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L'accès à une plus grande biodiversité de Musa pourrait permettre la création de nouvelles bananes pour répondre à la demande alimentaire croissante. Actuellement, une grande partie de la production mondiale repose sur un nombre limité de cultivars comestibles multipliés clonalement. Ces clones ont une diversité génétique étroite, sont sensibles aux principaux ravageurs et maladies et ne sont pas adaptés aux changements climatiques à venir. Dans deux projets, DiVBa financé par SEP2D et BforBB financé par la fondation Agropolis, nous avons cherché à caractériser la diversité des bananiers contenant du génome M. balbisiana (génome B), qui sont, à ce jour, sous-utilisés dans les programmes d'amélioration. Les ressources de M. balbisiana possèdent naturellement dans leur génome des séquences du Banana streak virus (eBSV). Certains eBSV sont capables de restituer spontanément des particules virales infectieuses, ce qui empêche l'utilisation des génomes M. balbisiana dans les programmes d'amélioration. Un génotypage approfondi des eBSV et un phénotypage BSV des ressources de M. balbisiana sont donc nécessaires, non seulement à des fins de sélection mais aussi pour renforcer la caractérisation de Musa. Notre groupe a en effet établi que les eBSV sont de bons marqueurs phylogénétiques qui peuvent être utilisés pour appréhender la structuration et l'origine géographique de M. balbisiana. Nous avons collecté 364 échantillons de bananiers sauvages ou cultivés localement dans la partie continentale de l'Asie du Sud-Est (nord du Vietnam, nord du Laos et province du Yunnan en Chine) qui représentent un hot spot de la biodiversité de Musa. Des données préliminaires de génotypage seront présentées. Les deux projets ont également contribué à la gestion durable et à la conservation de la biodiversité des bananiers sauvages et cultivées dans les pays visités où les collections ont été enrichies avec les nouveaux génotypes collectés.

To protect genome integrity against invasive nucleic acids, most eukaryotic organisms have evolved gene silencing mechanisms, often involving small interfering RNAs (siRNAs). Endogenous banana streak virus (eBSV) integrants derived from three distinct species, present in Musa balbisiana (B) but not Musa acuminata (A) banana genomes are able to reconstitute functional episomal viruses in interspecific triploid AAB banana hybrids, causing banana streak disease. In contrast, the seedy diploid (BB) parent line, which harbours identical eBSV loci, does not develop disease. Illumina sequencing revealed that eBSV loci produce low-abundance transcripts covering most of the viral sequence and generate predominantly 24-nt siRNAs, in contrast to predominantly 21–22 nt viral siRNAs derived from the respective episomal viruses in M. acuminata or AAB hybrid plants. Remarkably, siRNA production is restricted to duplicated and inverted viral sequences present in eBSV loci. Using bisulfite sequencing we found that both siRNA-producing and non-producing sequences in the eBSV loci are methylated at 100 % symmetric CG and CHG sites and up to 50% in non-symmetric CHH sites unlike episomal virus DNA previously found to be largely unmethylated. Our data suggest that eBSVs are controlled at the epigenetic level in the BB diploids. This regulation prevents both their awakening and systemic infection of the plant, but probably also serves to confer the resistant phenotype observed in BB plants against mealybug-transmitted viral particles. These findings are thus of relevance to other plant resources hosting integrated viruses.

Banana bunchy top virus (BBTV) is a six-component ssDNA nanovirus transmitted by aphids, causing severe disease of monocot Musa plants, which originates from South-East Asia. Asian BBTV isolates are often associated with self-replicating alphasatellites. By Illumina sequencing analysis of banana aphids and leafs collected in Congo, we discovered an alphasatellite of previously unknown genus, phylogenetically related to alphasatellites of dicot nanoviruses. It was encapsidated by BBTV coat protein and accumulated at high levels in plants and aphids, thereby reducing helper virus loads and altering relative abundance of its genomic components (formula). Consequently, the alphasatellite reduced virus transmission efficiency and delayed symptom appearance without impacting disease severity. BBTV and alphasatellite clones caused systemic infection in Nicotiana bentamiana, followed by recovery, and BBTV replication protein Rep (but not alphasatellite Rep) induced leaf chlorosis. Illumina sequencing revealed 21, 22 and 24 nucleotide small interfering (si)RNAs covering both strands entire viral of the genome, allowed to map monodirectional Pol II transcription units for each viral mRNA and uncovered pervasive transcription of each component and alphasatellite in both polarities, likely generating double-stranded precursors of viral siRNAs. Consistent with latter hypothesis, viral DNA formulas resembled viral siRNA (but not mRNA) formulas. In summary, following African invasion, BBTV got associated with an alphasatellite, likely originating from a banana neighbour plant and having a negative impact on BBTV replication and transmission. Molecular analysis of BBTV- and alphasatelliteinfected plants revealed novel mechanistic aspects of viral DNA transcription by Pol II and siRNA production by the antiviral silencing machinery.

