1 ZARC, Zanzibar, PO Box 1062, Tanzania
2 IRD, BP 64501, 34394 Montpellier cedex 5, France
3 INERA, 01 BP 476 Ouagadougou, Burkina Faso
4 CNRA, Man, Côte d'Ivoire
5 ENSAM, 34060 Montpellier cedex 1, France
6 NRI, Chatham, Kent ME4 4TB, UK
Correspondence
Denis Fargette
Denis.Fargette{at}mpl.ird.fr
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This paper is dedicated to the memory of our respected colleague, friend and co-author Placide N'Guessan,
who was killed while attempting to escape from fighting between rival forces during the current unrest in
Côte d'lvoire.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rice cultivation throughout the African continent is severely affected by RYMV. First reported in Kenya in 1966 (Bakker, 1974), RYMV has since been detected in most rice-growing countries of Africa and in Madagascar, but not elsewhere. The virus is considered to be among the most important rice pathogens within sub-Saharan Africa (Abo et al., 1998
). It is a member of the genus Sobemovirus (van Regenmortel et al., 2000
). The host range of RYMV is limited to grasses of the Oryzeae and Eragrostidae tribes (Abo et al., 1998
). RYMV is transmitted by several species of beetles (Coleoptera), most of which belong to the Chrysomelidae, and is not seed-borne (Fauquet & Thouvenel, 1977
; Konaté et al., 2001
). There are conflicting observations on the role and significance of abiotic transmission by mechanical means during cultural practices, through soil residues or by irrigation water (Abo et al., 2000
).
The diversity among RYMV isolates was first studied immunologically with polyclonal and monoclonal antibodies (mAbs) (Fauquet & Thouvenel, 1977; Mansour & Baillis, 1994
; Konaté et al., 1997
; N'Guessan et al., 2000
). Later, analyses of sequences of the coat protein gene were made (Pinel et al., 2000
; Traoré et al., 2001
; Fargette et al., 2002
). The coat protein gene was found to be a reliable part of the genome for assessing diversity, as congruent conclusions were obtained by sequencing other genes (Pinel et al., 2000
; Fargette et al., 2002
) or the full genome (A. Pinel & D. Fargette, unpublished results) of representative RYMV isolates. Both serological and molecular analyses showed a large virus diversity and a geographical distribution of the strains, with a split between isolates from East and West Africa, and with different strains in the savanna and forested regions of West Africa. Isolates from Central Africa were related to isolates of the savanna strain from West Africa (Traoré et al., 2001
).
However, the studies in East Africa have been done with few isolates, so the diversity has not been assessed in this region. Furthermore, analyses with an unbalanced number of isolates from different regions may lead to biased conclusions on the phylogenetic relationships between strains (Graur & Li, 2001). Consequently, additional work is needed in East Africa to allow a comprehensive phylogeographic assessment of RYMV in Africa. Accordingly, to complement earlier studies in West and Central Africa (N'Guessan et al., 2000
, 2001
; Pinel et al., 2000
; Traoré et al., 2001
; Fargette et al., 2002
), we have assessed the diversity of RYMV in Tanzania, which is one of the largest East African countries and one of the main African rice producers where RYMV has been known for several years (Rossel et al., 1982
; Ali & Abubakar, 1995
). Isolates were collected from the main rice-producing zones of Tanzania, their serological profile was determined using a range of mAbs and their coat protein gene was sequenced.
Subsequently, a balanced and representative sample of isolates from West, Central and East Africa was subjected to phylogeographical analyses. The results suggested that: (i) there was no evidence of long-distance spread; (ii) an earlier diversification occurred in East Africa, and a later strain radiation took place in West/Central Africa; (iii) the present genetic population structure in West/Central Africa reflected adaptation to different ecosystems, whereas divergence under isolated conditions was the main evolutionary factor in East Africa; and (iv) there were phylogenetic relationships between isolates from Madgascar and isolates from the Lake Victoria region for RYMV and for two other viruses.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Immunological typing.
A panel of mAbs has been previously prepared against an isolate of RYMV from Madagascar (N'Guessan et al., 2000). Six mAbs (A, B, C, D, E and G) directed against different epitopes reacted differently against the RYMV isolates in ELISA. These mAbs were used in a triple-antibody-sandwich (TAS)-ELISA to assess the immunological profile of the RYMV isolates, as described previously (N'Guessan et al., 2000
). Polyclonal antiserum against a Madagascan isolate was used as the primary antibody in the TAS-ELISA as it consistently detected all RYMV isolates. Monoclonal antibodies in culture supernatant fluids were used as secondary antibodies to discriminate among serotypes.
