Search

International audience; In Guadeloupe, a French overseas territory located in the Eastern Caribbean, infectious and non-infectious diseases, loss of biodiversity, natural disasters and global change threaten the health and well-being of animals, plants, and people. Implementing the “One Health” (OH) approach is crucial to reduce the archipelago's vulnerability to these health threats. However, OH remains underdeveloped in Guadeloupe, hampering efficient and effective intersectoral and transdisciplinary collaborations for disease surveillance and control. A multidisciplinary research group of volunteer researchers working in Guadeloupe, with collective expertise in infectious diseases, undertook a study to identify key attributes for OH operationalization by reviewing past and current local collaborative health initiatives and analyzing how much they mobilized the OH framework. The research group developed and applied an operational OH framework to assess critically collaborative initiatives addressing local health issues. Based on a literature review, a set of 13 opinion-based key criteria was defined. The criteria and associated scoring were measured through semi-directed interviews guided by a questionnaire to critically evaluate four initiatives in animal, human, plant, and environmental health research and epidemiological surveillance. Gaps, levers, and prospects were identified that will help health communities in Guadeloupe envision how to implement the OH approach to better address local health challenges. The methodology is simple, generic, and pragmatic and relies on existing resources. It can be transposed and adapted to other contexts to improve effectiveness and efficiency of OH initiatives, based on lessons-learned of local past or current multi-interdisciplinary and intersectoral initiatives.

International audience

International audience

Edible banana arose from combinations of species Musa acuminata, whose genome is denoted A, and M. balbisiana, whose genome is denoted B. The nuclear genome of all known diploid BB genitors harbors infectious alleles of endogenous Banana streak viruses (eBSV). Their activation by abiotic stresses leads to spontaneous infections by cognate viruses in diploid AB, triploid AAB and tetraploid AAAB interspecific hybrids. Banana streak viruses are naturally transmitted by several species of mealybug. A comprehensive risk assessment study of the activation of infectious eBSV alleles was undertaken in Guadeloupe. Firstly, a large scale prevalence survey was carried out for viral species BSOLV, BSGFV and BSIMV among the main cultivated banana types. Overall prevalence levels were low for BSOLV (0.85%) and BSGFV (0.99%). No sample was found infected by BSIMV. No impact of BSV in infected plants could be observed, with only one infected sample displaying typical BSV leaf streak symptoms. The comparison of prevalence levels monitored in AAB genotypes and in AAA genotypes suggests that BSOLV and BSGFV infections in AAB genotypes result preferentially from the activation of eBSV infectious alleles rather than from vector-borne transmission. Statistical analyses showed a positive correlation between prevalence levels and altitude. Secondly, the activation of eBSOLV and eBSGFV infectious alleles was monitored under field conditions over two production cycles in triploid plantains French Clair (AAB genotype) and Pelipita (ABB genotype), using a random block assay and plants originating from either cell culture or horticultural multiplication. Activation rates were significantly higher for infectious allele GF7 than for infectious allele OL1 and also higher in French Clair than in Pelipita. Results pointed to an influence of the mode of production of planting material on activation, and to an absence of impact of BSGFV and BSOLV infection on production.

