The Experts below are selected from a list of 1023 Experts worldwide ranked by ideXlab platform
Susan Seal - One of the best experts on this subject based on the ideXlab platform.
-
Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes
BIO-PROTOCOL, 2018Co-Authors: Moritz Bömer, Ajith I. Rathnayake, Goncalo Silva, Aliyu A. Turaki, P. Lava Kumar, Susan SealAbstract:Since the first discovery of Badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial Badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bomer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam Badnaviruses (Bomer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of Badnavirus diversity as well as the identification of potentially integrated Badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for Badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel Badnavirus genomes.
-
Complete genome sequence of a new member of the genus Badnavirus, Dioscorea bacilliform RT virus 3, reveals the first evidence of recombination in yam Badnaviruses.
Archives of Virology, 2017Co-Authors: Moritz Bömer, Ajith I. Rathnayake, Goncalo Silva, Paul Visendi, Susan SealAbstract:Yams (Dioscorea spp.) host a diverse range of Badnaviruses (genus Badnavirus, family Caulimoviridae). The first complete genome sequence of Dioscorea bacilliform RT virus 3 (DBRTV3), which belongs to the monophyletic species group K5, is described. This virus is most closely related to Dioscorea bacilliform SN virus (DBSNV, group K4) based on a comparison of genome sequences. Recombination analysis identified a unique recombination event in DBRTV3, with DBSNV likely to be the major parent and Dioscorea bacilliform AL virus (DBALV) the minor parent, providing the first evidence for recombination in yam Badnaviruses. This has important implications for yam breeding programmes globally.
-
PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm
Viruses, 2017Co-Authors: Aliyu A. Turaki, Moritz Bömer, Goncalo Silva, P. Lava Kumar, Susan SealAbstract:Badnaviruses (family Caulimoviridae, genus Badnavirus) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (Dioscorea spp.) plants are hosts to a diverse range of Badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of Badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam Badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex ‘fingerprints’ composed of multiple sequences of Dioscorea bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in Badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm.
-
A Sequence-Independent Strategy for Amplification and Characterisation of Episomal Badnavirus Sequences Reveals Three Previously Uncharacterised Yam Badnaviruses.
Viruses, 2016Co-Authors: Moritz Bömer, Goncalo Silva, Aliyu A. Turaki, P. Lava Kumar, Susan SealAbstract:Yam (Dioscorea spp.) plants are potentially hosts to a diverse range of Badnavirus species (genus Badnavirus, family Caulimoviridae), but their detection is complicated by the existence of integrated Badnavirus sequences in some yam genomes. To date, only two Badnavirus genomes have been characterised, namely, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV). A further 10 tentative species in yam have been described based on their partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences, generically referred to here as Dioscorea bacilliform viruses (DBVs). Further characterisation of DBV species is necessary to determine which represent episomal viruses and which are only present as integrated Badnavirus sequences in some yam genomes. In this study, a sequence-independent multiply-primed rolling circle amplification (RCA) method was evaluated for selective amplification of episomal DBV genomes. This resulted in the identification and characterisation of nine complete genomic sequences (7.4–7.7 kbp) of existing and previously undescribed DBV phylogenetic groups from Dioscorea alata and Dioscorea rotundata accessions. These new yam Badnavirus genomes expand our understanding of the diversity and genomic organisation of DBVs, and assist the development of improved diagnostic tools. Our findings also suggest that mixed Badnavirus infections occur relatively often in West African yam germplasm.
-
the prevalence of Badnaviruses in west african yams dioscorea cayenensis rotundata and evidence of endogenous pararetrovirus sequences in their genomes
Virus Research, 2014Co-Authors: Susan Seal, Emmanuelle Muller, Aliyu A. Turaki, Denis Filloux, Serge Galzi, Lava P Kumar, L Kenyon, Antonio Lopezmontes, Marieline IskracaruanaAbstract:Abstract Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other Badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of Badnaviruses, and determine if Badnavirus genomes are integrated in the yam genome. Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, were screened using generic Badnavirus PCR primers. Positive amplification of an expected ca. 579 bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from Badnavirus particles or putative endogenous Badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with Badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam Badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of Badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of Badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.