Molecular typing.
Genome fragments with the coat protein gene were transcribed and amplified by RT-PCR after extraction of total RNA from leaves. RT-PCR could not be performed directly on field isolates, probably because the virus concentration was too low, and virus multiplication on the susceptible cv. IR64 was necessary (see above). However, no modification of sequences was observed after passage on this host (A. Pinel & D. Fargette, unpublished results). The protocol was that of Pinel et al. (2000). Sequencing was carried out with the Taq terminator sequencing kit (Applied Biosystems) and analysed on an Applied Biosystems 373A sequencer. Two readings per base (3'
5' and 5'
3' directions) led to sequence accuracy >99·9 %. Sequences were assembled by Seqman (DNASTAR).
Phylogenetic analyses.
The following analyses were carried out. (i) The 720 bp nucleotide sequences of the coat protein gene of the 40 isolates were aligned using CLUSTAL W (Thompson et al., 1994) with default parameters, the alignment being checked by visual inspection. (ii) The genetic distances between the isolates were expressed in matrices of pairwise nucleotide divergence percentages (Nei & Kumar, 2000
). (iii) Relationships between genetic and geographical distances were assessed with the Mantel test implemented in Genetix 4.02 (Mantel, 1967
). (iv) The diversity index (
) was calculated as described in Nei & Kumar (2000)
and related to the surface areas of land from which the isolates had been collected, assuming that the surveys roughly covered the natural distribution of the virus. (v) The phylogenetic relationships between the 40 isolates were determined by the maximum-parsimony method and a distance method where nucleotide pairwise distances were corrected using the Kimura two-parameter method and trees reconstructed by the neighbour-joining method. Both methods were implemented in the PAUP program (Swofford, 2000
). One thousand bootstrap replicates were carried out for each data set to assess the robustness of the nodes. (vi) Relationships between East African isolates were further studied using amino acid pairwise sequence distances to reconstruct the phylogenetic trees. (vii) Sequences of SPFMV and SPCSV isolates were compared to assess relationships between isolates from Lake Victoria region and from Madagascar.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Relationships between genetic and geographical distances
There was a highly significant relationship (P<0·001 after the Mantel test; see Methods) between genetic and geographical distances of the 780 pairwise comparisons from the 40 isolates (Fig. 1). On average, the greater the geographical distance, the higher the genetic distance. The best regression fit between genetic and geographical distances was after logarithmic transformation of the geographical distances (data not shown). Geographical distances between 4000 and 8000 km were associated with genetic distances between 7 and 14 % in comparisons between East and West African isolates, and between East and Central African isolates. Geographical distances between 1000 and 4000 km corresponded to genetic distances between 4 and 14 %. In no instance was great distance (>1000 km) associated with low divergence (<2 %), so there was no evidence of long-distance dispersal of isolates. Geographical distances <1000 km were associated with divergence ranging from 0·1 to 13 %. Divergences up to 9 % resulted from comparisons between West, Central and East African isolates. Divergence above 9 % was found for East African isolate comparisons.
|
This ratio is likely to be an underestimate because of sampling differences. Only 20 isolates from East Africa, including the 15 from Tanzania, were sequenced, and additional sampling is likely to reveal greater diversity. In contrast, the selected sample of 20 West/Central African isolates was from a set of 100 isolates, which was itself selected from among
200 isolates tested serologically using strain-specific mAbs (D. Fargette & A. Pinel, unpublished results). It was representative of the current diversity, as new isolates from West/Central Africa invariably fell within the groups already known (D. Fargette & A. Pinel, unpublished results).
