A comparison of serological and nucleic acid techniques was used to investigate whether the PCRpositives 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 analyzed, 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, and breeding and multiplication of seed yams are discussed. IntroductionYam (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 Several surveys on yam viruses suggest that badnaviruses are the most prevalent globally (Bousalem et al., 2009; Eni et al., 2008a, b;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) Phillips et al., 1999). These viruses were reported to induce leaf distortions and veinal chlorosis , although others found that often infected plants show no marked symptoms (Kenyon et al., 2008;Seal and Muller, 2007). 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 (I...

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.

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.

Yam (Dioscorea species) and banana (Musa species) leaf sample contain high levels of polysaccharide and polyphenolic compounds. Extraction of good quality DNA from such leaves is usually problematic. Therefore, there is a need to extract good quality DNA in order to perform downstream DNA analysis, especially for the detection of viruses due to their low titration in some infected leaves. The cetyltrimethylammonium bromide (CTAB) DNA extraction method was selected in order to optimise DNA extraction from plant material with high level of polysaccharides. Four steps of the Lodhi et al. (1994) method were modified to be user friendly in less resource laboratories of developing countries. Yield and purity of the extracted DNAs were quantified using a NanoDrop 2000 spectrophotometer and by migration on an agarose gel after polymerase chain reaction (PCR) amplification. The results of the DNA yields and purity were in the range of 287.40±2.23 to 424.95±1.85 ng/ul and 2.10±0.05 to 2.19±0.04, respectively. Modification was found to yield DNA of reasonable quantity, quality and purity. Furthermore, the method can be used in a laboratory with less sophisticated waste disposal system.

[Abstract] 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;Bömer et al., 2016 andSukal 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 (Bömer 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. virus, also enabling the discrimination between integrated and episomal virus sequences (James et al., 2011) and was proposed to be used for DBV diagnostic purposes in yam (Umber et al., 2014). We uncovered several limitations to the usefulness of RCA in DBV diagnostics discussed in our previous study, including the amplification of putatively integrated sequences at lower frequencies and the

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Viruses of the genus Badnavirus (family Caulimoviridae) are double-stranded DNA-reverse transcribing (dsDNA-RT) plant viruses and have emerged as serious pathogens of tropical and temperate crops globally. Endogenous badnaviral sequences are found integrated in the genomes of several economically important plant species. Infection due to activation of replication-competent integrated copies of the genera Badnavirus, Petuvirus and Cavemovirus has been described. Such endogenous badnaviral elements pose challenges to the development of nucleic acid-based diagnostic methods for episomal virus infections and decisions on health certification for international movement of germplasm and seed. One major food security crop affected is yam (Dioscorea spp.). A diverse range of Dioscorea bacilliform viruses (DBVs), and endogenous DBV (eDBV) sequences have been found to be widespread in yams cultivated in West Africa and other parts of the world. This study outlines the development of multiplex PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE) to assist in the detection and analysis of eDBVs, through the example of analysing yam germplasm from Nigeria and Ghana. Primers targeting the three most prevalent DBV monophyletic species groups in West Africa were designed to improve DGGE resolution of complex eDBV sequence fingerprints. Multiplex PCR-DGGE with the addition of a tailor-made DGGE sequence marker enables rapid comparison of endogenous badnaviral sequence diversity across germplasm, as illustrated in this study for eDBV diversity in yam.

Thrombotic diseases are increasingly becoming among the prominent causes of death across the globe. Researchers are now turning attention towards fibrinolytic enzymes as potential alternative treatment for thrombolytic diseases. This present study focused on the production of extracellular fibrinolytic enzyme from soil Actinobacteria and evaluation of its hydrolytic activity on blood clot. The Actinobacteria was isolated from compost soil of semi-arid climate in Nigeria. Bacterial isolation was achieved using starch casein agar. Potent fibrinolytic enzyme producing Actinobacteria were identified and subjected to enzyme production using submerged fermentation method. The interactive effects of incubation time, temperature, pH and media components on enzyme production were analysed. Extracellular fibrinolytic enzyme produced by the selected Actinobacteria was partially purified by ammonium sulphate precipitation and subsequently assayed for blood clot lysis activity. Results of these studies indicated that fibrinolytic enzyme was produced optimally at pH 8 and temperature of 40 oC after 72 hour of fermentation. Partially purified fibrinolytic enzyme was able to degrade blood clot comparable to the positive control. These results shows that soil Actinobacteria of unexplored semi-arid climate of Nigeria present a prospect in search of novel microorganisms with potentials in the production of fibrinolytic enzyme that can serve as an alternative blood clot buster in treating thrombolytic diseases.