Emmanuelle Muller - One of the best experts on this subject based on the ideXlab platform.
-
Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin
Molecular Plant Pathology, 2020Co-Authors: Matthieu Chabannes, Serge Galzi, Marc Gabriel, Abderrahmane Aksa, Jean‐françois Dufayard, Marie‐line Iskra‐caruana, Emmanuelle MullerAbstract:Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although Badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing Badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.
-
Cacao Swollen Shoot Virus (CSSV): History, Biology, and Genome
Cacao Diseases, 2016Co-Authors: Emmanuelle MullerAbstract:Cacao swollen shoot virus (CSSV) is the only virus disease of cacao that is prevalent and damaging. CSSV is a dsDNA virus of the genus Badnavirus and the family Caulimoviridae transmitted by several species of mealybugs. The historical emergence of the disease is closely associated with the establishment of cacao cultivation in West Africa as it appeared soon after the introduction of the cacao in West Africa and remains endemic to this area. The disease is likely due to several host shifts from indigenous hosts. We can additionally conclude from the high molecular variability of the virus that the disease consists of a complex of viral species. Although the disease spreads slowly, eradication campaigns have failed to contain the disease which continues to emerge in new West African regions.
-
Improved diagnostic tools for the detection Badnaviruses in yams unveil the existence of endogenous sequences of extant Badnavirus species in yams. [O.36]
2015Co-Authors: Marie Umber, Emmanuelle Muller, Marie-line Caruana, Nathalie Laboureau, Philippe Roumagnac, Claudie Pavis, Denis Filloux, Serge Galzi, Rose-marie Gomez, Pierre-yves TeycheneyAbstract: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. (Resume d'auteur)
-
A scenario of co-evolution between Badnaviruses and Musa sp.. [P.22]
2015Co-Authors: Marie-line Iskra Caruana, Nathalie Laboureau, Serge Galzi, Pierre-olivier Duroy, Matthieu Chabannes, Emmanuelle MullerAbstract:The banana streak disease is due to a complex of distinct Banana streak viruses (BSVs) species showing a wide genetic diversity. Banana streak viruses (BSVs) are double stranded DNA pararetroviruses belonging to the family Caulimoviridae, genus Badnavirus. Outbreaks of BSVs causing banana streak disease have been recorded worldwide where Musa spp. is grown during the last 20 years with no convincing evidence of epidemics. Epidemics were previously reported in Uganda where BSV is currently endemic. In addition, the banana genome (Musa sp.) is invaded by numerous Badnavirus sequences. The majority of these viral integrants is mostly defective as a result of pseudogenisation driven by the host genome evolution. They are just called BEV (banana endogenous virus sequences) because episomal particles corresponding to the integrated counterparts have not been identified so far. Conversely, only few viral integrants named endogenous BSV (eBSVs) can release a functional BSV genome following stresses. All the badnaviral sequences described so far are spread among the three main clades of the Badnavirus genus diversity. Our group established that BSVs are distributed among Clade I and Clade III. Clade I gathers BSV species distributed worldwide whereas Clade III is dedicated to BSV species only present in Uganda. eBSVs exclusively correspond to BSV species of Clade I and are endogenous to the Musa balbisiana (B) genomes only. We elucidated their sequence and organization for three BSV species (BSOLV, BSGFV and BSIMV) present in the seedy banana diploid PKW (BB). In addition, we established that all BEVs sequences belong to Clade II. All together our results allowed us to propose an evolutionary scheme of Badnavirus and banana co-evolution, which is presented here. (Resume d'auteur)
-
the prevalence of Badnaviruses in west african yams dioscorea cayenensis rotundata and evidence of endogenous pararetrovirus sequences in their genomes
Virus Research, 2014Co-Authors: Susan Seal, Emmanuelle Muller, Aliyu A. Turaki, Denis Filloux, Serge Galzi, Lava P Kumar, L Kenyon, Antonio Lopezmontes, Marieline IskracaruanaAbstract:Abstract Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other Badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of Badnaviruses, and determine if Badnavirus genomes are integrated in the yam genome. Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, were screened using generic Badnavirus PCR primers. Positive amplification of an expected ca. 579 bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from Badnavirus particles or putative endogenous Badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with Badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam Badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of Badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of Badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.
