Biochemical characterization of the cotton bollworm Helicoverpa armigera resistance to pyrethroids in Burkina Faso.

The bollworm Helicoverpa armigera is cotton plant main pest in most parts of the world. The mechanisms of the resistance of the bollworm to the pyrethroid deltamethrin were studied by comparing field strains to the reference and susceptible strain (BK77). Resistance to deltamethrin was studied using bio-assays. Results showed that the field collected strains had susceptibility 11 to 43 fold lower than that of the susceptible standard strain BK77. Activities of two types of enzymes i.e., oxidases and Glutathione-S-Transferases (GST) were significantly higher in field strains, whereas esterase activities were lower compared to that of standard strain. The increase of oxidases and GST activities and the decrease of esterase activity are at least in part, responsible for the development of resistance of H. armigera to pyrethroids.

Recombination, selection and clock-like evolution of Rice yellow mottle virus.

The clock-like diversification of Rice yellow mottle virus (RYMV), a widespread RNA plant virus that infects rice in Africa, was tested following a three-step approach with (i) an exhaustive search of recombinants, (ii) a comprehensive assessment of the selective constraints over lineages, and (iii) a stepwise series of tests of the molecular clock hypothesis. The first evidence of recombination in RYMV was found in East Africa, in the region most favorable to co-infection. RYMV evolved under a pronounced purifying selection, but the selection pressure did vary among lineages. There was no phylogenetic evidence of transient deleterious mutations. ORF2b, which codes for the polymerase and is the most constrained ORF, tends to diversify clock-like. With the other ORFs and the full genome, the departure from the strict clock model was limited. This likely reflects the dominant conservative selection pressure and the clock-like fixation of synonymous mutations.

A reassessment of the epidemiology of Rice yellow mottle virus following recent advances in field and molecular studies.

The available knowledge on the epidemiology of Rice yellow mottle virus (RYMV) is reassessed in the light of major advances in field and molecular studies of the disease it causes in rice. Previously un-described means of transmission by mammals and through leaf contact have been discovered recently. Several agricultural practices, including the use of seedbed nurseries, have also contributed to a massive build-up of RYMV inoculum. Phytosanitation is now known to be critical to reduce disease incidence in rice. A new model of the ecology of RYMV in which man plays a central role has emerged. Furthermore, estimates of the evolutionary rate of change of RYMV provided a time-frame for its epidemiology, the first attempt for a plant virus. Earlier interpretations of the patterns of virus diversity which assumed a long-term evolution, and assigned a major role to adaptive events had to be discarded. In contrast, a wave-like model of dispersal of RYMV, which postulates its initial diversification in East Africa, followed by westward spread across the continent, was developed, refined and dated. The most salient -- and largely unexpected -- finding is that RYMV emerged recently and subsequently spread rapidly throughout Africa in the last two centuries. Diversification and spread of RYMV has been concomitant with an extension of rice cultivation in Africa since the 19th century. This major agro-ecological change increased the encounters between primary hosts of RYMV and cultivated rice. It also modified the landscape ecology in ways that facilitated virus spread.

Abiotic transmission of Rice yellow mottle virus through soil and contact between plants.

The roles of guttation fluid, irrigation water, contact between plants and transplantation into contaminated soil in the transmission of Rice yellow mottle virus (RYMV) were assessed. RYMV presence and infectivity were tested by Enzyme-Linked Immunosorbent Assay (ELISA) and by inoculation to susceptible rice cultivar BG90-2. The virus was readily detected in guttation fluid collected from infected rice plants. Transmission tests from this fluid led to high disease incidence (86.6%). Irrigation water collected at the base of infected plants growing in pots was less infectious, as inoculations led to disease incidences below 40%. No virus was detected and could be transmitted from field-irrigation water. Up to 44% healthy rice plants whose leaves were in contact with those of infected plants became infected but, no transmission occurred through intertwined roots. Transplantation of rice seedling into virus-contaminated soil also led to plant infection. However, virus survival in the soil decrease rapidly and infectivity was completely lost 14 days after soil contamination. Altogether, these results indicated that high planting densities of rice are likely to favour secondary spread of rice yellow mottle disease. Transplantation of rice seedlings not earlier than 2 weeks after soil preparation should prevent soil transmission of the virus. Although guttation fluid is highly infectious its contribution to virus infectivity in irrigation water is negligible as field-irrigation water was not found to be an infectious source for RYMV.

Rice yellow mottle virus, an RNA plant virus, evolves as rapidly as most RNA animal viruses.

