The prevalence of badnaviruses in West African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous pararetrovirus sequences in their genomes
Introduction
Yam (Dioscorea species) is the fourth most important food tuber crop in the world after potato, sweet potato, and cassava (FAO, 2012). In West Africa, it is the second most important food crop after cassava by value and production (FAO, 2012, Scarcelli et al., 2006). It plays an essential role in food security and income generation for smallholders, particularly in West Africa which produces about 95% of the world's total yam production (Asiedu and Sartie, 2010, IITA, 2012, Mignouna et al., 2008). The white Guinea yam, Dioscorea rotundata, is the predominant yam species grown in this region, in contrast to other popular yam species such as D. alata and D. esculenta which predominate in the South Pacific (Kenyon et al., 2008). The yellow Guinea yam, D. cayenensis is also cultivated widely in West Africa, and like D. rotundata, is an African domesticated species originating from wild Dioscoreaceae of the Enantiophyllum Uline section. It should be noted that the classification of Guinea yam into either D. rotundata Poir. or D. cayenensis Lam. has, however, been confused for a long time, and they have in the past few decades often been referred to collectively as members of the D. cayenensis-rotundata species complex (Dumont et al., 2006).
Yam is generally propagated vegetatively through its tubers. This facilitates the accumulation of pathogens, particularly viruses of which there are at least 26 different species belonging to nine virus genera reported in yams worldwide to date (Bousalem et al., 2009, Kenyon et al., 2001). These virus infections have the potential to reduce tuber yields and quality, and impede yam germplasm movement and thus hinder international exchange of selected improved varieties (Bousalem et al., 2009, Kenyon et al., 2008). The scarcity and associated high expense of ‘clean seed’ yam has been identified as one of the most important critical constraints to increasing yam production and productivity in West Africa (IITA, 2012).
Several surveys on yam viruses suggest that badnaviruses are the most prevalent globally (Bousalem et al., 2009, Eni et al., 2008a, Eni et al., 2008b, Eni et al., 2009, Galzi et al., 2013, Kenyon et al., 2008). Badnavirus particles were first reported in yam in association with a flexuous virus, causing internal brown spot disease in D. alata and D. cayenensis in the Caribbean (Harrison and Roberts, 1973, Mantell and Haque, 1978). Two decades later yam badnaviruses were characterised by their nucleic acid and serological properties; particles isolated from D. alata and D. bulbifera were partially characterised and named informally as Dioscorea alata bacilliform virus (DaBV) and Dioscorea bulbifera bacilliform virus (DbBV) (Briddon et al., 1999, Phillips et al., 1999). These viruses were reported to induce leaf distortions and veinal chlorosis (Phillips et al., 1999), although others found that often infected plants show no marked symptoms (Kenyon et al., 2008, Seal and Muller, 2007).
Current taxonomic criteria (King et al., 2012) recognise only two species of yam badnavirus, for which complete genome sequence data (ca. 7.2–7.4 kb) exist, namely Dioscorea bacilliform alata virus (DBALV) isolated from D. alata in Nigeria (Briddon et al., 1999), and Dioscorea bacilliform sansibarensis virus (DBSNV) present in a wild D. sansibarensis from Benin (Seal and Muller, 2007). At least a further 10 putative Badnavirus species are indicated to be present in Dioscorea species globally through possessing partial (529 bp) RT-RNaseH nucleotide sequences that differ by more than the International Committee on Taxonomy of Viruses (ICTV) recommended species demarcation threshold for this region of >20% (Bousalem et al., 2009, King et al., 2012).
Sensitive virus diagnostic tests are required to enable the identification of virus-free seed yams, and will underpin current efforts in West Africa to generate and multiply disease-free yam planting material (IITA, 2012), as well as being essential to generate meaningful data in field surveys and epidemiology. Of the three virus genera (Badnavirus, Cucumovirus and Potyvirus) known to be of economic importance to yams in West Africa, reliable diagnostic tests exist for detection of yam potyviruses and cucumoviruses (Eni et al., 2008b, Eni et al., 2009, Mumford and Seal, 1997, Wylie et al., 1993). The serological and genetic heterogeneity of yam badnaviruses, however, poses a challenge for the development of diagnostic tests, as also experienced for badnaviruses in a wide range of other crops (Harper et al., 2005, Kenyon et al., 2008, Lockhart, 1986, Muller et al., 2011). Furthermore, the discovery of DNA sequences of the genus Badnavirus as integrated sequences in their plant host genome complicates the use of nucleic-acid based diagnostics, as illustrated by the challenges experienced in reliable detection of virus particles of banana streak viruses (BSVs) in Musa species (Harper et al., 1999b, Ndowora et al., 1999, Le Provost et al., 2006). Such integrated sequences appear to be a common phenomenon within genera of the family Caulimoviridae, and are termed endogenous pararetroviruses (EPRVs) (Geering et al., 2010, Mette et al., 2002, Staginnus et al., 2009).