Denis Filloux - One of the best experts on this subject based on the ideXlab platform.
-
Genetic diversity within yam Badnavirus species and their relationship with endogenous Dioscorea bacilliform virus sequences (eDBV) into yam genome. P61
2017Co-Authors: Marie Umber, Claudie Pavis, Denis Filloux, Rose-marie Gomez, Pierre-yves TeycheneyAbstract: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. (Resume d'auteur)
-
Improved diagnostic tools for the detection Badnaviruses in yams unveil the existence of endogenous sequences of extant Badnavirus species in yams. [O.36]
2015Co-Authors: Marie Umber, Emmanuelle Muller, Marie-line Caruana, Nathalie Laboureau, Philippe Roumagnac, Claudie Pavis, Denis Filloux, Serge Galzi, Rose-marie Gomez, Pierre-yves TeycheneyAbstract: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. (Resume d'auteur)
-
the prevalence of Badnaviruses in west african yams dioscorea cayenensis rotundata and evidence of endogenous pararetrovirus sequences in their genomes
Virus Research, 2014Co-Authors: Susan Seal, Emmanuelle Muller, Aliyu A. Turaki, Denis Filloux, Serge Galzi, Lava P Kumar, L Kenyon, Antonio Lopezmontes, Marieline IskracaruanaAbstract:Abstract Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other Badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of Badnaviruses, and determine if Badnavirus genomes are integrated in the yam genome. Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, were screened using generic Badnavirus PCR primers. Positive amplification of an expected ca. 579 bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from Badnavirus particles or putative endogenous Badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with Badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam Badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of Badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of Badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.
-
The genome of African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct Badnavirus species
Molecular Plant Pathology, 2014Co-Authors: Marie Umber, Marie-line Iskra-caruana, Emmanuelle Muller, Nathalie Laboureau, Philippe Roumagnac, Pierre-yves Teycheney, Denis Filloux, Serge Galzi, Claude Pavis, Susan E. SealAbstract: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.
-
Towards a better characterization of endogenous Badnavirus sequences of yams (Dioscorea spp.)
2013Co-Authors: Marie Umber, Emmanuelle Muller, Marie-line Caruana, Nathalie Laboureau, Philippe Roumagnac, Claudie Pavis, Pierre-yves Teycheney, Denis FillouxAbstract:Yams, and more generallytubers, are very important crops for food security in tropical and subtropical countries.They are propagated vegetativelytherefore they accumulate viruses over long periods of time. Viruses are currently the main constraint for yam production and yam germplasm conservation and distribution. A wide range of Badnavirus sequences belonging to 13 distinct viral species were amplified from genomic DNA of severalyam species when using Badnavirus degenerate primers [1; 2].However, we consistently observed that the proportion of amplification products raised by PCR performed on total genomic DNA is significantly higher than that raised by direct binding PCR, which has been designed to detect episomal forms of yam Badnaviruses.Both observations have fueled suspicion that yams might host endogenous Badnavirus sequences, and possibly infectious ones like bananas [3]. Therefore, search for endogenous Badnavirus sequences was undertaken in yam accessions conserved in the germplasm collection of the Guadeloupe Tropical Plant Biological Ressource Center (CRB-PT) and the yam quarantine facility in Montpellier (France). Southern blots performed on genomic DNA extracted from uninfected Dioscoreatrifidaand using parts of yam Badnavirus genomes as probes confirmed the suspicion of endogenous Badnavirus sequences in yam genomes. Furthermore, PCR performed on genomic DNA extracted from healthy seedlings of D. alata and D. rotundata using Badnavirus degenerate primers raised amplification products whose sequences fit in the current phylogeny of Badnaviruses. Amplification products raised from several of these DNA samples by long-PCR displayedrearrangements such asduplications andreversions which are reminiscent of endogenous Badnavirus sequences encountered in the genome of other crops such as banana. Similarly rearranged sequences were raised by rolling circle amplification, which is known to sometime amplify chromosomal sequences. These results suggest that yams do host endogenous sequences of several distinct Badnavirus species. (Texte integral)
Aliyu A. Turaki - One of the best experts on this subject based on the ideXlab platform.
