A Multi-assay molecular toolkit for rapid and sensitive detection of Ustilaginoidea virens the causative agent of rice false smut using PCR, qPCR, RPA, and LAMP assays.

Ustilaginoidea virens, the causative agent of rice false smut, poses a significant threat to global rice production, reducing grain yield and quality. Our objective was to develop rapid and sensitive detection assays that enable timely detection and management. BLASTn analysis was conducted on the whole genome of U. virens Uv-8b against NCBI NR database in order to find sequences exclusive to U. virens. Only candidate genes that were 100% identical to the genomes of U. virens and had no homology to non-target species were retained. Finally, the Uv-05919 gene was selected and used to develop conventional PCR, quantitative PCR, Recombinase Polymerase Amplification (RPA), and Loop-Mediated Isothermal Amplification (LAMP) assays. The specificity of the assays was evaluated using multiple U. virens isolates as well as non-U. virens pathogens. All the developed assays exhibited high specificity to U. virens with no cross-reactivity with non-target organisms. The sensitivity thresholds were 100 pg / 50 µL in RPA, 10 pg / 25 µL in LAMP, 12 plasmid copies/ 20 µL in qPCR, and 100 pg/ 25 µL in conv-PCR. All assays reliably detected U. virens in infected rice panicles but not in healthy controls, demonstrating their field applicability.