Topology of the RYMV tree
The molecular diversity of RYMV was high and reached up to 14·3 % between an isolate from Pemba island in the Indian Ocean and one from Mali 5500 km westward. Phylogenetic trees derived from parsimony and distance analyses of sequences were congruent and only distance trees are presented (Fig. 3). The topology of the phylogenetic tree was quite unbalanced. West/Central African isolates belonged to the same phylogenetic group and different clusters could be distinguished clearly following the bifurcation pattern of the internal nodes. The group could first be divided into two clusters, one consisting mostly of isolates from the savanna regions, the other mostly from forest regions, termed savanna and forest clusters, respectively. The savanna cluster could be further divided into two groups, later regarded as strains. One included isolates from West Africa (
=2·6), the other isolates from Central Africa (
=4·1). The forest cluster was made up of one major strain (
=1·5) and a numerically minor strain (
=1·0) consisting of isolates from Sierra Leone at the far west of Africa and originating from mangrove habitats (Fomba, 1984
).
|
In Tanzania, there was a significant link between genetic and geographical distances (P<0·001). In contrast, there was no relationship between genetic and geographical distances in West Africa (P=0·14). This reflected the specific and restricted geographical range of each Tanzanian strain, and the large and partially overlapping geographical distributions of West African strains.
Phylogenetic relationships between East African strains
The proportion of observed synonymous (Ds) and non-synonymous (Dn) substitutions after the JukesCantor correction for multiple hits was calculated on nine isolates representing each of the nine strains. The average of all pairwise comparisons indicated that Ds was 0·29 and Dn 0·04. The ratio Ds/Dn was 7·3, indicating a high conservative selection pressure on the coat protein gene. Then, in an attempt to resolve uncertainties over the phylogenetic relationships among East African strains, phylogenetic trees were reconstructed from amino acid distances, since amino acids sequences are more conserved than nucleotide sequences and thus provide useful information on the long-term evolution of genes (Nei & Kumar, 2000). Actually, the amino acid distance tree suggested phylogenetic relationships between strains from Mwanza and Mbeya (Fig. 4
). Madagascan and Kenyan isolates fell within the East African phylogenetic group shared with Mwanza and Mbeya isolates, but Madagascan isolates belonged to a subcluster within this group (Fig. 4
). Similar relationships were found with non-synonymous substitution distances (data not shown) and were consistent with the serological relationships, as isolates from Mwenza and Mbeya regions and from Madagascar all shared the Ser 4 serological profile (Table 2
). In contrast, these phylogenetic relationships were not apparent using distances based on synonymous nucleotide differences (data not shown) or all differences (Fig. 3
). Similar phylogenetic relationships between virus isolates from Madagascar and from the Lake Victoria region were found with SPFMV and SPCSV. SPFMV and SPCSV isolates from Uganda at the north of Lake Victoria and isolates from Madagascar belonged to the same phylogenetic group, suggesting a common ancestor not shared with isolates outside East Africa (Fig. 5
).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The genetic structure of the RYMV population in Tanzania was characterized by an exceptionally high diversity, which was apparent through the number of strains, the level of divergence between the strains, the diversity index and the diversity index per area sampled. This greater diversity and the topology of the phylogenetic tree suggested an earlier virus diversification in East Africa. The monophyletic origin of West/Central African isolates suggested a distinct and later strain radiation in this region. The close relationship between RYMV strains and their geographical origins has been progressively elucidated. In West/Central Africa, a link has been established between specific strain and specific ecological conditions (forest, savanna, mangrove) (N'Guessan et al., 2001).
Tanzania offered a contrasting picture with highly divergent strains of RYMV in each of the different agro-ecologies, all of which differed from those in West/Central Africa. Furthermore, each Tanzanian strain had a specific and restricted geographical range, whereas West African strains had large and partially overlapping geographical distributions. Actually, this could reflect strain adaptation to different regional ecosystems: mid-altitude plateau, lake regions (Mwanza, Mbeya), valleys (Morogoro) and island (Pemba). However, a more parsimonious' scenario based on divergence, reinforced by isolated conditions, can also explain the RYMV situation in Tanzania. The high ratio of synonymous to non-synonymous substitution reflects the strong conservation pressure on the genome. It indicates that RYMV evolution has resulted from genetic drift under conservative selection, as for most animal and plant viruses (Domingo et al., 2001; García-Arenal et al., 2001
). Physical barriers such as mountain chains, lakes and sea arms in Tanzania have created isolations that have allowed divergence between strains.