-
Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes
BIO-PROTOCOL, 2018Co-Authors: Moritz Bömer, Ajith I. Rathnayake, Goncalo Silva, Aliyu A. Turaki, P. Lava Kumar, Susan SealAbstract:Since the first discovery of Badnaviruses (family Caulimoviridae, genus Badnavirus) in yam (Dioscorea spp.) germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial Badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al., 2008; Bousalem et al., 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al., 1999; Seal and Muller, 2007; Bomer et al., 2016 and 2017; Sukal et al., 2017; Umber et al., 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm. We have employed this approach successfully revealing three novel episomal yam Badnaviruses (Bomer et al., 2016). We proposed this to be a complementary method to denaturing gradient gel electrophoresis, which enables a rapid indication of Badnavirus diversity as well as the identification of potentially integrated Badnavirus sequences in the host genome (Turaki et al., 2017). Here, we describe the step-by-step protocol to screen yam germplasm for Badnavirus infections using RCA as an efficient research tool in the amplification and characterization of novel Badnavirus genomes.
-
PCR-DGGE Analysis: Unravelling Complex Mixtures of Badnavirus Sequences Present in Yam Germplasm
Viruses, 2017Co-Authors: Aliyu A. Turaki, Moritz Bömer, Goncalo Silva, P. Lava Kumar, Susan SealAbstract:Badnaviruses (family Caulimoviridae, genus Badnavirus) have emerged as serious pathogens especially affecting the cultivation of tropical crops. Badnavirus sequences can be integrated in host genomes, complicating the detection of episomal infections and the assessment of viral genetic diversity in samples containing a complex mixture of sequences. Yam (Dioscorea spp.) plants are hosts to a diverse range of Badnavirus species, and recent findings have suggested that mixed infections occur frequently in West African yam germplasm. Historically, the determination of the diversity of Badnaviruses present in yam breeding lines has been achieved by cloning and sequencing of polymerase chain reaction (PCR) products. In this study, the molecular diversity of partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences from yam Badnaviruses was analysed using PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE). This resulted in the identification of complex ‘fingerprints’ composed of multiple sequences of Dioscorea bacilliform viruses (DBVs). Many of these sequences show high nucleotide identities to endogenous DBV (eDBV) sequences deposited in GenBank, and fall into six monophyletic species groups. Our findings highlight PCR-DGGE as a powerful tool in Badnavirus diversity studies enabling a rapid indication of sequence diversity as well as potential candidate integrated sequences revealed by their conserved nature across germplasm.
-
A Sequence-Independent Strategy for Amplification and Characterisation of Episomal Badnavirus Sequences Reveals Three Previously Uncharacterised Yam Badnaviruses.
Viruses, 2016Co-Authors: Moritz Bömer, Goncalo Silva, Aliyu A. Turaki, P. Lava Kumar, Susan SealAbstract:Yam (Dioscorea spp.) plants are potentially hosts to a diverse range of Badnavirus species (genus Badnavirus, family Caulimoviridae), but their detection is complicated by the existence of integrated Badnavirus sequences in some yam genomes. To date, only two Badnavirus genomes have been characterised, namely, Dioscorea bacilliform AL virus (DBALV) and Dioscorea bacilliform SN virus (DBSNV). A further 10 tentative species in yam have been described based on their partial reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences, generically referred to here as Dioscorea bacilliform viruses (DBVs). Further characterisation of DBV species is necessary to determine which represent episomal viruses and which are only present as integrated Badnavirus sequences in some yam genomes. In this study, a sequence-independent multiply-primed rolling circle amplification (RCA) method was evaluated for selective amplification of episomal DBV genomes. This resulted in the identification and characterisation of nine complete genomic sequences (7.4–7.7 kbp) of existing and previously undescribed DBV phylogenetic groups from Dioscorea alata and Dioscorea rotundata accessions. These new yam Badnavirus genomes expand our understanding of the diversity and genomic organisation of DBVs, and assist the development of improved diagnostic tools. Our findings also suggest that mixed Badnavirus infections occur relatively often in West African yam germplasm.