The European Virus Archive (EVA) was created in 2008 in response to the need for a coordinated and readily accessible collection of human and animal viruses that could be made available to academia, public health organisations and industry. The current virus pandemic has demonstrated once more the need for an international network that enables the sharing of resources for diagnosis and the development of antiviral strategies as well as a digital platform for easy access. The European virus Archive - Global (EVA-GLOBAL; https://www.european-virus-archive.com/) is a non profit organisation involving a global network with expertise in virology, consisting of 38 partner laboratories both based in EU member states and abroad (Romette et al., Antiviral Res 2018;158:127–134; Coutard et al., Biopreserv Biobank 2020;18:561-569). This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 871029. The aim of the project is to collect, amplify, characterize, standardize, authenticate, and distribute viral isolates and derived products (antisera, cloned cDNAs, nucleic acid extracts...). In EVA-Global the distributed collections assembled during previous EVA projects will be expanded to include plant viruses. Additionally to the growing number of animal and human viruses, plant viruses, derived products and associated services are constantly being added to the online catalog and can be ordered online. As in October 2021 already 68 plant virus isolates comprising RNA and DNA viruses as well as 15 antisera for virus detection and 6 services or training modules (e.g. HTS, diagnostic tools, virus indexing) are available at the online catalogue. EVA-GLOBAL is conceived to be an open access entity aiming at developing synergies and complementary capabilities so as to offer an improved access to researchers and industry. A single-entry point, the web-based catalogue (https://www.european-virus-archive.com/evag-portal), provides easy access for the end user to the collections including viruses, derived materials, services and engineered cells. The consortium ensures that the available materials meet the highest scientific standards in terms of safety, quality and characterization. While the property of the provided materials remains with the originators, specific EU funding provides the option for the free-of-charge access to viral resources towards public research institutions. Besides various forms of virus material, antibodies, molecular detection assays, cutting edge methodology for virus cultivation, preservation and analyses are available. All partners agreed on implementing a quality management system. As part of this, 3 different quality standards (Silver, Gold and Platinum) are defined for available plant virus isolates, targeting e.g. the availability of sequence information and purity of isolates. The large number of distinct viruses and isolates enables the concept of preparedness that has been beneficial to researchers worldwide during the current SARS-CoV-2 pandemic. Moreover, the project will represent an essential contribution to the preservation of virus biodiversity.

Plant DNA and RNA extraction methods are well established, with a wide range of protocols, depending on the purposes of each laboratory/research. Nowadays, quick, inexpensive and easy plant DNA and RNA extraction methods are highly sought after. We developed an optimized protocol for plant DNA and RNA extraction that uses an inexpensive bench drill and plastic bags and does not require liquid nitrogen. DNA from leaves and RNA from leaves and roots of banana, pineapple, citrus, papaya, passion fruit and cassava, were extracted using a basic cetyltrimethylammonium bromide method. Both nucleic acids were quantified and evaluated for quality based on agarose gel electrophoresis. The DNA and RNA extractions were successful for all species, and RNA quality in pellets was maintained after storage at room temperature for three weeks. This protocol can reduce costs considerably in laboratories with ongoing routine activities of DNA and RNA extraction for genetic diversity and gene expression analyses, where other conventional methods have not been successful due to explant, condition of samples and quantity and quality of nucleic acids. This is especially relevant for many laboratories in developing countries where the cost and availability of liquid nitrogen may be a constraint.

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (eBSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové – BSIMV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. Using deep sequencing of total siRNAs of PKW we underlined the presence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSIMV by blasting sequences against the 3 BSV species genomes. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. vsRNA are enriched in 24-nt class which represent about 75% of the total 21-24nt siRNA matching eBSV. We also demonstrated that eBSV are highly methylated in the three different sequence contexts (CG, CHH and CHG) throughout the whole sequence of eBSVs with no difference in methylation profile between siRNA producing and non producing areas. Interestingly, methylation patterns of all three eBSV are similar whereas they are located in different genomic context; eBSOLV being in a TE rich area whereas eBSIMV and eBSGFV are in genes rich region. It seems that eBSV are controlled mainly by epigenetic mechanisms similar to those described for transposable elements (TE). All together, our data indicate that eBSVs in PKW genome are likely silenced at the transcriptional level and this is probably responsible for the natural resistance of this genotype to the activation of such infectious eBSV as well as infection by external BSV particles.

