Medium-term and long-term in vitro conservation and safe international exchange of yam ( Dioscorea spp.) germplasm

Yam edible tubers feed million of peoples in the intertropical area, where they represent 12% of human feeding. However, as a vegetatively propagated crop, yam is seriously affected by an accumulation of pathogens. Establishing in vitro germplasm collection is a process that cleans the plants from all diseases but viruses. It gives a good control on the preservation of the yam genetic resources and facilitates international exchanges of healthy plant material. Two kinds of in vitro germplasm preservation were considered : slow growth condition culture for mid-term preservation, and cryopreservation using the encapsulation/dehydration technique for long-term preservation. Virus eradication was approached by meristem culture and chemo and thermotherapy. Production of virus-free plants was controlled by ELISA. We succeeded in the introduction and maintenance of 20 yam species, under slow growth conditions. Cryopreservation was applied successfully on two edible yam species, Dioscorea. alata L and D. bulbifera L. Virus-free plants were obtained by meristem culture in D. cayenensis-D. rotundata complex and D. praehensilis. Indexation allowed the detection of different virus (poty-, potex-, badna- and cucumovirus), where the most important potyvirus was YMV. Mid-term conservation of yam germplasm is used routinely, and from these conditions a direct acclimatization is possible. On the cryopreservation aspect, experiments are under way to apply the optimized protocol to genotypes which are more representative of the diversity, to insure a routinely use. More work can be conducted now on virus eradication, based on knowledge accumulated on potyvirus diversity, on several tests available for yam indexing (ELISA, rt/PCR, monoclonal antibodies) and on new sanitation techniques

Différenciation électrophorétique, immunologique et moléculaire des isolats du Plum Pox Potyvirus. Mise au point et évaluation de techniques de caractérisation.

*INRA, Centre de Montpellier (FRA) Diffusion du document : INRA, Centre de Montpellier (FRA) Diplôme : Dr. d'Universit

Molecular evidence for a new potyvirus species in yam (Dioscorea spp.) on the island of Guadeloupe

International audienc

First report and molecular characterization of YMMV on Dioscorea alata on the island of Martinique

umr bgpi equipe 6International audienceNaturally infected Dioscorea alata plants showing mild mosaic were collected in 1998 on the island of Martinique in the Caribbean. Isolates were first screened by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies raised against Yam mosaic virus (YMV) and antigen-coated plate ELISA with universal potyvirus monoclonal antibodies (Agdia, Elkhart, IN). A positive reaction was obtained only with the universal potyvirus antiserum. Immunocapture reverse-transcriptase polymerase chain reaction was performed for specific detection of Yam mild mosaic virus (YMMV [3]) and YMV. A product with the predicted size of 249 bp was obtained with YMMV primers. YMMV is a recently recognized distinct potyvirus infecting D. alata in West Africa and the South Pacific (2–4). It was originally described as Yam virus I and is synonymous with Dioscorea alata virus (4). To characterize the YMMV Martinique isolate, total RNA was extracted, and universal potyvirus degenerate primers (1) were used to amplify a 700-bp fragment that included the core and C-terminal region of the coat protein (CP) and 3' untranslated region (3'UTR). Sequence information generated (EMBL AJ250336) from the cloned fragment was compared with sequences of other yam potyviruses. Sequence comparisons of the partial CP (453 nt) showed a similarity of 94.6% (amino acids [aa]) with the YMMV isolate from Papua New Guinea (EMBL AB022424 [2]); 72.2% (aa) with the Japanese yam mosaic virus (JYMV) isolate (EMBL AB016500); and 67 to 73% (aa) with 27 YMV isolates. These sequences are most diverse in the 3'UTR, which showed a similarity of 72.8% with the YMMV Papua New Guinea isolate, 30% with the JYMV isolate, and 26% with the YMV isolates. These results confirm, as previously shown by S. Fuji et al. (2), that YMMV should be classified as a new potyvirus of yam. This is the first report of the natural occurrence of YMMV in the Caribbean

Isolation, characterization and cross-species amplification of microsatellite DNA loci in the tropical Americam yam Dioscorea trifida

International audienceWe developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence-enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species

The use of phylogenetic data to develop molecular tools for the detection and genotyping of Yam mosaic virus. Potential application in molecular epidemiology

UMR BGPI Equipe 6International audienceMolecular detection of Yam mosaic virus (YMV), the most important potyvirus for yam plants (Dioscorea spp.) has been limited by its high genetic diversity. According to recent phylogenetic data, suitable molecular tools were developed for detection and genotyping. A reliable and rapid molecular test is described using single-tube immunocapture (IC)-RT-PCR combining the use of a monoclonal antibody able to recognise all YMV serotypes and primers designed to take into account the whole variability of YMV. Phylogenetic data were then used to develop an uncoupled IC-RT-PCR procedure adapted to the genotyping of the three major phylogenetic groups and the two recombinants implicated in the YMV epidemics in the Caribbean and French Guiana. A third exploitation of phylogenetic data was carried out with the development of a typing test based on the analysis of short nucleotide sequences. The direct sequencing of a 450 nts fragment constituted a reliable complementary typing tool as the resulting information was quite similar to that obtained with sequences of the complete coat protein gene. The approach, consisting in a robust study of YMV variability followed by the use of phylogenetic data to develop reliable detection and genotyping tools, offers new perspectives for powerful molecular epidemiological studies

Potentialité d'invasion, en Méditerranée occidentale, par des potyvirus proches du Papaya ringspot virus (PRSV)

Supplément 1National audienc

Choix de techniques simples pour distinguer des variants du plum pox virus

International audienc

Origin, world-wide dispersion, bio-geographical diversification, radiation and recombination : an evolutionary history of Yam mild mosaic virus (YMMV)

BGPI - Equipe 6International audienceWe developed an evolutionary epidemiological approach to understand the regional and world-wide dispersion of Yam mild mosaic virus (YMMV) by retracing its evolutionary history. Analyses of the distribution and the prevalence of YMMV in the Caribbean islands of Guadeloupe and Martinique, and in French Guyana revealed that YMMV has a wide repartition and different prevalence on Dioscorea alata L. (Asian and Oceanic origin), on D. cayenensis Lam.–D. rotundata Poir. (African origin) and on D. trifida L. (Amazon and the Caribbean origin) in this region. Considering the data on the current dispersion of the virus and the evolution and the history of the yams, the phylogenetic analysis of the 3′ terminal part of the YMMV genome gave a consistent support of the Asian-Pacific origin of YMMV from D. alata species. The YMMV phylogenetic tree is star-like, suggesting an early split of the genetic lineages. An important part of the clades is constituted by a single lineage arisen by recombination. The largest emerging monophyletic group illustrates well YMMV geographical dispersion. This evolutionary pattern contrasts with the one revealed by the African distinct lineages and by the second significant monophyletic group, for which a host adaptation to D. trifida is suggested. The analysis of the pattern of nucleotide substitutions in the CP gene revealed that purifying selection dominates the evolution of the CP of potyviruses and strongly operates on the YMMV. Switching events, radiation, host and geographical adaptation and recombination events are proposed as major traits of YMMV evolutionary history

Correlation entre trois techniques permettant de différencier des isolats de plum pox virus

International audienc