-
the prevalence of Badnaviruses in west african yams dioscorea cayenensis rotundata and evidence of endogenous pararetrovirus sequences in their genomes
Virus Research, 2014Co-Authors: Susan Seal, Emmanuelle Muller, Aliyu A. Turaki, Denis Filloux, Serge Galzi, Lava P Kumar, L Kenyon, Antonio Lopezmontes, Marieline IskracaruanaAbstract:Abstract Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other Badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of Badnaviruses, and determine if Badnavirus genomes are integrated in the yam genome. Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, were screened using generic Badnavirus PCR primers. Positive amplification of an expected ca. 579 bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from Badnavirus particles or putative endogenous Badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with Badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam Badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of Badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of Badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.
-
Characterisation of Badnaviruses and endogenous pararetroviruses in West African yam breeding lines
2013Co-Authors: Aliyu A. Turaki, Emmanuelle Muller, Susan Seal, P. Lava Kumar, Denis Filloux, Serge Galzi, Antonio Lopez-montes, Marie-line Iskra CaruanaAbstract:Cultivated yams (Dioscorea spp.) are propagated vegetatively through their tubers, which results in the accumulation of tuber-borne virus infections, and their perpetuation from one crop to the next. These infections reduce the productivity of the plants and are an impediment to the international movement and exchange of yam germplasm. The only effective method of controlling these virus diseases is to use virus-free planting material. The 26 virus species that have been reported to infect yams worldwide fall into nine taxonomic genera, but only three of these (Badnavirus, Potyvirus and Cucumovirus) have been shown to be widespread in recent surveys across West Africa. Badnaviruses were detected in over 95% of landraces and breeding lines suggesting its wide distribution in West Africa. Analysis of >150 partial PCR-amplified Badnavirus RT-RNaseH sequences has grouped them into 12 species clusters each sharing
Serge Galzi - One of the best experts on this subject based on the ideXlab platform.
-
Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin
Molecular Plant Pathology, 2020Co-Authors: Matthieu Chabannes, Serge Galzi, Marc Gabriel, Abderrahmane Aksa, Jean‐françois Dufayard, Marie‐line Iskra‐caruana, Emmanuelle MullerAbstract:Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although Badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing Badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.