Yams (Dioscorea spp.) are important staple food worldwide, particularly in West Africa and the South Pacific, and also in the Caribbean. Badnaviruses (family Caulimoviridae) are studied extensively due to their high prevalence and economic impact on important tropical crops such as cocoa, banana, sugarcane, citrus and yams, and to their high molecular diversity [1]. Until recently, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV) are the only yam-infecting badnaviruses whose genomes have been entirely sequenced [2, 3]. DBALV and DBSNV sequences were used for phylogenetic studies, together with partial nucleotide sequences generated by PCR using badnavirus-specific. These analyses unveiled the existence of 12 distinct badnavirus groups of species in yams [4, 5]. However, three additional species and four new episomal genomes have been characterized by three recent and independent studies. The use of the RCA (Rolling Circle Amplification) to distinguish episomal and endogenous badnaviral sequences highlighted that African yams (complex of species Dioscorea cayenensis-rotundata) were infected by three new Badnavirus species which were never been identified yet, belonging to groups 13, 14 and 15 [6]. Two of them have been fully sequenced and called DBRTV1 (Dioscorea bacilliform rotundata virus 1; group 13) and DBRTV2 (Dioscorea bacilliform rotundata virus 2; group 14). At the same time, a badnavirus prevalence study of the yam collection in the French West Indies Biological Resources Centre for Tropical Plants (BRC-TP) showed that America yams (D.trifida) were infected by only two distinct species from groups 8 and 9 [7]. This study used immunocapture-PCR (IC-PCR) to avoid false positive due to endogenous badnaviral sequences. Nucleotide sequences from group 9 were only reported as endogenous forms in D. cayenensis-rotundata genomes, so this species has been sequenced and called DBTRV (Dioscorea bacilliform trifida virus). Furthermore, in silico analyses of shotgun sequences from a D.alata genome displayed a complete badnavirus genome from group 15, showing that this plant was likely infected by a new uncharacterized species unveil however by RCA. Thus, three different approaches have led to the characterization of four new episomal sequences of yam badnavirus and pointed out that yams from different geographical areas were infected by distinct Badnavirus species. Moreover, the absence of DBTRV-infected plants among D. cayenensis-rotundata accessions studied in two first studies could be explained by the presence of eDBV9 sequences in these yam species that could trigger silencing-based resistance against DBTRV.

Several species of Banana streak virus (BSV; family Caulimoviridae, genus Badnavirus) occur in banana (Musa spp). They cause a wide range of symptoms on the leaves, pseudostem and fruits of infected plants, although the impact of infections on yield and fruit quality has never been properly assessed. BSVs are naturally transmitted by mealybugs. However, infections can also occur spontaneously in interspecific varieties following stress-induced activation of infectious endogenous BSV sequences (eBSVs) integrated in B (Musa balbisiana) genomes. A 484 plants experimental plot using a random block design was set up in Guadeloupe in order to study the kinetics and impact of BSV infections resulting from the activation of infectious eBSVs in two triploid interspecific plantain varieties : French Clair (AAB genotype), and Pelipita (ABB genotype). Both varieties harbor similar infectious endogenous sequences of Banana streak OL virus (BSOLV) and Banana streak GF virus (BSGFV). Planting material was multiplied either by cell culture (vitroplants) or by a corm splitting technique (PIF). Activation of infectious eBSOLV and eBSGFV sequences was monitored every three months by immunocapture-PCR indexing [1, 2] of all the plants of the plot. Data gathered over a 15 months period showed that activation of infectious eBSGFVs occured in both varieties, although activation rates were significantly higher in French Clair. Activation of infectious eBSOLVs occurred only in French Clair, at a much lower rate. These results suggest that infectious eBSGFVs and eBSOLVs are more prone to activation by abiotic stresses under field conditions in French Clair than in Pelipita, pointing to a role of plant genetic background in the activation process. They also suggest that in French Clair, infectious eBSGFVs are more prone to activation than infectious eBSOLVs, confirming previous observations made in cell culture and pointing to differential activation potentials among infectious eBSVs [3]. Our data also showed that activation of eBSGFVs in French Clair was higher in plants originating from PIF than in plants originating from vitroplants. No significant impact of BSOLV and BSGFV infection on plant growth and fruit production was observed, and none of the infected plant did express BSV symptoms.

Several species of Banana streak virus (BSV) occur in banana. They cause a wide range of symptoms on leaves, pseudostem and fruits, although the impact of infections on yield and fruit quality has never been properly assessed. BSVs are naturally transmitted by mealybugs. However, spontaneous infections occur in interspecific varieties such as plantains, following stress-induced activation of infectious endogenous BSV sequences (eBSVs) integrated in B (Musa balbisiana) genomes. The kinetics of activation of infectious eBSVs was monitored in Guadeloupe in an experimental plot, using a random block design. It showed that infectious eBSVs display differential activation potentials in plantain varieties French Clair and Pelipita, pointing to a role of plant genetic background in the activation process. It also showed that the multiplication mode of planting material influences activation levels monitored under field conditions and that infection had no significant impact on plant growth and fruit production of both varieties. A wide range prevalence study of BSVs undertaken throughout Guadeloupe's plantations, Creole gardens, abandoned fields and wild areas among varieites representative of the main dessert banana and plantain types grown in Guadeloupe showed that overall BSV prevalence were low in dessert banana and cooking banana. Compared with a similar survey carried out in 2006, prevalence was very similar for dessert banana but significantly lower for plantains, which carry eBSVs that interfere with molecular diagnostic and cause frequent false positives. It is likely that the recent optimization of BSV molecular diagnostic increased the accuracy of detection. Overall, these results suggest that BSVs have a low prevalence and unmeasurable impact on dessert banana and plantain in Guadeloupe, owing to low vector-borne transmission and low activation of infectious eBSVs. These results also lead to recommendation regarding the management of BSVs through safe multiplication modes of plantain planting material.