Com o avanço das técnicas de Biologia molecular, surge a necessidade de se aprimorar protocolos de extração de DNA para que sejam mais rápidos e baratos, possibilitando a inclusão de novas metodologias que tendem a beneficiar seus usuários. Nos protocolos de rotina, o uso do nitrogênio líquido é indispensável para o rompimento da parede celular e retirada dos ácidos nucleicos do interior das células durante o processo de maceração. No entanto, sabe-se que o custo do nitrogênio líquido é bastante elevado. As furadeiras de bancada fazem a mesma função do nitrogênio líquido e são extremamente baratas, duráveis e fáceis de serem utilizadas e o seu uso em relação ao nitrogênio e permite uma economia de R$20.000-R$30.000,00/ano a depender do número de amostras quando se compara aos gastos em laboratórios ao final do ano. Portanto, o objetivo deste trabalho foi validar protocolo de extração de DNA com o uso da furadeira de bancada na extração de DNA das principais culturas da Embrapa Mandioca e Fruticultura. Após a coleta do material vegetal, foram realizadas as extrações de DNA das seguintes culturas: bananeira, abacaxi, citros, mamão, maracujá e mandioca. Para cada cultura foram utilizados três genótipos, totalizando três repetições cada. A quantificação para verificar a quantidade e a qualidade do DNA foi feita em gel de agarose 1% por meio de eletroforese. Em seguida foram realizadas as amplificações com dez marcadores moleculares, sendo: cinco marcadores SSR (Simple Sequence Repeats) e cinco ISSR (Inter-simple Sequence Repeats), com o intuito de validar o protocolo. A extração de ácidos nucleicos com uso da furadeira de bancada foi idealizada no CIRAD – Baillarguet em Montpellier – França e CIP-Lima Peru. O método de extração de DNA com auxílio da furadeira de bancada foi eficiente na obtenção de DNA de qualidade em comparação com o método tradicional que faz uso do nitrogênio liquido e sua validação foi confirmada por meio das amplificações via marcadores ISSR e SSR. A nova metodologia irá contribuir para a redução de custos nos laboratórios de Biologia Molecular, principalmente em épocas de recursos escassos para a pesquisa.

Cultivated bananas mainly stem from intra- and interspecific crosses between two species: Musa acuminata (A genome) and Musa balbisiana (B genome). The B genome harbors viral sequences of Banana streak virus (BSV), inducing the banana streak disease and affecting growth and fruits production. The plantain is a natural hybrid (AAB) containing these integrations. BSV integrations can release functional virus following stresses, leading to spontaneous infections. However, no epidemic has been reported so far. We suspect the plantain to control this viral infection using a RNA silencing mechanism, resulting from the co-evolution between viral integrations and B genome. My PhD work aims to investigate BSV infections in plantain, from eBSV activation to complete recovery (qPCR, immunolabelling); as well as the defense mechanism set up by the host (Northern blot, NGS, in situ hybridization). I aim to propose a BSV infection evolution profile in plantain, to further test the virus transmission to a healthy plant. Indeed, other banana trees among which Cavendish cultivar (AAA, producing “dessert bananas”), without B genome, are extremely susceptible to BSV. The plantain culture intensification, associated with global changes, could threat the dessert bananas culture. My project will help answering this arising question of BSV epidemic risk.

The majority of cultivated bananas are intra and interspecific hybrids of the two main species Musa acuminata (denoted genome A) and Musa balbisiana (denoted genome B). The Banana streak virus (BSV) is responsible of the banana streak mosaic disease and causes severe damage on the Musa acuminata hybrids, which are extremely susceptible. The virus is mealybugs- vector transmitted from plant to plant. Curiously, BSV also exists as endogenous sequences named (eBSV) within all B genomes and can give rise active viruses leading to a systemic infection in banana plants. The banana plantain is a natural triploid interspecific hybrid AAB and harbors viral sequences for two BSV species within its B genome. It spontaneously develops BSV infections following abiotic stresses by releasing active viral genome from eBSVs. However, no epidemic has been reported so far, whereas several plantains regularly show infections in the fields. We suspected that the banana plantain regulates such endogenous viral infections by a gene silencing-based plant defense mechanism resulting from a co-evolution process. Our project aims understanding how this pathosystem works by following after an endogenous infection the kinetic of the virus distribution in the plant via molecular (IC-PCR, qPCR) and histologic (serological detection) markers as well as the small viral RNA profiles involved in gene silencing (TGS, PTGS) via Northern blots, NGS and LNA probes.