-
Improved diagnostic tools for the detection Badnaviruses in yams unveil the existence of endogenous sequences of extant Badnavirus species in yams. [O.36]
2015Co-Authors: Marie Umber, Emmanuelle Muller, Marie-line Caruana, Nathalie Laboureau, Philippe Roumagnac, Claudie Pavis, Denis Filloux, Serge Galzi, Rose-marie Gomez, Pierre-yves TeycheneyAbstract: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. (Resume d'auteur)
-
A scenario of co-evolution between Badnaviruses and Musa sp.. [P.22]
2015Co-Authors: Marie-line Iskra Caruana, Nathalie Laboureau, Serge Galzi, Pierre-olivier Duroy, Matthieu Chabannes, Emmanuelle MullerAbstract:The banana streak disease is due to a complex of distinct Banana streak viruses (BSVs) species showing a wide genetic diversity. Banana streak viruses (BSVs) are double stranded DNA pararetroviruses belonging to the family Caulimoviridae, genus Badnavirus. Outbreaks of BSVs causing banana streak disease have been recorded worldwide where Musa spp. is grown during the last 20 years with no convincing evidence of epidemics. Epidemics were previously reported in Uganda where BSV is currently endemic. In addition, the banana genome (Musa sp.) is invaded by numerous Badnavirus sequences. The majority of these viral integrants is mostly defective as a result of pseudogenisation driven by the host genome evolution. They are just called BEV (banana endogenous virus sequences) because episomal particles corresponding to the integrated counterparts have not been identified so far. Conversely, only few viral integrants named endogenous BSV (eBSVs) can release a functional BSV genome following stresses. All the badnaviral sequences described so far are spread among the three main clades of the Badnavirus genus diversity. Our group established that BSVs are distributed among Clade I and Clade III. Clade I gathers BSV species distributed worldwide whereas Clade III is dedicated to BSV species only present in Uganda. eBSVs exclusively correspond to BSV species of Clade I and are endogenous to the Musa balbisiana (B) genomes only. We elucidated their sequence and organization for three BSV species (BSOLV, BSGFV and BSIMV) present in the seedy banana diploid PKW (BB). In addition, we established that all BEVs sequences belong to Clade II. All together our results allowed us to propose an evolutionary scheme of Badnavirus and banana co-evolution, which is presented here. (Resume d'auteur)
-
the prevalence of Badnaviruses in west african yams dioscorea cayenensis rotundata and evidence of endogenous pararetrovirus sequences in their genomes
Virus Research, 2014Co-Authors: Susan Seal, Emmanuelle Muller, Aliyu A. Turaki, Denis Filloux, Serge Galzi, Lava P Kumar, L Kenyon, Antonio Lopezmontes, Marieline IskracaruanaAbstract:Abstract Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international movement of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes of some other Badnaviruses are known to be integrated in their host plant species. However, it was not clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of Badnaviruses, and determine if Badnavirus genomes are integrated in the yam genome. Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, were screened using generic Badnavirus PCR primers. Positive amplification of an expected ca. 579 bp fragment, corresponding to a partial RT-RNaseH region, was detected in 47 (81%) of 58 samples analysed from CIRAD collections, and 100% of the 127 IITA D. rotundata samples. All the D. cayenensis and D. rotundata samples from the CIRAD and IITA collections tested PCR-positive, and sequencing of a selection of the PCR products confirmed they were typical of the genus Badnavirus. A comparison of serological and nucleic acid techniques was used to investigate whether the PCR-positives were sequences amplified from Badnavirus particles or putative endogenous Badnavirus sequences in the yam genome. Protein A sandwich-enzyme-linked immunosorbent assay (PAS-ELISA) with Badnavirus polyclonal antisera detected cross-reacting viral particles in only 60% (92 of 153) of the CIRAD collection samples analysed, in contrast to the aforementioned 81% by PCR. Immunosorbent electron microscopy (ISEM) of virus preparations of a select set of 16 samples, representing different combinations of positive and negative PCR and PAS-ELISA results, identified bacilliform particles in 11 of these samples. Three PCR-positive yam samples from Burkina Faso (cv. Pilimpikou) were identified in which no viral particles were detected by either PAS-ELISA or ISEM. Southern hybridisation results using a yam Badnavirus RT-RNaseH sequence (Gn155Dr) as probe, supported a lack of Badnavirus particles in the cv. Pilimpikou and identified their equivalent sequences to be of plant genome origin. Probe Gn155Dr, however, hybridised to viral particles and plant genomic DNA in three D. rotundata samples from Guinea. These results represent the first data demonstrating the presence of integrated sequences of Badnaviruses in yam. The implications of this for virus-indexing, breeding and multiplication of seed yams are discussed.
-
The genome of African yam (Dioscorea cayenensis-rotundata complex) hosts endogenous sequences from four distinct Badnavirus species
Molecular Plant Pathology, 2014Co-Authors: Marie Umber, Marie-line Iskra-caruana, Emmanuelle Muller, Nathalie Laboureau, Philippe Roumagnac, Pierre-yves Teycheney, Denis Filloux, Serge Galzi, Claude Pavis, Susan E. SealAbstract: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.