French West Indies Biological Resources Centre for Tropical Plants (CRB-PT) maintains several germplasm collections of tropical crops and wild relatives, including a collection of 480 yam accessions (Dioscorea spp.) in vitroculture. The purpose of this Centre is to conserve this biodiversity and distribute virus-free germplasm to end users. To this aim, virus populations infecting conserved accessions have been characterized and the diversity of intra and inter-species highlighted through the SafePGR project. Thus, three new virus generi have been found out (Ampelovirus, Macluravirus and a Secoviridae member) besides those existing (Badnavirus [1], Potyvirus [2] and Potexvirus [3]). Efficient extraction method and appropriate detection tools have been created and/or optimized, then implemented for an initial diagnosis of the in vitro collection. Prevalence shown that more than 75% of yam accessions are infected by Potyvirus, 80% of D. alata, the most cultivated yam, are infected by the new yam virus Ampelovirus and Macluravirus are only present in this yam species. Only 19 accessions remained free of viruses, but the majority of the collection contained one, two or more of viruses, so the necessity to sanitize the infected germplasm. The sanitation process consists to submit shoots to thermotherapy at 34°C, then excise apical meristem and obtain the regeneration of a new plant, expecting to be free of viruses. Using optimized detection tools, each new plant is diagnosed for the six viral generi. Up to now, the sanitation process leads to the achievement of 10 accessions completely virus-free. The development and yield of this improved plant material have to be evaluated by comparison of infected and sanitized accessions. Macluravirus, which only infects D. alata with a low prevalence, are removed quite easily (100% of sanitation). YMV, which leads the strongest loss of yield in yam, has a good sanitation rate of 56% and YMMV, that infects 76% of the collection, has a sanitation rate of only 16%. The sanitation is a time-consuming process, not quite efficient, but necessary for yam germplasm international exchange.

National audience; Plant genetic resources are instrumental in the adaptation of agriculture to social and environmental change. They are the backbone of research and breeding programs aimed at the development and transfer of new crop varieties best suited to consumers’ needs and tastes and to new farming systems. To this aim, plant germplasm collections have been constituted worldwide. Securing such collections requires substantial human and financial investments that can prove difficult to maintain on the long run for small countries and territories such as most Caribbean countries. In Guadeloupe and Martinique, INRA and CIRAD have constituted large plant germplasm collections of tropical crops over several decades. They joined forces in 2010 to create the Tropical Plant Biological Resource Center of the French West Indies (CRB-PT), which is affiliated to both institutions. In this paper, we describe CRB-PT’s collections, services provided to end users and research programs as well as scientific and technical networking strategy.

Breeding new interspecific banana hybrid varieties with pests and disease resistance characters is instrumental for the development of pesticide-free agroecological banana farming systems. Such breeding relies on the combined use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of M. balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus (BSV) in interspecific hybrids harbouring both M. acuminata and M. balbisiana genomes. In this work, we show that M. balbisiana diploid genitors available for breeding host at least one infectious eBSV. We also show how we segregated infectious and non-infectious eBSV alleles in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. This work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.

