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.

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28Yam mosaic virus (YMV; genus Potyvirus) is considered to cause the most economically important 29 viral disease of yams (Dioscorea spp.) in West Africa which is the dominant region for yam 30 production globally. 31Yams are a vegetatively propagated crop and the use of virus-free planting material forms an essential 32 component of disease control. Current serological and PCR-based diagnostic methods for YMV are 33 time consuming involving a succession of target detection steps. 34In this study, a novel assay for specific YMV detection is described that is based on isothermal 35 reverse transcription-recombinase polymerase amplification (RT-exoRPA). This test has been shown 36 to be reproducible and able to detect as little as 14 pg/μl of purified RNA obtained from an YMV-37 infected plant, a sensitivity equivalent to that obtained with the reverse transcription-polymerase chain 38 reaction (RT-PCR) in current general use. The RT-exoRPA assay has, however, several advantages 39 over the RT-PCR; positive samples can be detected in less than 30 min, and amplification only 40 requires a single incubation temperature (optimum 37°C). These features make the RT-exoRPA assay 41 a promising candidate for adapting into a field test format to be used by yam breeding programmes or 42 certification laboratories.

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA).The developed method, named ‘Direct RT-RPA’, detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation.The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-017-3605-9) contains supplementary material, which is available to authorized users.

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.

In vitro culture offers many advantages for yam germplasm conservation, propagation and international distribution. However, low virus titres in the generated tissues pose a challenge for reliable virus detection, which makes it difficult to ensure that planting material is virus-free. In this study, we evaluated next-generation sequencing (NGS) for virus detection following yam propagation using a robust tissue culture methodology. We detected and assembled the genomes of novel isolates of already characterised viral species of the genera Badnavirus and Potyvirus , confirming the utility of NGS in diagnosing yam viruses and contributing towards the safe distribution of germplasm.

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