Banana streak viruses (BSV), which infect bananas and plantains (Musa spp.) worldwide, became a couple of decade ago, the major constraint for banana breeding programmes and Musa germplasm exchange. Indeed, the movement of germplasm and propagation of virus-free planting material have been restricted due to the discovery, in the genome of Musa balbisiana (noted B genome), of infective BSV integrations (eBSV) which can spontaneously release functional BSV virions following stresses. Our objective is to both study the mechanisms underlying the regulation of eBSV and gain knowledge on the BSV biology. Unfortunately to date, BSV is known to infect banana only which is a difficult/complex plant to work with because the life cycle is long, mutant databases are not available, transient and stable transformations are difficult and laborious for instance. To overcome most of those difficulties, we tried to infect the model plant Arabidopsis thaliana (A.t.). We firstly stably transformed two different At ecotypes with a viral clone containing more than one length copy (1,5) of BSOLV DNA genome via agrobacterium. This clone, previously selected as infectious in banana plants, contains twice the promoter region because of the poly A signal is located in front of the promoter. Out of the thirty T2 transformants we analysed, none of them showed abnormal phenotype or BSV symptoms. Besides, RT-PCR experiments showed that viral transcription was undetectable suggesting that T-DNA expression was inhibited at the transcriptional level. Using methylation sensitive restriction enzymes we indeed evidenced that the T-DNA was methylated. To overcome this problem, we transformed several A.t. mutants affected at different stage of the epigenetic RNA silencing pathway. Screening and characterization of primary transformants is on the way but preliminary results on dcl234 triple mutant background indicate that BSV transcription is detectable. Immuno-capture PCR experiments and electron microscopy observations will be soon carried out to look for the presence of the virus. (Texte intégral)

Controlling an invasive species, in Agriculture, is a context variable activity, borrowing the technical advances of numerous sciences, including the insect sciences. The Banana bunchy top disease (BBTD), caused by the bunchy top virus (BBTV), is spread between regions through planting infected suckers and locally by the banana aphid feeding off infected plants. It is present in 16 countries in Sub Saharan Africa; and causes accelerating production losses and reduced access to clean seed. Although yield decline is more rapid in some cultivars, none is resistant. Our work towards recovery of banana production in nine BBTD-affected sites in eight countries; thus includes a cross talk between different natural sciences, social sciences and policy studies, relying on existing knowledge and technologies: reliable clean seed supply system and tactics to reduce re-infection pressure, vector entomology, virus detection, epidemiology, imaging and mapping. We developed a non-intrusive DNA extraction methods and used it to amplify the mtCOI gene to differentiate the two species of banana aphids, Pentalonia nigronervosa and P. caladii while preserving voucher specimens for morphological studies. ELISA, key diagnostic approach should be supplemented by early symptom recognition. We also studied cropping systems and banana diversity as potential options and indicators of long term BBTD control, as varieties showed different attractiveness to the vector and human preferences. Seed systems studies revealed that clean seed systems (like other control approaches) need to incorporate diversity, vector, human behaviour and policy. BBTD is a spreading invasive disease, a potential model in regional invasive species management. What then is our role as Insect Scientists working in this region in limiting of the spread of BBTD? How can we foster an interplay between entomology, ecology, other natural scientists, social sciences and regulatory services and the private sector to prevent its spread to the banana growing areas in Africa.