Evidence is presented that isolates of RYMV from the Lake Victoria region and from Madagascar form sister groups within an East African monophyletic group. Similar relationships were found with isolates of SPFMV and SPCSV. Overall, for each of the three viruses whatever their genus or ecology isolates from Madagascar and the Lake Victoria region had a common ancestor not shared with isolates from other geographical origins in Africa. Continental islands such as Madagascar support flora and fauna closely related to the nearby mainland East Africa, which have diverged since separation between the island and the continent in the Cretaceous period 100 million years ago (Brown & Lomolino, 1998
). Accordingly, vicariance events may be responsible for the disjunction between the Tanzanian and the Madagascan strains of each of the three plant viruses, RYMV, SPFMV and SPCSV. This hypothesis applies preferentially to RYMV for which no evidence of long-distance spread has been detected. Palaeovirological speculations on co-evolution between viruses and their hosts over geological time have been made previously for Turnip yellow mosaic virus (Blok et al., 1987
), potyviruses (Ward et al., 1994
), geminiviruses (Rybicki, 1994
) and tobamoviruses (Gibbs, 1999
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abo, M., Alegbejo, M., Sy, A. & Misari, S. (2000). An overview of the mode of transmission, host plants and methods of detection of rice yellow mottle virus. J Sustain Agric 17, 1936.
Ali, F. (1999). The epidemiology of rice yellow mottle virus disease in Tanzania. PhD thesis. University of Greenwich, UK.
Ali, F. & Abubakar, Z. (1995). Incidence of RYMV in Zanzibar. In First International Symposium on Rice yellow mottle virus, p. 9. Bouaké, Côte d'Ivoire.
Alicai, T., Fenby, N., Gibson, R., Adipala, E., Vetten, H., Foster, G. & Seal, S. (1999). Occurrence of two serotypes of sweet potato chlorotic stunt virus in East Africa and their associated differences in coat protein and HSP70 homologue gene sequences. Plant Pathol 48, 718726.[CrossRef]
Bakker, W. (1974). Characterisation and ecological aspects of rice yellow mottle virus in Kenya. Agric Res Rep (Wageningen) 829, 1152.
Blok, J., Mackensie, A., Guy, P. & Gibbs, A. (1987). Nucleotide sequence comparisons of turnip yellow mosaic virus isolates from Australia and Europe. Arch Virol 97, 283295.[Medline]
Bousalem, M., Douzery, E. & Fargette, D. (2000). High genetic diversity, distant phylogenetic relationships and intraspecies recombination events among natural populations of Yam mosaic virus: a contribution to understanding potyvirus evolution. J Gen Virol 81, 243255.
Brown, J. & Lomolino, H. (1998). Biogeography, 2nd edn. Sunderland, MA: Sinauer.
Domingo, E., Biebricher, K., Eigen, M. & Holland, J. (2001). Quasispecies and RNA Virus Evolution: Principles and Consequences. Georgetown: Landes Bioscience.
Fargette, D., Pinel, A., Halimi, H., Brugidou, C., Fauquet, C. & van Regenmortel, M. (2002). Comparison of molecular and immunological typing of isolates of Rice yellow mottle virus. Arch Virol 147, 583596.[CrossRef][Medline]
Fauquet, C. & Thouvenel, J.-C. (1977). Isolation of the rice yellow mottle virus in Ivory Coast. Plant Dis Rep 61, 443446.
Fenby, N., Foster, G., Gibson, R. & Seal, S. (2002). Partial sequence of HSP70 homologue gene shows diversity between West African and East African isolates of sweet potato chlorotic stunt virus. Trop Agric 79, 2630.
Fomba, S. (1984). Rice disease situation in mangrove and associated swamps in Sierra Leone. Trop Pest Manag 30, 7381.
García-Arenal, F., Fraile, A. & Malpica, J. (2001). Variability and genetic structure of plant virus populations. Annu Rev Phytopathol 39, 157186.[CrossRef][Medline]
Gibbs, A. (1999). Evolution and origins of tobamoviruses. Philos Trans R Soc Lond Ser B Biol Sci 354, 593602.[CrossRef][Medline]
Graur, D. & Li, W. (2001). Fundamentals of Molecular Evolution, 2nd edn. Sunderland, MA: Sinauer.
Konaté, G., Traoré, O. & Coulibaly, M. (1997). Characterisation of rice yellow mottle virus isolates in Sudano-Sahelian areas. Arch Virol 142, 11171124.[CrossRef][Medline]
Konaté, G., Sarra, S. & Traoré, O. (2001). Rice yellow mottle virus is seed-borne but not seed transmitted in rice seeds. Eur J Plant Pathol 107, 361364.[CrossRef]
Krause-Sakate, R., Le Gall, O., Fakhfakh, H. & 8 other authors (2002). Molecular and biological characterisation of Lettuce mosaic virus (LMV) isolates reveals a distinct and widespread type of resistance-breaking isolate: LMV-Most. Phytopathology 92, 563572.
