Development and application of recombinase polymerase amplification for rapid detection of rice false smut pathogen (Ustilaginoidea virens)

Ustilaginoidea virens (Cke.) Tak. is the causal pathogen of rice false smut, a grain disease which affects both grain quality and yield. In this study, a recombinase polymerase amplification (RPA) assay protocol was developed to detect U. virens directly from rice spikelet. Three pairs of primer were designed from the U. virens GTP binding protein beta subunit (UvG-beta 1) gene to function both in RPA and in polymerase chain reaction (PCR). The developed RPA assay efficiently detected U. virens from both genomic DNA and mycelial crude extract. A comparison of sensitivity of RPA with PCR and loop-mediated isothermal amplification (LAMP) revealed that the latter two methods failed to detect U. virens from crude extract. The specificity of the RPA primers was evaluated using DNA from other rice pathogens, as well as, by sequencing the RPA amplicons. For validation, the RPA assay was evaluated on 32 different U. virens isolates from eastern and north-eastern India, as well as, on field samples. The developed RPA assay was able to detect U. virens directly from crude extract prepared from infected rice spikelet. The developed RPA assay will be effective for field monitoring of U. virens.