French West Indies Biological Resources Centre for Tropical Plants (CRB-PT) maintains several germplasm collections of tropical crops and wild relatives, including a collection of more than 450 yam accessions (Dioscorea spp) in vitroculture. The purpose of this Centre is to conserve this biodiversity and distribute virus-free germplasm to end users. To this aim, virus populations infecting conserved accessions have been characterized and the diversity of intra and inter-species highlighted through the SafePGR project. Thus, three new virus generi have been found out (Ampelovirus, Macluravirus and Sadwavirus) besides those existing (Badnavirus [1], Potyvirus [2] and Potexvirus [3]). Efficient extraction method and appropriate detection tools have been created and/or optimized, then implemented for an initial diagnosis of the in vitro collection. Prevalence shown that more than 75% of yam accessions are infected by Potyvirus, 80% of D. alata, the most cultivated yam, are infected by the new yam virus Ampelovirus and Macluravirus are only present in D. alata. Only 14 accessions remained free of viruses, but the majority of the collection contained one, two or more of viruses, so the necessity to sanitize the infected germplasm. The sanitation process consists to submit shoots to thermotherapy at 34°C, then in excising apical meristem to obtain the regeneration of a new plant, expecting to be free of viruses. Using optimized detection tools, each new plant is diagnosed for the six viral generi. Up to now, the sanitation process leads to the achievement of 8 accessions completely virus-free. The development and yield of this improved plant material have to be evaluated by comparison of infected and sanitized accessions.

Depuis plusieurs années, des programmes de création de variétés d’ignames performantes, initiés à la demande des professionnels, ont abouti à la création de seize nouvelles variétés adaptées aux contraintes de production de Guadeloupe et conformes aux attentes des producteurs et des consommateurs. D'autres variétés sont attendues dans les années à venir. Ces dernières ont déjà été pré-évaluées en stations expérimentales et sont maintenant prêtes à être diffusées chez les producteurs. Plus récemment, de nouvelles variétés de madères ont été introduites en Guadeloupe afin de diversifier l’offre sur le territoire. Ces variétés, introduites sous formes de vitroplants sains, ont commencé à être multipliées pour être diffusées. Aujourd’hui, il est indispensable de multiplier, diffuser et promouvoir ces nouvelles variétés d’ignames et de madères auprès des agriculteurs, sous forme de matériel végétal de qualité. Pour y arriver, deux grands objectifs sont poursuivis dans le cadre de ce projet : i) Assurer la production et la diffusion de semences d’ignames de qualité. Ceci pourra être réalisé directement, ou par la mise en place d’un dispositif durable et économiquement viable, qui associe l’ensemble des acteurs de la filière. ii) Développer et promouvoir le portefeuille variétal. Pour cela, il est prévu de diffuser et caractériser ces nouvelles variétés dans les différentes zones de production, dans le cadre d’un réseau participatif. Ceci permettra d'accompagner les choix des producteurs, et de diffuser également des variétés traditionnelles, sous forte pression sanitaire, pour lesquelles l’accès aux semences par les professionnels est très limité. L’enjeu à moyen terme de ce projet est donc de mettre en place un dispositif de production de plants d’ignames et de madères de qualité en Guadeloupe, qui repose sur une forte implication des professionnels agricoles. Dans le même temps, le projet vise à accompagner le développement d’une structure locale de production de vitroplants de qualité, l’EARL CAVI.

Blast annotations of 44,164 CAP3-assembled sequences of public ESTs (NCBI) revealed the presence of a putative new macluravirus species infecting South-East Asian tropical yam species D. alata in Nigeria. Based on this new information, the diversity of macluraviruses was investigated in tropical yam germplasm using four complementary approaches: 1) Sanger sequencing of cloned yam-macluravirus-specific RT-PCR amplicons, 2) virion-associated nucleic acids (VANA), 3) dsRNA and 4) siRNA deep sequencing. Primers were designed in the cp gene of yam macluraviruses, based on the alignment of ESTs sequences and sequences of known viral species (Chinese yam necrotic mosaic virus and Yam chlorotic necrotic mosaic virus), and used to screen the worldwide yam germplasm collection maintained by Guadeloupe's Biological Resource Centre for Tropical Plants (CRB-PT), by RT-PCR performed on total nucleic acids. Additional samples from India and South Pacific Islands were also screened. Sanger sequencing of the RT-PCR amplicons revealed the existence of two novel macluravirus species in tropical yams: one species tentatively named Dioscorea alata macluravirus was identified in D. alata and D. nummularia, whereas a distinct species, tentatively named Dioscorea esculenta macluravirus, was identified in D. esculenta. These two species appear to be present in Nigeria, Guadeloupe, India and in some South Pacific Islands (Palau, Papua New Guinea, Tonga, Vanuatu), suggesting a worldwide distribution. siRNA sequences from a D. alata plant collected in India were assembled in contigs, leading, after Blast annotation, to the complete genomic sequence of a strain of Dioscorea alata macluravirus, which was confirmed by Sanger sequencing of RT-PCR and 3'-RACE amplicons. Using 454 pyrosequencing of VANA or cDNA synthesized from purified viral dsRNAs, RT-PCR and RACE analyses, the complete genomic sequence of another strain of Dioscorea alata macluravirus was obtained.