Kreuze, J., Karyeija, R., Gibson, R. & Valkonen, J. (2000). Comparisons of coat protein gene sequences show that East African isolates of Sweet potato feathery mottle virus form a genetically distinct group. Arch Virol 145, 567574.[CrossRef][Medline]
Makinen, K., Tamm, T., Naess, V., Truve, E., Purand, U., Munthe, T. & Saarma, M. (1995). Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses. J Gen Virol 76, 28172825.[Abstract]
Mansour, A. & Baillis, K. (1994). Serological relationships among rice yellow mottle virus isolates. Ann Appl Biol 125, 133140.
Mantel, N. (1967). The detection of disease clustering and a generalized regression approach. Cancer Res 27, 209220.[Medline]
Nei, M. & Kumar, S. (2000). Molecular Evolution and Phylogenetics. Oxford: Oxford University Press.
N'Guessan, P., Pinel, A., Caruana, M., Frutos, R., Sy, A., Ghesquière, A. & Fargette, D. (2000). Evidence of the presence of two serotypes of rice yellow mottle sobemovirus in Côte d'Ivoire. Eur J Plant Pathol 106, 167178.[CrossRef]
N'Guessan, P., Pinel, A., Sy, A., Ghesquière, A. & Fargette, D. (2001). Distribution, pathogenicity and interactions of two strains of Rice yellow mottle virus in forested and savannah zones of West Africa. Plant Dis 85, 5964.
Ohshima, K., Yamaguchi, Y., Hirota, R. & 10 other authors (2002). Molecular evolution of Turnip mosaic virus: evidence of host adaptation, genetic recombination and geographical spread. J Gen Virol 83, 15111521.
Pfosser, M. & Baumann, H. (2002). Phylogeny and geographical differentiation of zucchini yellow mosaic virus isolates (Potyviridae) based on molecular analysis of the coat protein and part of the cytoplasmic inclusion protein genes. Arch Virol 147, 15991609.[Medline]
Pinel, A., N'Guessan, P., Bousalem, M. & Fargette, D. (2000). Molecular variability of geographically distinct isolates of Rice yellow mottle virus in Africa. Arch Virol 145, 16211638.[CrossRef][Medline]
Reckhaus, P. & Andriamasintseheno, H. (1997). Rice yellow mottle virus in Madagascar and its epidemiology in the northwest of the island. Z Pflanzenkr Pflanzenschutz 104, 289295.
Ridley, M. (1996). Evolution. Oxford: Blackwell Scientific Publications.
Roossinck, M., Lee, Z. & Hellwald, K. (1999). Rearrangements in the 5' nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups. J Virol 73, 67526758.
Rossel, H., Thottapilly, G. & Buddenhagen, I. (1982). Occurrence of rice yellow mottle in two important rice growing areas of Nigeria. FAO Plant Prot Bull 30, 137139.
Rybicki, E. (1994). A phylogenetic and evolutionary justification for three genera of Geminiviridae. Arch Virol 139, 4977.[Medline]
Swofford, D. L. (2000). PAUP: Phylogenetic Analysis Using Parsimony, version 4. Sunderland, MA: Sinauer.
Thompson, J., Higgins, D. & Gibson, T. (1994). CLUSTAL W. Improving the sensitivity of the progressive multiple sequence alignment through sequence weighting, positions specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
Traoré, O., Pinel, A., Fargette, D. & Konaté, G. (2001). First report and characterization of Rice yellow mottle virus in Central Africa. Plant Dis 85, 920.
van Regenmortel, M., Fauquet, C., Bishop, D. & 8 other editors (2000). Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses. San Diego: Academic Press.
Ward, C., Weiller, G., Shukla, D. & Gibbs, A. (1994). Molecular systematics of the Potyviridae, the largest plant virus family. In Molecular Basis of Virus Evolution, pp 477500. Edited by A. Gibbs, C. H. Calisher & F. García-Arenal. Cambridge: Cambridge University Press.
Received 7 August 2002;
accepted 17 October 2002.