O BSV é um vírus de DNA, apresenta partícula baciliforme não envelopada e possui a capacidade de integrar o seu DNA no genoma B da bananeira. Sendo assim, a depender das condições de estresse, o genoma do vírus pode ser ativado e o fato da bananeira ser propagada vegetativamente agrava ainda mais esse quadro, uma vez que a micropropagação pode causar estresse às plantas e com isso ativar as partículas endógenas do vírus, podendo levar ao aparecimento da doença. Os principais sintomas do BSV em Musa spp., são: estrias foliares cloróticas que evoluem para estrias necróticas, apodrecimento do pseudocaule, deformação dos frutos, diminuição do vigor da planta e da produção de frutos. A banana é uma das frutas mais consumidas no mundo, apresentando relevante papel sócio-econômico em inúmeros países tropicais. O Brasil difere do cenário mundial por concentrar sua produção em cultivares do subgrupo Prata, em especial a 'Prata-Anã' e 'Pacovan', genótipos triploides com genoma AAB. São comuns os relatos de sintomas de BSV em bananais que fazem uso dessas cultivares, localizados nos principais polos de produção da fruta no Brasil, o que leva a concluir-se pela presença do vírus integrado ao genoma B ou a infecção local por vetores, em especial as cochonilhas. Para o agronegócio brasileiro da banana é fundamental o uso de cultivares livres de sequencias completas do vírus integrado no genoma B, demanda que pode ser atendida por meio do melhoramento genético baseado em cruzamentos e seleção nas progênies. Portanto, é de suma importância ter-se disponível ferramentas moleculares capazes de identificar partículas virais com potencial de causar doença no germoplasma de banana brasileiro, para subsidiar a seleção de parentais livres do BSV para uso em cruzamentos. Com base nisso, o objetivo desse trabalho foi avaliar a diversidade genética das espécies de BSV presentes em acessos de banana mantidos no banco ativo de germoplasma da Embrapa Mandioca e Fruticultura, localizado em Cruz das Almas (BA), utilizando primers espécies-especificos desenvolvidos pelo Centro de cooperação internacional em pesquisa agronômica para o desenvolvimento (CIRAD). Foram analisados 303 acessos para detecção de três espécies de virus (BSGFV-Goldfinger, BSOLV-Obino L´Ewai e BSIMV-Imové), dos quais 141 acessos demonstraram resultados positivo para a existência de integrações virais em seus genomas, o que corresponde a 46% do germoplasma avaliado. Todos os acessos das espécies selvagens de bananeira e aqueles com apenas o genoma A foram negativos; resultado que corrobora com a literatura internacional que vincula a integração do vírus apenas ao genoma B. As espécies virais mais frequentes na coleção de germoplasma foram BSOLV e BSGFV, com 43% e 41%, respectivamente. Além dos primers especies-especificos foram utilizadas enzimas de restricões para detectar quais acessos com genoma B apresentam alelos virais infectivos ou não infectivos. Os resultados destas análises demonstraram que os acessos com genoma BB estão livres do alelo infectivo. Estes genótipos tem potencial de uso em cruzamentos, em especial com autotetraploides com background genético similar as cultivares de banana do subgrupo Prata e de plátanos desenvolvidos pela Embrapa Mandioca e Fruticultura. Além disso, estas analises confirmaram que todas as cultivares desenvolvidas pelo programa de melhoramento genético de banana da Embrapa Mandioca e Fruticultura, por meio da hibridação, estão livres de alelos infectivos de BSV, fato de suma importância para o agronegócio de banana brasileiro.

BACKGROUND and OBJECTIVES Viruses are major constraints to the production and exchange of banana and plantain (Musa spp.) germplasm in the world. Several viruses are recognized to affect Musa spp: Banana bunchy top virus (BBTV), Banana streak GF virus (BSGFV), Banana streak OL virus (BSOLV), Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Cucumber mosaic virus (CMV), Banana bract mosaic virus, (BBrMV). To date, in Democratic Republic of Congo (DRC), except BBTV, others banana viruses are not yet reported. The main aim of this work was to elucidate the epidemiological situation, genetic variability and origin of Musa spp. viruses in DRC. MATERIALS and METHODS Epidemiological surveys were conducted from 2011-2013, in DRC, to search main viruses of Musa spp. Up to 666 samples collected from 122 locations; Samples were analysed using PCR based method. Detected viruses were sequenced and phylogenetic studies were performed. RESULTS BBTV is the most widespread virus (frequency=44,8%) in DRC. Molecular analyses support infrequent BBTV dispersal events between continents. This study reveals a low genetic variability of DRC BBTV isolates. They are close, gather in the South Pacific group and are linked with isolates of other sub-Saharan Africa (SSA) countries, but no with Egyptian isolates. Since the earliest BBTV report in SSA was in DRC in 1950, it is likely that BBTV spread from DRC to other SSA countries rather than from Egypt and that at least two BBTV introductions occurred in Africa. Haplotypes analysis based on the coding region m-Rep protein showed 38 haplotypes (haplotype diversity value of 0.944±0.013). In Bas Congo, symptoms of Banana streak disease were associated to the presence of BSV species (frequency=19,9%); the most widespread were BSGFV (frequency=67,2%) and (BSOLV) (frequency=24,2%). The sequenced BSOLV isolate from Musa acuminata, gather in the clade I recognized to gathering the BSV species with endogenous counterpart. CMV was also detected (frequency=9,3%). Genetic diversity analysis reveals the high similarity between DRC CMV isolates with a South African isolate). BBrMV was not detected in collected samples. CONCLUSION DRC is a place of high prevalence of banana viruses, indicating the need for an improved quality of planting material. DRC seems to be the primary center of BBTV spread in Africa. BSV species data revealed the activation of endogenous BSV and the transmission of BSV species from plantain (B genome) to banana (A genome), in natural conditions. While for CMV, data suggest the hypothesis of a unique introduction through infected planting material and the distribution of a single strain of CMV. The BBrMV still absent in DRC. (Texte intégral)