National audience

A comparative assay of 8 viral metagenomic protocols based on next generation sequencing (NGS) was performed on 6 tropical and subtropical crops (banana, garlic, sugarcane, sweet potato, vanilla and yam) originating from the Azores, Guadeloupe, Madeira and Réunion. Roche 454-based sequencing of purified dsRNA and virion-associated nucleic acids (VANA) proved the best strategies for recovering plant virus sequences following a first screening performed on a set of 96 plant samples. These two metagenomics approaches were further used on a total of 740 plant samples of the 6 targeted crops. Following demultiplexing, reads were either mapped against viral references or assembled into contigs and annotated using Blast analyses, leading to the identification and molecular characterization of 5 new viral species in garlic, 10 in yams, 7 in sugarcane, and 3 in vanilla. Sequences hinting at the possible presence of additional novel agents were also identified. In addition, a systematic search was performed on publicly available EST resources for the 6 targeted crops in order to identify yet uncharacterized viral agents. For all newly identified viruses, corresponding EST sequences were downloaded, assembled in contigs and taxonomic relationships were established. Sequences assembled from NGS reads and EST datamining were used to design specific primers for the detection of the novel viral species, allowing studies of their prevalence and diversity in germplasm collections conserved in European outermost regions and providing insights into transmission modes of some of these novel viruses. The potential of metagenomics for plant virus discovery and diagnostics will be discussed, together with the risk of complicating the exchange of plant materiel following the discovery of new virus species in conserved plant germplasm.

Three new Potexvirus species infecting yams were identified by RT-PCR performed on total nucleic acids (TNAs) prepared from yams of the Guadeloupe Centre for Biological Resources (CRB PT) germplasm collection [1], using degenerate primers potex2 and potex5 [2] which target the RdRp domain of potexviruses ORF1. However, two different amplification products were obtained with some samples. One product had a size smaller than expected. Its sequence is closely related to those of sadwaviruses RNA1 and clearly belongs to a yet undescribed viral species. Additional data resulting from yam ESTs datamining point the existence of a sadwavirus species infecting yams. Primers specific for the RNA1 and RNA2 of this novel virus species were designed based on the PCR fragment and on yam ESTs, respectively, and used in RACE experiments. Additional nucleotide sequences corresponding to the same agent were gathered from deep sequencing reads performed on yam accessions from CRB PT's germplasm collection, leading to the assembly of the complete genome of a novel sadwavirus, for which the name Yam necrosis virus (YNV) is proposed. Yam ESTs datamining also retrieved sequences of viruses of the family Closteroviridae. Complete genomes of two distinct viral species were assembled from 454 and Illumina deep sequencing reads obtained on yam accessions from CRB PT's germplasm collection: a novel member of the Ampelovirus genus, for which the name Yam ampelovirus 1 (YAV-1) is proposed, and Cordyline virus 1 (CoV-1), a member of the tentative genus Velarivirus previously reported in Hawaï on Cordyline fruticosa, an evergreen flowering plant in the Asparagus family [3]. Specific primers to YNV and YAV-1 were designed and used, together with published CoV-1 specific primers [3], to assess the prevalence of these three viruses in CRB PT's germplasm collection providing insight into the diversity and epidemiology of these viruses.

