Elsevier

Virus Research

Volume 186, 24 June 2014, Pages 144-154
Virus Research

The prevalence of badnaviruses in West African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous pararetrovirus sequences in their genomes

https://doi.org/10.1016/j.virusres.2014.01.007Get rights and content

Highlights

  • First study to reveal endogenous pararetrovirus (EPRV) sequences in a yam genome.

  • Dioscorea cayenensis-rotundata samples all test PCR-positive for badnavirus sequences.

  • ELISA reveals badnavirus infections to be prevalent (27–95%) in West African yam.

  • Existing diagnostic tools are inadequate for indexing yam for badnavirus infections.

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.

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

References (60)

  • R.W. Briddon et al.

    Analysis of the sequence of Dioscorea alata bacilliform virus; comparison to other members of the badnavirus group

    Virus Genes

    (1999)
  • M. Chabannes et al.

    Three infectious viral species lying in wait in the banana genome

    Journal of Virology

    (2013)
  • M. Chabannes et al.

    Endogenous pararetroviruses—a reservoir of virus infection in plants

    Current Opinion in Virology

    (2013)
  • F.X. Cote et al.

    Micropropagation by tissue culture triggers differential expression of infectious endogenous banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains

    Molecular Plant Pathology

    (2010)
  • S. Dallot et al.

    Evidence that the proliferation stage of micropropagation procedure is determinant in the expression of banana streak virus integrated into the genome of the FHIA 21 hybrid (Musa AAAB)

    Archives of Virology

    (2001)
  • M. Delanoy et al.

    Development of real-time PCR for the rapid detection of episomal Banana streak virus (BSV)

    Plant Disease

    (2003)
  • R. Dumont et al.

    Biodiversity and domestication of yams in West Africa

    Traditional practices giving rise to Dioscorea rotundata Poir

    (2006)
  • A.O. Eni et al.

    Sequence diversity among badnavirus isolates infecting yam (Dioscorea spp.) in Ghana, Togo, Benin and Nigeria

    Archives of Virology

    (2008)
  • A.O. Eni et al.

    Survey of the incidence and distribution of five viruses infecting yams in the major yam-producing zones in Benin

    Annals of Applied Biology

    (2008)
  • A.O. Eni et al.

    Survey of the incidence and distribution of viruses infecting yam (Dioscorea spp.) in Ghana and Togo

    Annals of Applied Biology

    (2009)
  • FAO

    Food and Agricultural Organisation of the United Nations FAO Statistics 2012. Rome, Italy

    (2012)
  • S. Galzi et al.

    Abstract in the 14íémes Rencontres de Virologie Végétale meeting

    Assessment and characterisation of the diversity of viruses infecting cultivated yams (Dioscorea spp.) in Haïti

    (2013)
  • P. Gayral et al.

    Phylogeny of Banana streak virus reveals recent and repetitive endogenization in the genome of its banana host (Musa sp.)

    Journal of Molecular Evolution

    (2009)
  • P. Gayral et al.

    A single banana streak virus integration event in the banana genome as the origin of infectious endogenous pararetrovirus

    Journal of Virology

    (2008)
  • P. Gayral et al.

    Evolution of endogenous sequences of Banana streak virus: what can we learn from banana (Musa sp.) evolution?

    Journal of Virology

    (2010)
  • A.D.W. Geering et al.

    Banana contains a diverse array of endogenous badnaviruses

    Journal of General Virology

    (2005)
  • A.D.W. Geering et al.

    The classification and nomenclature of endogenous viruses of the family Caulimoviridae

    Archives of Virology

    (2010)
  • C. Goudou-Urbino et al.

    Aetiology and ecology of a yam mosaic disease in Burkina Faso

    Tropical Science

    (1996)
  • G. Harper et al.

    The diversity of banana streak virus isolates in Uganda

    Archives of Virology

    (2005)
  • G. Harper et al.

    Viral sequences integrated into plant genomes

    Annual Review of Phytopathology

    (2002)
  • Cited by (0)

    1

    Present address: AVRDC, PO Box 42, Shanhua, Tainan 74199, Taiwan.

    View full text