BACKGROUND and OBJECTIVES Banana bunchy top virus (BBTV), is the most devastating and widespread banana virus. Banana and plantain (Musa spp.) are major crops in terms of household income and food security in Democratic republic of Congo (DRC) (1). Despite the large territory under banana and plantain cultivation in the country, the genetic characterization of BBTV isolates has been undertaken for two provinces. The main objectives of this work were to understand the genetic variability of DRC BBTV isolates and to determine their origin. MATERIALS and METHODS In this study, genetic variation in BBTV was assessed from 52 BBTV isolates collected in five out of 11 provinces in DRC (Bandundu, Bas-Congo, Katanga, Kinshasa and Kasai Oriental). 52 full sequences of DNAR and 30 full sequences of all BBTV components were analysed. RESULTS Full genome sequencing of DNA-R components was performed revealing a low genetic variation (98-100% nucleotide identity) amongst the BBTV isolates detected in seven of 11 DRC provinces. The phylogenetic tree showed that all DRC isolates gather a unique clade in the South Pacific group of BBTV. Based on the coding region for replication initiator protein, haplotype diversity was estimated to be 0.944±0.013, with 30 different haplotypes from 68 isolates in DRC. In addition, fives isolates were selected from each province for total genome sequencing, confirming low genetic variation among isolates from seven provinces (97-100% nucleotide identity). CONCLUSION This study strengthens the hypothesis of a single BBTV introduction some time ago, followed by the spread of the virus in the country. Analysis of the geographical dispersion reveals specific haplotypes groups according to the different agro-environmental conditions.

Following the open comment period for MusaNet and ProMusa members in September 2015, the ''BSV Position Paper'' was finalized and is now available here. The purpose of the position paper was to propose a strategy for the distribution of germplasm containing infective endogenous BSV in the B genome, while minimising any risk associated with the distribution of BSV to the recipient country. Comments on the paper were well received and addressed by the taskforce. Please click here for a summary of the comments and responses. The next step is for the International Transit Centre (ITC) to develop an official procedure for the distribution of eBSV infected germplasm based on the position paper. This will be discussed in the MusaNet Expert Committee and reviewed by the taskforce (pictured below) that developed the position paper. The strategy will be implemented after all of the ITC accessions containing the B genome are screened and cleaned if necessary. This is currently being carried out by CIRAD and the University of Liege, Gembloux Agro-Biotech, and is expected to be completed by the end of 2016. If you have any questions or comments, please contact Rachel Chase at r.chase@cgiar.org.

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.

Banana bunchy top virus (BBTV) is one of the most severe and widespread virus limiting produc- tion and distribution of planting material of banana (Musa spp.) crops in the world. In Democratic Republic of Congo (DRC), these crops play a major role in daily life of almost 70% of citizen. Many factors influence banana production negatively such as Banana bunchy top disease. Epidemiol- ogical survey was conducted in experimental stations and farmers’ fields for two consecutive sea- sons covering 72 sites in five provinces of south western of RDC. The objective of this study was to evaluate the presence and distribution of the Banana bunchy top virus in five provinces of South Western of DRC, with emphasis on the agro-ecological factors. A total of 174 Musa spp. leaves samples were collected and analyzed by PCR. The results revealed the presence of BBTV in all provinces investigated. The frequency of BBTV was 6.3% in Bandundu, 12.1% in Kasaï Oriental, 17.8% Bas Congo, 1.1% in Katanga and 7.5% Kinshasa Urban and Peri-urban. Results also re- vealed that BBTV occurred in experimental station and farmers’ fields, both having all cooking and dessert bananas. The high prevalence of BBTV seemed to be linked to multiple introductions of planting materials in the Bas Congo province during 1990 and 2002. However, the province of Katanga had not experienced the introduction of planting material. This factor would explain the lowest prevalence of Banana bunchy top virus in this province. The results indicated that there was a real need to facilitate access to genetically improved and healthy certified planting material in these provinces.