Le projet avait pour objectif de sécuriser la conservation et intensifier la connaissance et la diffusion des collections de plantes cultivées du Centre de Ressources Biologiques des Antilles. Pour cela, l'acquisition d'équipements de laboratoire complémentaires a été nécessaire et les serres de confinement ont dû être rénovées afin de pouvoir protéger les plants pendant le processus d’assainissement ou d'introduction. Les grands objectifs suivant ont été poursuivis : Acquérir de nouvelles variétés, en accord avec les règles de quarantaine. Poursuivre les caractérisations, en mettant l’accent sur le génotypage et sur les traits fonctionnels d’intérêt. Cela permettra de mieux connaître la diversité des collections, et de les utiliser à moyen terme dans des programmes d’amélioration génétique. Poursuivre l’assainissement des pieds-­mères de maladies virales qui rendent impossible tout échange de matériel. Poursuivre les recherches sur les virus intégrés dans le génome de la plante. Certifier le dispositif selon la nouvelle norme en vigueur (NF S-­96 900). Gérer et porter à connaissance les informations afférentes aux collections.

Les Antilles hébergent un riche patrimoine végétal, que ce soit dans les espaces naturels, au sein des exploitations agricoles, ou ex situ dans des collections de ressources génétiques. Ces ressources biologiques diverses de par leur nature et leur origine constituent un patrimoine valorisable pour le développement agricole et pour l'agro-transformation. Ces ressources permettent et permettront d’élaborer et/ou diffuser des variétés végétales et itinéraires techniques associés, permettant de répondre aux évolutions qui surviennent dans nos régions. Ces évolutions sont liées aux maladies et ravageurs émergents, aux changements du climat, des façons de produire, transformer et commercialiser, changements des habitudes et souhaits des consommateurs et des politiques, de la réglementation... Face à ces évolutions, l'ensemble des filières agricoles et leurs acteurs doivent s'adapter, notamment en s'appuyant sur de nouveaux idéotypes variétaux. Certaines ressources génétiques végétales du CRB INRA-CIRAD Plantes Tropicales des Antilles mobilisées en innovation variétale dans le projet CAVALBIO peuvent contenir des allèles d'adaptation à ces changements. Une meilleure valorisation des ressources génétiques devrait permettre d'offrir des systèmes de production plus diversifiés en termes d’espèces et de gammes variétales. Cette diversification des cultures a été identifiée comme incontournable aux Antilles françaises pour répondre à l'enjeu de l’augmentation de l'auto-suffisance et d’une alimentation plus équilibrée. Elle contribue également à faciliter la protection intégrée des cultures. Il est aujourd’hui nécessaire de consolider et pérenniser le dispositif, afin qu’il soit en mesure de conserver à long terme ses ressources génétiques, à enrichir les collections, et à les diffuser plus largement. Les objectifs du projet s’inscrivent dans les missions des CRB, qui consistent à enrichir, conserver, et mettre à la disposition des utilisateurs de la recherche, des acteurs du développement et de l’enseignement, les ressources biologiques et les informations associées à ces dernières (données d’origine, caractéristiques génétiques et agronomiques).

French West Indies Biological Resources Centre for Tropical Plants (CRB-PT) maintains several germplasm collections of tropical crops and wild relatives, including a collection of more than 450 yam accessions (Dioscorea spp). The purpose of this Centre is to conserve and distribute virus-free germplasm to end users. To this aim, virus populations infecting conserved accessions are characterized and appropriate detection tools are created or optimized, then implemented for the sanitation of infected germplasm. Several badnavirus species have been reported in yams. Recently, endogenous Dioscorea badnaviral sequences (eDBVs) were described in the genome of African yams of the D. cayenensis-rotundata complex [1, 2]. These sequences interfere with badnavirus PCR-based detection methods and are therefore a constraint for the accurate diagnostic of badnaviruses in yams. To address this problem, an analysis of the diversity of eDBVs was carried out in D. rotundata, using virus-free seedlings. It showed that sequences from at least four distinct Badnavirus species are integrated in yam genomes, and that these viral species belong to the groups 5, 8, 9 and 12 defined by Kenyon et al. [3]. This finding enabled the development and implementation of a reliable technique for the specific detection of badnavirus episomal forms in yams. Using this method, we showed that the CRB-PT yam collection hosts different strains of Dioscorea bacilliform AL virus (DBALV; group 8) and yet unreported episomal forms of one badnavirus species of group 9, for which only endogenous forms were known so far. The genome of this new yam badnavirus species was amplified from an infected Dioscorea trifida accession, cloned and sequenced. Its organization is similar to that of DBALV and Dioscorea bacilliform SN virus (DBSNV; group 4). However, phylogenetic analyses showed that it is a novel and distinct badnavirus species, for which the name Dioscorea bacilliform TR virus (DBTRV) is proposed. This first report of the occurrence of endogenous sequences (eDBV8 and eDBV9) from extant badnavirus species in yams should suggest that some eDBVs could be infectious. Conversely, molecular evidence supporting the role of these eDBVs in antiviral defense will also be presented.