The rate of evolution of an RNA plant virus has never been estimated using temporally spaced sequence data, by contrast to the information available on an increasing range of animal viruses. Accordingly, the evolution rate of Rice yellow mottle virus (RYMV) was calculated from sequences of the coat protein gene of isolates collected from rice over a 40-year period in different parts of Africa. The evolution rate of RYMV was estimated by pairwise distance linear regression on five phylogeographically defined groups comprising a total of 135 isolates. It was further assessed from 253 isolates collected all over Africa by Bayesian coalescent methods under strict and relaxed molecular clock models and under constant size and skyline population genetic models. Consistent estimates of the evolution rate between 4 x 10(-4) and 8 x 10(-4) nucleotides (nt)/site/year were obtained whatever method and model were applied. The synonymous evolution rate was between 8 x 10(-4) and 11 x 10(-4) nt/site/year. The overall and synonymous evolution rates of RYMV were within the range of the rates of 50 RNA animal viruses, below the average but above the distribution median. Experimentally, in host change studies, substitutions accumulated at an even higher rate. The results show that an RNA plant virus such as RYMV evolves as rapidly as most RNA animal viruses. Knowledge of the molecular clock of plant viruses provides methods for testing a wide range of biological hypotheses.

Host range of rice yellow mottle virus in Sudano-Sahelian Savannahs.

In the present study, we investigated on the experimental host range of RYMV among plant species most of which are frequently encountered in rice-growing environments of west and central African savannahs. Only seven out of 66 plant species inoculated were infected by RYMV. All susceptible plant species belonged to the Poaceae family and three of them (Chloris prieuri, Eragrostis cilianensis and Shoenefeldia gracilis) were reported for the first time. Symptoms were conspicuous and persistent in most species but disappeared totally in older plants of some host species such as S. gracilis and Eragrostis tenella. Therefore, surveys for identification ofRYMV wild hosts should be conducted before the flowering stage. Virus-host Interactions were studied between 15 RYMV isolates of different strains and 10 wild host species. Differential reactions were obtained in the crow-foot grass Dactyloctenium aegyptium which was susceptible to five of the fifteen isolates. All other plants were susceptible to the whole set of virus isolates. Altogether, this study underlined the narrowness of RYMV host range and pointed out the complexity of interactions between the virus and its hosts, especially the rationale behind overcoming host barriers.

Effect of cowpea seeds contamination rate by the Cowpea aphid borne mosaic virus on epidemics development.

Cowpea aphid borne mosaic virus (CABMV) diseased seeds provide at seedling, virus infected plants which are the only source of primary inoculum. Secondary infections are bequeathed by aphids. The objective of this research is to study the development of the secondary infection in field. Therefore, eight cowpea varieties with different seed contamination rate (0, 0.05, 0.25, 0.5, 1, 5%) were used over consecutive four years. The infected plants were recorded every week from the tenth day after sowing and over seven weeks. In the same way, aphids' population were evaluated in plots 30 days after sowing. There was no difference for the incidence rate between the average of plots sown with virus free-seeds and those sown with infected seeds with a rate of 0, 5%. In any case, the disease progressed lowly leading to incidences less than 50% at the post-flowering period in spite of a relatively high initial contamination rate of seed. For this group of varieties, the low progression of the disease indicated a high level of resistance to the infection. The high levels of infection especially observed with the varieties with high level of virus transmission to seed, translated the need to reduce aphids' population density notably by the use of insecticides during cowpea growing cycle. The high number of aphids and inoculum availability in the neighbouring plots were undoubtedly at the source of this result. This situation laid out the problematic of the use of seeds then little or not contaminated by the virus.

Molecular ecology and emergence of tropical plant viruses.

An appreciation of the risks caused by emergent plant viruses is critical in tropical areas that rely heavily on agriculture for subsistence and rural livelihood. Molecular ecology, within 10 years, has unraveled the factors responsible for the emergence of several of the economically most important tropical plant viruses: Rice yellow mottle virus (RYMV), Cassava mosaic geminiviruses (CMGs), Maize streak virus (MSV), and Banana streak virus (BSV). A large range of mechanisms--most unsuspected until recently--were involved: recombination and synergism between virus species, new vector biotypes, genome integration of the virus, host adaptation, and long-distance dispersal. A complex chain of molecular and ecological events resulted in novel virus-vector-plant-environment interactions that led to virus emergence. It invariably involved a major agricultural change: crop introduction, cultural intensification, germplasm movement, and new genotypes. A current challenge is now to complement the analysis of the causes by an assessment of the risks of emergence. Recent attempts to assess the risks of emergence of virulent virus strains are described.

Processes of diversification and dispersion of rice yellow mottle virus inferred from large-scale and high-resolution phylogeographical studies.

Phylogeography of Rice yellow mottle virus (RYMV) was reconstructed from the coat protein gene sequences of a selection of 173 isolates from the 14 countries of mainland Africa where the disease occurred and from the full sequences of 16 representative isolates. Genetic variation was linked to geographical distribution and not to host species as isolates from wild rice always clustered with isolates from cultivated rice of the same region. Genetic variation was not associated to agro-ecology, viral interference and insect vector species. Distinct RYMV lineages occurred in East, Central and West Africa, although the Central African lineage included isolates from Benin, Togo and Niger at the west, adjacent to countries of the West African lineage. Genetic subdivision at finer geographical scales was apparent within lineages of Central and West Africa, although less pronounced than in East Africa. Physical obstacles, but also habitat fragmentation, as exemplified by the small low-lying island of Pemba offshore Tanzania mainland, explained strain localization. Three new highly divergent strains were found in eastern Tanzania. By contrast, intensive surveys in Cote d'Ivoire and Guinea at the west of Africa did not reveal any new variant. Altogether, this supported the view that the Eastern Arc Mountains biodiversity hotspot was the centre of origin of RYMV and that the virus spread subsequently from east to west across Africa. In West Africa, specific strains occurred in the Inner Niger Delta and suggested it was a secondary centre of diversification. Processes for diversification and dispersion of RYMV are proposed.

