., 1999
; Fig. 2
). Although a larger number of isolates should be analysed for a more accurate estimation of BPYV genetic diversity, we can conclude that with respect to the genomic region analysed, geographically distant BPYV isolates are genetically very similar. Analysis of eight Ugandan isolates of another Crinivirus, Sweet potato chlorotic stunt virus (SPCSV; Alicai et al., 1999
), showed a small genetic diversity for HSP70 and CP genes (Fig. 2
). In contrast, a partial CP gene sequence of 20 Californian isolates of Citrus tristeza virus (CTV), a member of the genus Closterovirus (the other genus within the family Closteroviridae), showed a significantly greater genetic diversity (Fig. 2
) despite the low dN/dS ratio observed (0·02739), suggesting high negative selection pressure (unpublished data). The different genetic variation for CTV and CYSDV might be caused in part by differences in factors such as host plants and/or modes of transmission. CTV has perennial host plants that can be infected for years whereas CYSDV hosts are annual and infections are generally less than 60 days old. CYSDV is transmitted only by its whitefly vector, whereas CTV is also transmitted by vegetative propagation, a means which could remove selective constraints related to insect transmission.
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Acknowledgments |
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Footnotes |
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References |
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Received 13 November 2000;
accepted 15 December 2000.