International audience; Several endogenous viral elements (EVEs) have been identified in plant genomes, including endogenous pararetroviruses (EPRVs). Here, we report the first characterization of EPRV sequences in the genome of African yam of the Dioscorea cayenensis-rotundata complex. We propose that these sequences should be termed ‘endogenous Dioscorea bacilliform viruses' (eDBVs). Molecular characterization of eDBVs shows that they constitute sequences originating from various parts of badnavirus genomes, resulting in a mosaic structure that is typical of most EPRVs characterized to date. Using complementary molecular approaches, we show that eDBVs belong to at least four distinct Badnavirus species, indicating multiple, independent, endogenization events. Phylogenetic analyses of eDBVs support and enrich the current taxonomy of yam badnaviruses and lead to the characterization of a new Badnavirus species in yam. The impact of eDBVs on diagnosis, yam germplasm conservation and movement, and breeding is discussed.

Infections of banana and plantain by banana streak viruses (BSV) can occur in the absence of vector-mediated transmission, through the activation of infectious endogenous BSV sequences (eBSVs). Such infectious eBSVs are present in the genome of Musa balbisiana spp, which are important progenitors for breeding improved banana varieties. Once activated by biotic or abiotic stresses, these viral integrants cause spontaneous infection in both natural and synthetic interspecific hybrids harbouring the M. balbisiana (B) genome. Therefore, the presence of infectious eBSVs within B genomes is the main constraint for breeding banana and plantain interspecific hybrids and for exchanging Musa germplasm. The sequence and organization of eBSVs in the diploid M. balbisiana genitor Pisang Klutuk Wulung (PKW) was elucidated for BSV species Obino l'EwaI (BSOLV), Goldfinger (BSGFV) and ImovO (BSIMV). This work showed that integration of BSGFV and BSOLV is di-allelic, with one infectious and one non-infectious allele, whereas that of BSImV is monoallelic. Allele-specific molecular markers were developed and used to genotype M. balbisiana germplasm, unveiling important differences between accessions and the presence of modified eBSV alleles in several accessions. Breeding improved M. balbisiana progenitors devoid of infectious eBSGFV and/or eBSOLV alleles was achieved through self-pollination and chromosome doubling of haploid lines. Both approaches lead to M. balbisiana cultivars devoid of infectious eBSOLV and/or eBSGFV resulting from the segregation of eBSOLV and eBSGFV alleles. These results pave the way to the safe use of M. balbisiana in breeding programs, and open new perspectives for breeding improved banana and plantain hybrid varieties. (Texte intégral)

National audience

International audience; Since 2010, INRA and CIRAD have jointly set up the Biological Resources Centre (BRC) for ‘Tropical Plants’ in the French West Indies. The BRC is a germplasm repository, which carries out its activities with quality control procedures in the fields of acquisition, conservation and provision of plant material. The purpose of the BRC is to secure conservation of Banana, Mango, Pineapple, Sugarcane and Yam germplasm collections and to provide users such as research institutions, extension services and the private sector with healthy plant material. The BRC currently hosts a total of 1 800 accessions conserved in vitro and/or in the field, including 450 yam accessions belonging to the main cultivated species and wild relatives. Databases and a web portal (http://collections.antilles.inra.fr/) provide access to the conserved accessions and related information. Besides BRC’s core activities, scientific projects are developed aiming to enhance safety of plant material movements through sanitation and improved viral diagnostic. On yam species, the molecular diversity of the main viral families present in the germplasm is being analyzed, including endogenous viral elements. In addition, classical diagnostic methods are being optimized, taking into account the new data on viral diversity thus generated, with the objective to develop new multi-pathogen diagnostic methods based on metagenomics and deep-sequencing technologies.

