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.
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.
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