The structure of EPRV sequences can be complex, and generally consists of rearranged patterns showing tandem repeats, fragmentations, inversions and duplications of the viral genome or parts thereof (Chabannes et al., 2013, Gayral et al., 2008, Ndowora et al., 1999, Richert-Pöggeler et al., 2003). Although most EPRVs reported seem to be simply neutral components in their host plant genomes, there have been three host examples to date which are of concern to breeding and virus-indexing programmes as they are ‘activatable’, i.e. episomal virus infections can be initiated de novo from these sequences integrated in their host plant genomes (Chabannes et al., 2013, Lockhart et al., 2000, Richert-Pöggeler et al., 2003). The activatable EPRVs represent three species of the genus Badnavirus discovered in banana genomes of Musa balbisiana species, namely Banana streak OL virus (BSOLV), Banana streak Imové virus (BSImV), and Banana streak GF virus (BSGFV) (Chabannes et al., 2013, Gayral et al., 2008, Harper et al., 1999a, Iskra-Caruana et al., 2014, Ndowora et al., 1999), as well as the petuvirus Petunia vein clearing virus (PVCV) in petunia (Richert-Pöggeler et al., 2003), and solendovirus Tobacco vein-clearing virus (TVCV) in tobacco (Jakowitsch et al., 1999, Lockhart et al., 2000). Activation is considered in banana to be triggered by the epigenetic modifications that occur during hybridisation of parental genomes as well as environmental stresses (e.g. wounding, tissue culture, and drought) (Dallot et al., 2001, Cote et al., 2010, Harper et al., 2002). Episomal virus has been suggested to be generated and released from EPRV sequences through mechanisms involving either homologous recombination between repeat regions, and/or by direct reverse transcription (Chabannes and Iskra-Caruana, 2013, Harper et al., 2002, Iskra-Caruana et al., 2010, Iskra-Caruana et al., 2014, Ndowora et al., 1999, Richert-Pöggeler et al., 2003).
Previous studies on yam badnaviruses have reported unusually high levels (91–96%) of badnavirus PCR-positive samples within Dioscorea rotundata collections from both Benin and Guadeloupe (Bousalem et al., 2009), in comparison to much lower ELISA-positives (26–35%) from South Pacific yam samples (Kenyon et al., 2008). This study was initiated to investigate whether the unusually high PCR-positive results in D. rotundata samples represented virus particle infections, or might be the result of PCR amplification of previously unidentified endogenous Dioscorea bacilliform virus sequences (termed eDBVs, according to the nomenclature proposed by Staginnus et al., 2009) in this yam species genome. Data revealed some samples from the D. cayenensis-rotundata complex to contain eDBVs. A high prevalence of serologically and genetically diverse badnaviruses was also detected in the West African yam collections. These findings have implications for the generation of high quality breeder and foundation yam planting material, and for the maintenance of its virus-free status when cultivated in the field.
Section snippets
Plant samples
Yams (n = 153) from the CIRAD-IRD collections were maintained and leaves collected from glasshouses as described previously (Bousalem et al., 2009). They were of eight species: D. abyssinica, D. alata, D. cayenensis, D. dumetorum, D. nummularia, D. rotundata, D. sansibarensis and D. trifida. The IITA collection samples (n = 127) consisted of first filial (F1) generations of breeding lines of D. rotundata (n = 112) and D. rotundata landraces (n = 15), which were collected from screen houses at the
PCR amplification of badnavirus sequences
Forty seven of the 58 CIRAD samples (81%) tested were scored as PCR-positive (Fig. 2, Table 1, and Supplementary materials). All 35 samples tested from the collections from West Africa (10 from Benin, 14 from Guinea, and 11 from IRD-Africa) were PCR-positive, in contrast to only 9 of 14 (64%), and 3 of 9 (33%) yam samples from Central and Southern America and the South Pacific (Vanuatu) respectively. A striking difference between the composition of these collections is that 29 of the 35 samples
Identification of endogenous Dioscorea bacilliform virus sequences (eDBV) in D. cayenensis-rotundata genomes
The data presented are the first results demonstrating the integration of badnavirus sequences in yam genomes of the D. cayenensis-rotundata species complex. EPRV sequences have not previously been reported for the plant family Dioscoreaceae, and support the proposition that EPRVs are widely distributed in plant genomes across a diverse range of plant families, including other cultivated hosts such as banana, petunia, rice, and tobacco (Geering et al., 2010, Iskra-Caruana et al., 2010).
Conclusions
This first discovery of integrated badnavirus sequences (eDBVs) in yam (Dioscorea spp.) has led to the realisation that PCR diagnostic techniques are not adequate for enabling decisions to be made on the suitability of yam germplasm for wide-scale cultivation or international exchange between yam breeding programmes. This study has also demonstrated that existing serological techniques for yam badnaviruses are inadequate in failing to cross-react sufficiently to some isolates. These findings
Acknowledgements
The authors thank Mustapha Bousalem and Camara Fadjimba for providing access to yam plants collected and maintained at CIRAD, Montpellier. Funding for this project was from a combination of sources; authors were generally ‘core’-funded by their associated employing institutions, with the exception of Aliyu Turaki who received funding from Kebbi State Government, Nigeria as well as partial research support funding from a Bill and Melinda Gates Foundation-funded project entitled ‘Yam Improvement
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