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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.

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.

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.

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.

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)

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)

Infectious endogenous sequences of several Banana streak virus species 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 sequences can cause infection in natural and synthetic interspecific hybrids harbouring the M. balbisiana genome. Therefore, infectious endogenous BSV (eBSV) sequences are currently the main constraints for breeding much needed banana and plantain interspecific hybrids and for exchanging Musa germplasm. The full genomic and genetic organization of eBSV sequences present in diploid M. balbisiana cultivar Pisang Klutuk Wulung (PKW) was recently elucidated, showing that endogenous BSGFV, BSOLV and BSImV sequences in PKW result from a single integration event. This work also showed that integration of eBSGFV and eBSOLV is di-allelic with only one infectious allele whereas that of infectious eBSImV is monoallelic. We have undertaken the characterization of eBSOLV, eBSGFV and eBSImV in the M. balbisiana accessions of CIRAD Guadeloupe's germplasm collection. This work unveiled important differences between accessions, which were shown to harbor all combinations of infectious and non infectious alleles for one or several distinct BSV species. These results were confirmed biologically, by following the kinetics of expression of infectious eBSV sequences in various genotypes during cell culture, which is the most potent abiotic stress activating the expression of infectious eBSV sequences. Breeding improved M. balbisiana progenitors devoid of infectious eBSGfV and/or BSOLV sequences was achieved in several selfed and/or double haploid lines by segregating the corresponding infectious and non infectious alleles. This work paves the way to the elimination of infectious eBSV sequences from M. balbisiana progenitors, and to the safe use of M. balbisiana genitors for creating interspecific banana and plantain hybrids. (Texte intégral)

Infectious endogenous sequences of at least 3 distinct Banana streak virus species are present in the genome of Musa balbisiana spp [1, 2], which are important progenitors for breeding improved banana varieties. Once activated by biotic or abiotic stresses, these sequences can cause infection in natural and synthetic interspecific hybrids harbouring the M. balbisiana genome [3]. Therefore, infectious endogenous BSV (eBSV) sequences are currently the main constraint for breeding much needed banana and plantain interspecific hybrids and for exchanging Musa germplasm. The full genomic and genetic organization of eBSV sequences present in diploid M. balbisiana cultivar Pisang Klutuk Wulung (PKW) was recently elucidated [1, 4], showing that endogenous BSGFV, BSOLV and BSImV sequences in PKW result from a single integration event. This work also showed that integration of infectious eBSGFV and eBSOLV is di-allelic whereas that of infectious eBSImV is monoallelic. In the case of eBSGFV and eBSOLV, only one allele is infectious. Taking advantage of recently designed primer sets [1, 3; Baurens, unpublished], we have undertaken the characterization of eBSOLV, eBSGFV and eBSImV in the M. balbisiana accessions of CIRAD Guadeloupe's germplasm collection. This work unveiled important differences between accessions, which were shown to harbor all combinations of infectious and non infectious alleles for one or several distinct BSV species. Some accessions were even found to be exempt from eBSImV. These results were confirmed biologically, by following the kinetics of expression of infectious eBSV sequences in various genotypes during cell culture, which is the most potent abiotic stress activating infectious eBSV sequences. Based on these results, breeding improved M. balbisiana progenitors devoid of infectious eBSGfV and/or BSOLV sequences was undertaken by segregating the corresponding infectious and non infectious alleles. This was achieved in several selfed and/or double haploid lines. This work paves the way to the elimination of infectious eBSV sequences from M. balbisiana progenitors, and to the safe use of M. balbisiana genitors for creating interspecific banana and plantain hybrids. (Texte intégral)

Banana streak viruses (BSV) infect bananas and plantains worldwide. They are naturally transmitted by mealybugs; however infections can also occur in the absence of vector-mediated 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. 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 for the three BSV species Obino l'Ewaï (BSOLV), Goldfinger (BSGFV) and Imové (BSImV). This work showed that integration of infectious eBSGFV and eBSOLV is di-allelic, with one infectious and one non-infectious allele, whereas that of infectious eBSImV is monoallelic. Taking advantage of the development of allele-specific molecular markers, eBSV signatures were established for all M. balbisiana genitors of the CIRAD Guadeloupe Musa collection, unveiling important differences between accessions. All combinations of infectious and non-infectious alleles were observed for all 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.

The presence of infectious endogenous Banana streak virus alleles (eBSV) in the genome of Musa balbisiana progenitors hampers breeding efforts aimed at the development of much needed banana interspecific hybrids. We report on the characterization of eBSVs in M. balbisiana progenitors and on the development and implementation of strategies for relieving several of these M. balbisiana progenitors from infectious eBSV alleles. Our results pave the way to improved strategies for breeding banana interspecific hybrids with no risk of activation of infectious eBSV alleles. Key words: breeding; interspecific hybrids; endogenous Banana streak virus; infectious allele; segregation.