The Musa balbisiana banana genome (B genome) forms part of the genotype of many important banana cultivars, such as the famous plantain subgroup that is a staple food for millions of people in Africa and Latin America. Moreover, it is often associated with desirable traits of agronomic interest such as vegetative vigour, biotic and abiotic stress tolerance. However, it became recently the main constraint for breeding banana and plantain interspecific hybrids and for exchanging Musa germplasm as it harbors integrated copies of Banana streak virus (BSV) named endogenous BSV (eBSV) that are able to release infectious virus. We recently fully characterized integrants of three BSV species-Goldfinger (eBSGFV), Imové (eBSImV) and Obino l'Ewai (eBSOLV)-in the seedy Musa balbisiana Pisang klutuk wulung (PKW) by studying their molecular structure, genomic organization, genomic landscape and infectious capacity [1 ; 2]. This thorough characterisation led to the production of eBSV specific molecular markers which are used to widely screen banana hybrids, genitors and germplasm to future crop-oriented breeding programmes aimed at producing safe interspecific banana hybrids but also to estimate and limit the risk of BSV outbreak on natural hybrids spread intensively in developing countries as a food source.

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)

Cacao swollen shoot virus (CSSV) is a member of the family Caulimoviridae, genus Badnavirus naturally transmitted to Theobroma cacaoby several mealybug species. Typical symptoms of the disease on cocoa trees are red vein banding of young leaves, mosaic on older leaves and swelling of the orthotropic shoots. The virus, restricted to West Africa whereas the cacao tree originates from the Western Hemisphere, could therefore most probably have an indigenous origin on the West African subcontinent. The disease has caused enormous economical damage in Ghana since the1930s but was only restricted to small areas in Togo and Côte d'Ivoire until recently. Now, renewed outbreaks in the main producing areas in Côte d'Ivoire, Ghana and Togo cause serious problems. The knowledge of the viral biodiversity in the different outbreaks will in turn help to provide a better understanding of the development of the epidemics, and of the evolution of viral populations and may permit to retrace the emergence and dispersal of CSSV. CSSV diversity is genetically structured in at least eight different species according to ICTV recommendations. Only group B was detected in the three countries and in most of the outbreaks, whereas the other groups have a more restricted geographic repartition. To understand such extend of CSSV variability compared to its very short evolutionary history on cocoa trees, we used BEAST software. The results suggest the existence of many emergences from native hosts to cacao trees in the various countries of West Africa. (Texte intégral)

Banana streak virus (BSV) is a double stranded DNA pararetrovirus belonging to the genus Badnavirus and triggering necrotic mosaic lesions on banana plants. BSV exhibits a higher biodiversity than other badnaviruses as a consequence of two phenomena: the epidemic process of the disease through contamination of plant to plant by vector and the release of infectious viral genomes by the banana genome harboring endogenous sequences. BSV sequences described so far are spread among the three groups of the badnavirus diversity. BSV-like sequences belonging to group 2 seem to be all integrated into the banana genome since no infectious episomal particles have been identified so far. We describe here the characterization of such integrations in thirty-four plant samples belonging to different families of the Zingiberale order with a banana sampling representative of the Musa diversity. We have firstly looked for group 2 sequences using PCR with 7 sets of primers which are specific of the 7 BSV-like species identified so far in this group. We then used Southern blot approach with viral probes corresponding to each BSV-like species. Sequenced PCR products of the RT-RNase H region of the viral genome have been used for the construction of a phylogenetic tree. We found all the 7 BSV-like species within the A genomes whereas only 4 species were within B genomes. Interestingly, two new species have been discovered in M. balbisiana genomes only. Additionally, four BSV-like species seem to be integrated in M. schizocarpa, M. basjoo, M. ornata and M. itinerans but these results have to be confirmed. The integration patterns observed by Southern blot analysis show multiple and different integrations of BSV-like species into M. acuminata plants conversely to M. balbisiana plants where patterns are highly conserved. The data suggest that BSV-like integrations likely occurred for some of them before the speciation M. acuminata/M. balbisiana whereas others occurred afterwards. (Texte intégral)

The genome of banana (Musa sp.) harbours multiple integrations of Banana streak virus (eBSV), whereas this badnavirus does not require integration for the replication of its ds DNA genome. Some endogenous BSV sequences (eBSV), only existing in the Musa balbisiana genome, are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. The structure of these eBSV is much longer than a single BSV genome, composed of viral fragments duplicated and more or less extensively rearranged. Wild M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV without any visible virus particles. In collaboration with the group headed by M. Pooggin (Basel, Switzerland), a deep sequencing of total siRNAs of PKW was performed using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSImV by blasting sequences against the 3 BSV species genomes. vsRNA are enriched in 24-nt class thus eBSV in PKW genome are likely silenced at the transcriptional level. Interestingly, we show that hot and cold spots of vsRNA generation do not target similar viral sequences from one eBSV species to the other but are directly correlated with the structure of the integration. All together, those data seem indicate these eBSV induce a natural resistance driven by gene silencing mechanisms based on their complex molecular re-arranged structure which could lead to dsRNA formation. (Texte intégral)