Banana streak viruses (BSV) infect bananas and plantains worldwide. They are naturally transmitted by mealybugs; however infections can also occur in the absence of vectormediated transmission, through the activation of infectious endogenous BSV sequences (eBSVs). Infectious eBSVs are present in the genome of Musa balbisiana spp, which are important progenitors for breeding improved banana varieties. Once activated by biotic or abiotic stresses, these viral sequences cause spontaneous infection in both natural and synthetic interspecific hybrids harbouring the M. balbisiana genome, denoted B [1]. Therefore, the presence of infectious eBSVs within B genomes is currently the main constraint for breeding banana and plantain interspecific hybrids and for exchanging Musa germplasm. The sequence and organization of eBSVs in the diploid M. balbisiana genitor Pisang Klutuk Wulung (PKW) was elucidated [2], showing that integration of infectious eBSGFV [3] and eBSOLV is di-allelic, with one infectious and one non-infectious allele, whereas that of infectious eBSImV is monoallelic [2]. Taking advantage of the development of allele-specific molecular markers [2; 4], eBSV signatures were established for all M. balbisiana genitors of the CIRAD Guadeloupe Musa collection. This work unveiled important differences between accessions. All combinations of infectious and non-infectious alleles were observed for the three BSV species, as well as complete and uncomplete integrants when compared to those described in PKW. Breeding improved M. balbisiana progenitors devoid of infectious eBSGFV and/or eBSOLV alleles was undertaken through self-pollination and chromosome doubling of haploid lines. Both approaches successfully lead to M. balbisiana cultivars devoid of infectious eBSOLV and/or eBSGFV resulting from the segregation of eBSOLV and eBSGFV alleles. Improved lines of one particular M. balbisiana cultivar, cv. Honduras, originally free of eBSImV, were shown to be free of infectious eBSV. These results pave the way to the safe use of M. balbisiana in breeding programs, and open new perspectives for breeding improved banana and plantain hybrid varieties. (Texte intégral)

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak viruses(eBSVs), although integration is not required for the replication of cognate viruses. In the past 20 years, BSV outbreaks monitored in banana-producing areas resulted mostly from the activation of infectious eBSVs in newly created interspecific banana hybrids. Recently, we established that these problematic infectious eBSVs are present in theMusa balbisiana genomes,one of the main group of progenitors used for breeding hybrid varieties [1-4]. We also elucidated the sequence and organization of eBSVs in the model diploid M. balbisiana cultivar PisangKlutukWulung (PKW), showing that integration of infectious eBSGFV[5] and eBSOLV is di-allelic, with one infectious and one non-infectious allele, whereas that of infectious eBSImV is monoallelic[6].Based on the sequences and the structure of these eBSV we have developed several PCR and Derived Cleaved Amplified Polymorphic Sequences (deCAPS) markers for genotyping eBSVs[4 ; 6].To this aims, different markers were developed: (i) integration markers targeting eBSV insertion sites, (ii) structure markers targeting internally reorganized eBSV structures and (iii) allelic markers allowing the distinction between alleles of the same eBSV species. Markers were assessed by screening the same banana genotypes in two different laboratories located in Montpellier and Guadeloupe respectively. A larger scale validation was undertaken through the screening ofall Musa balbisiana genotypes available fromCIRAD Musa collection in Guadeloupe, newly created hybrids from CARBAP in Cameroon and germplasm collection provided by Bioversity International in Belgium. The use of these molecular tools is now a prerequisite not only for future crop-oriented breeding programmes aimed atproducing safe interspecific banana hybrids but also for assessing the risk of BSV outbreaksresulting from the activation of infectious eBSVs in natural hybrids that have been widely distributed in developing countries. (Texte intégral)