Molecular epidemiology of the RNA satellite of Rice yellow mottle virus in Africa.

Satellite RNA was sought in 51 isolates of Rice yellow mottle virus (RYMV) representative of the geographical, molecular and pathogenic variability of the virus in Africa. Three-quarters of the isolates from cultivated rice and wild gramineaceous hosts supported a satellite RNA. The prevalence of RYMV isolates that were associated with a satellite differed among regions, being c. 100% in West and Central Africa and c. 36% in East Africa. The RYMV satellite showed a low diversity as only seven of the 220 sequenced positions were variable. One insertion also occurred after serial host passages of the satellite. Two forms of the satellite differed by six substitutions forming three base pairs in one branch of the predicted RNA secondary structure. There was no evidence of intermediates between these two forms, but double-infection occurred. Each form had a specific geographical distribution: one occurred in Central Africa, the other elsewhere in Africa. There was no relation between the occurrence or the forms of the satellite and the phylogeny of the helper virus. The satellite was not involved in symptom modulation or ability to break host-plant resistances to the disease.

First Report and Characterization of Rice yellow mottle virus in Central Africa.

Rice yellow mottle virus (RYMV) of the genus Sobemovirus is the main virus infecting rice (Oryza sativa) in Africa. First reported in Kenya (East Africa), RYMV was later found in most countries of East and West Africa where rice is grown, and in Madagascar in the Indian Ocean. In Central Africa however, the disease had never been reported in rice fields. Ninety-eight field samples with typical yellow mottle symptoms from cultivated rice and two wild rice species (Oryza longistaminata and O. barthii) were collected in the Soudano-Sahelian zones, in the north of Cameroon and the south of Chad (Central Africa) in September 2000. RYMV was detected by ELISA with polyclonal antisera (1) in all samples. All virus isolates were also mechanically transmitted to rice cv. BG 90-2, which is highly susceptible to RYMV. Tests with monoclonal antibodies showed that most isolates from Central Africa were of the SI serotype, which is widespread in the Soudano-Sahelian zones of West Africa (1). The coat protein gene of 7 isolates was amplified by RT-PCR and the expected 720 bp fragment was obtained. Resulting sequences (AJ306735, AJ317949, AJ317950, AJ317951, AJ317952, AJ317953, AJ317954) shared over 95% sequence identity. They were compared to a set of sequences of RYMV isolates from cultivated rice of different geographical origins (2). Phylogenetic analyses by maximum parsimony (PAUP 4) showed that isolates from Central Africa belonged to a monophyletic group, a sister group of West African isolates from the Soudano-Sahelian zones, further supporting the geographic basis of RYMV diversity (2). RYMV incidence was generally less than 10% but reached 20% in some irrigated plots in the two countries. References: (1) G. Konaté et al. Arch Virol. 142:1117, 1997. (2) A. Pinel et al. Arch. Virol. 145:1621, 2000.

Characterization of rice yellow mottle virus isolates in Sudano-Sahelian areas.

The use of a panel of polyclonal antisera and monoclonal antibodies (MAbs) raised against West African isolates of rice yellow mottle virus (RYMV) in ELISA resulted in separation of 73 RYMV isolates into three distinct serogroups. Using a set of differential rice varieties, the serogroups could be correlated to two RYMV pathotypes. A relationship was found between serological properties of the RYMV isolates and their probable ecological origin. It was concluded that RYMV isolates originating in closely related agroecological zones displayed variability in coat protein and pathogenicity. This should be taken into account in developing tolerant or resistant rice varieties.

Extension of the ELISA method to the measurement of the specific radioactivity of viruses in crude cellular extracts.

The double-antibody sandwich method of ELISA, which allows accurate quantitative determination of plant viruses, was extended to a radiochemical procedure which permits direct measurement of the specific radioactivity of virus labelled in vivo and present in very crude plant homogenates. Evidence is presented showing that 20 to 50% of the virus introduced in the polystyrene wells during the antigen incubation step could be trapped in the sandwich. The percentage of virus bound increased with the concentration of the coating antibody and was almost proportional to the concentration of the antigen and to the incubation time of the antigens. Complete dissociation of the double-antibody sandwich was achieved by incubation with 0.2 M KOH or NaOH (pH 13.3), and the label carried by the virus was measured by scintillation counting of the solubilization fluid. The ratio infected/healthy was much higher for the radiochemical procedure than for the immunosorbent assay itself since binding of the virus to the coating antibody was not accompanied by any nonspecific trapping of radioactive contaminants in the double-antibody sandwich. The procedure was highly sensitive since the background corresponded to the scintillation counting background. The detection of label carried by tobacco mosaic virus was possible when the tobacco samples contained at least 5 ng of virus carrying a label as low as 40 dpm 3H or 20 dpm 14C.