Banana streak virus (BSV), the causative agent of banana streak disease, is a plant pararetrovirus belonging to the family Caulimoviridae, genus Badnavirus. The genome of BSV is a circular double ? stranded DNA of 7.4 kbp made of three ORFs and like other pararetroviruses replicates via reverse transcription of viral pregenomic RNA (Lockhart, 1990). In the mid-ninetys Ndowora et al. (1999), revealed the presence of multiple integrations of Banana streak virus in the genome of banana (Musa sp.) whereas this badnavirus does not require integration for its replication. Some endogenous BSV (eBSV) sequences in the Musa balbisiana genome are infectious by releasing a functional viral genome following stresses such as those existing in in vitro culture and interspecific crosses context. Seedy M. balbisiana diploid genotypes (BB) such as Pisang Klutuk Wulung (PKW) harbour such infectious eBSV belonging to three widespread species of BSV (Goldfinger -BSGFV, Imové - BSImV and Obino l'Ewai - BSOLV) but are nevertheless resistant to any multiplication of BSV whatever the viral origin. We postulated this resistance is mediated by gene silencing and performed deep sequencing of total small RNAs of PKW using the Illumina ultra-high-throughput technology. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from eBSOLV, eBSGFV and eBSImV by blasting small RNAs against the viral genome counterparts. VsRNAs are systematically enriched in 24-nt class. Interestingly, we showed that hot and cold spots of vsRNA generation do not target similar viral sequences but are directly correlated with the structure of each integration. Finally, preliminary data using Southern blotting coupled with methylation-dependent restriction enzyme (McrBC) digestion indicated that each eBSV is heavily methylated. All together, our findings indicate that eBSVs in PKW are likely silenced at the transcriptional level. In parallel, we deep sequenced small RNAs from Cavendish banana plants (lacking eBSV integrations) infected independently by BSOLV, BSGFV, BSImV and 3 other distinct BSV species in order to evaluate silencing-ased regulation of episomal virus infection. Abundunt vsRNAs were found to be enriched in 21-nt class and most of the hot spots of siRNA production are located in the coding sequences. Our methylation analysis indicated that episomal BSV DNA is not methylated and therefore, is likely silenced only at the post-transcriptional level.

Banana streak virus (BSV), the causative agent of banana streak disease, is a plant pararetrovirus belonging to the family Caulimoviridae, genus Badnavirus. The genome of BSV is a circular double-stranded DNA of 7.4 kbp made of three ORFs and like other pararetroviruses replicates via reverse transcription of viral pregenomic RNA (Lockhart, 1990). While the first two ORFs encode two small proteins of unknown function, the third ORF (~210 kD) encodes a polyprotein that can be cleaved to yield the viral coat protein and proteins with homology to aspartic protease, reverse transcriptase and RNaseH. Little information is available about antiviral defense response of the host plant on BSV or other members of Caulimoviridae. RNA silencing, also known as RNA interference (RNAi), is an ancient gene regulation and cell defense mechanism, which exists in most eukaryotes (Xie and Qi, 2008). Plants have adapted the RNA silencing machinery into an antiviral defense system. Interestingly, Arabidopsis plants infected with Cauliflower mosaic virus (CaMV), a type member of the genus Caulimovirus in the family Caulimoviridae, accumulate siRNAs of 21, 22 and 24 nt size classes, where the 24 nt species are the most predominant ones (Blevins et al., 2006; Moissiard and Voinnet, 2006). Further analysis showed that, the leader region (600 nt) of CaMV pregenomic RNA produces massive amounts of siRNAs with several hot and cold spots of siRNA generation (Blevins et al., 2011) to function as a decoy for the RNA silencing defense system of the plant. To determine whether the viral decoy strategy was universally used among viruses belonging to the family Caulimoviridae, we have performed a deep sequencing of total siRNAs of 6 Cavendish banana plants infected independently with one of the 6 BSV species we own in the laboratory. We obtained for the first time, experimental evidence of virus-derived small RNA (vsRNA) from those 6 BSV species by blasting sequencing data against the 6 BSV species genomes. vsRNA are enriched in 21-nt class thus BSV are likely silenced at the post-transcriptional level. Besides, our data unequivocally show that the decoy strategy used by the CaMV is not employed by the BSV since most of the hot spots of siRNA production are located in ORF1 and 2. Information generated about siRNAs derived from BSV genome could help us to design silencing-based transgenic and non-transgenic (RNA vaccination) approaches to obtain BSV resistance in banana crop. (Texte intégral)