G Model VIRUS 96199 1–11 ARTICLE IN PRESS Virus Research xxx (2014) xxx–xxx Contents lists available at ScienceDirect Virus Research jo urnal homepage: www.elsevier.com/locate/virusres 1 The prevalence of badnaviruses in West African yams 2 (Dioscorea cayenensis-rotundata) and evidence of endogenous 3 pararetrovirus sequences in their genomes a,b,∗ a b c a,1 4 Q1 Susan Seal , Aliyu Turaki , Emmanuelle Muller , P. Lava Kumar , Lawrence Kenyon , b b c b 5 Denis Filloux , Serge Galzi , Antonio Lopez-Montes , Marie-Line Iskra-Caruana a 6 Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK b 7 CIRAD, UMR BGPI, F-34098 Montpellier, France c 8 International Institute of Tropical Agriculture (IITA), Oyo Road PMB 5320, Ibadan, Nigeria 9 10 a r t i c l e i n f o a b s t r a c t 11 12 Article history: Yam (Dioscorea spp.) is an important vegetatively-propagated staple crop in West Africa. Viruses are Available online xxx pervasive in yam worldwide, decreasing growth and yield, as well as hindering the international move- 13 ment of germplasm. Badnaviruses have been reported to be the most prevalent in yam, and genomes 14 Keywords: of some other badnaviruses are known to be integrated in their host plant species. However, it was not 15 Badnavirus clear if a similar scenario occurs in Dioscorea yam. This study was conducted to verify the prevalence of 16 Dioscorea yam badnaviruses, and determine if badnavirus genomes are integrated in the yam genome. 17 ELISA Leaf samples (n = 58) representing eight species of yam from global yam collections kept at CIRAD, 18 Endogenous pararetroviruses France, and 127 samples of D. rotundata breeding lines (n = 112) and landraces (n = 15) at IITA, Nigeria, 19 Immunosorbent electron microscopy 20 PCR 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 ana- lysed 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 serolog- ical 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 antis- era 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 prepa- rations 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, and breeding and multiplication of seed yams are discussed. © 2014 Published by Elsevier B.V. Abbreviations: DaBV, Dioscorea alata bacilliform virus; DbBV, Dioscorea bulb- 1. Introduction 21 ifera bacilliform virus; DBALV, Dioscorea bacilliform alata virus; DBSNV, Dioscorea bacilliform sansibarensis virus; DBV, Dioscorea bacilliform virus; eDBV, endogenous Dioscorea bacilliform virus; EPRV, endogenous pararetrovirus; PVCV, Petunia vein- Yam (Dioscorea species) is the fourth most important food tuber 22 clearing virus; TVCV, Tobacco vein-clearing virus. crop in the world after potato, sweet potato, and cassava (FAO, 23 ∗ Corresponding author at: Natural Resources Institute, University of Greenwich, 2012). In West Africa, it is the second most important food crop 24 Q2 Central Avenue, Chatham Maritime, Kent ME4 4TB, UK. Tel.: +44 1634 880088; after cassava by value and production (FAO, 2012; Scarcelli et al., 25 fax: +44 1634 883379. 2006). It plays an essential role in food security and income gener- 26 E-mail address: [email protected] (S. Seal). 1 Present address: AVRDC, PO Box 42, Shanhua, Tainan 74199, Taiwan. ation for smallholders, particularly in West Africa which produces 27 0168-1702/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.virusres.2014.01.007 Please cite this article in press as: Seal, S., et al., The prevalence of badnaviruses in West African yams (Dioscorea cayenensis-rotundata) and evidence of endogenous pararetrovirus sequences in their genomes. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.01.007 G Model VIRUS 96199 1–11 ARTICLE IN PRESS 2 S. Seal et al. / Virus Research xxx (2014) xxx–xxx 28 about 95% of the world’s total yam production (Asiedu and Sartie, 2011). Furthermore, the discovery of DNA sequences of the genus 94 29 2010; IITA, 2012; Mignouna et al., 2008). The white Guinea yam, Badnavirus as integrated sequences in their plant host genome com- 95 30 Dioscorea rotundata, is the predominant yam species grown in this plicates the use of nucleic-acid based diagnostics, as illustrated by 96 31 region, in contrast to other popular yam species such as D. alata the challenges experienced in reliable detection of virus particles of 97 32 and D. esculenta which predominate in the South Pacific (Kenyon banana streak viruses (BSVs) in Musa species (Harper et al., 1999b; 98 33 et al., 2008). The yellow Guinea yam, D. cayenensis is also culti- Ndowora et al., 1999; Le Provost et al., 2006). Such integrated 99 34 vated widely in West Africa, and like D. rotundata, is an African sequences appear to be a common phenomenon within genera of 100 35 domesticated species originating from wild Dioscoreaceae of the the family Caulimoviridae, and are termed endogenous pararetro- 101 36 Enantiophyllum Uline section. It should be noted that the classifi- viruses (EPRVs) (Geering et al., 2010; Mette et al., 2002; Staginnus 102 37 cation of Guinea yams into either D. rotundata Poir. or D. cayenensis et al., 2009). 103 38 Lam. has, however, been confused for a long time, and they have in The structure of EPRV sequences can be complex, and generally 104 39 the past few decades often been referred to collectively as mem- consists of rearranged patterns showing tandem repeats, fragmen- 105 40 bers of the D. cayenensis-rotundata species complex (Dumont et al., tations, inversions and duplications of the viral genome or parts 106 41 2006). thereof (Chabannes et al., 2013; Gayral et al., 2008; Ndowora et al., 107 42 Yam is generally propagated vegetatively through its tubers. 1999; Richert-Pöggeler et al., 2003). Although most EPRVs reported 108 43 This facilitates the accumulation of pathogens, particularly viruses seem to be simply neutral components in their host plant genomes, 109 44 of which there are at least 26 different species belonging to nine there have been three host examples to date which are of concern 110 45 virus genera reported in yams worldwide to date (Bousalem et al., to breeding and virus-indexing programmes as they are ‘activat- 111 46 2009; Kenyon et al., 2001). These virus infections have the poten- able’, i.e. episomal virus infections can be initiated de novo from 112 47 tial to reduce tuber yields and quality, and impede yam germplasm these sequences integrated in their host plant genomes (Chabannes 113 48 movement and thus hinder international exchange of selected et al., 2013; Lockhart et al., 2000; Richert-Pöggeler et al., 2003). 114 49 improved varieties (Bousalem et al., 2009; Kenyon et al., 2008). The activatable EPRVs represent three species of the genus Bad- 115 50 The scarcity and associated high expense of ‘clean seed’ yam has navirus discovered in banana genomes of Musa balbisiana species, 116 51 been identified as one of the most important critical constraints to namely Banana streak OL virus (BSOLV), Banana streak Imové virus 117 52 increasing yam production and productivity in West Africa (IITA, (BSImV), and Banana streak GF virus (BSGFV) (Chabannes et al., 118 53 2012). 2013; Gayral et al., 2008; Harper et al., 1999a; Iskra-Caruana et al., 119 54 Several surveys on yam viruses suggest that badnaviruses are this issue; Ndowora et al., 1999), as well as the petuvirus Petunia 120 55 the most prevalent globally (Bousalem et al., 2009; Eni et al., vein clearing virus (PVCV) in petunia (Richert-Pöggeler et al., 2003), 121 56 2008a,b, 2009; Galzi et al., 2013; Kenyon et al., 2008). Badnavirus and solendovirus Tobacco vein-clearing virus (TVCV) in tobacco 122 57 particles were first reported in yam in association with a flexu- (Jakowitsch et al., 1999; Lockhart et al., 2000). Activation is consid- 123 58 ous virus, causing internal brown spot disease in D. alata and D. ered in banana to be triggered by the epigenetic modifications that 124 59 cayenensis in the Caribbean (Harrison and Roberts, 1973; Mantell occur during hybridisation of parental genomes as well as envi- 125 60 and Haque, 1978). Two decades later yam badnaviruses were char- ronmental stresses (e.g. wounding, tissue culture, and drought) 126 61 acterised by their nucleic acid and serological properties; particles (Dallot et al., 2001; Cote et al., 2010; Harper et al., 2002).