Molecular virulotyping and advancing the detection of Magnaporthe oryzae using a CRISPR-Cas12a-based diagnostic tool

Rice blast, caused by Magnaporthe oryzae, is a major threat to global rice production. The disease affects various growth stages of rice including leaves, nodes, and panicles leading to significant yield loss. Understanding the genetic diversity and early detection of M. oryzae is essential for developing effective intervention and disease management. In current study, 30 isolates of M. oryzae were collected from major rice-growing areas in Karnataka, Tamil Nadu, Andhra Pradesh, and Telangana, revealing considerable morphological and genetic diversity. Molecular characterization using ITS, LSU, and actin primers confirmed all isolates as M. oryzae, showing 100% sequence homology to reference strains. Phylogenetic analysis revealed regional clustering of isolates, with notable diversity observed in populations from Tamil Nadu and Karnataka. Pathogenicity assays identified significant variability in disease severity, with isolates MOK1, MOTN4, and MOK10 being highly virulent. Virulotyping revealed the widespread presence of critical pathogenicity genes, with Avr-Pik, Avr-Pita, MPS1, SLP1 and TYR1 as prominent contributors. Disease severity is highly correlated with the presence of EXO 70. The tyrosinase gene remains well conserved among all the isolates and was used as a target for CRISPR cas12a based detection. A novel CRISPR-Cas12a detection system, coupled with recombinase polymerase amplification (RPA), demonstrated high sensitivity and specificity for M. oryzae DNA, detecting concentrations as low as 10⁻¹/µL of DNA copies. Field testing successfully identified the pathogen in leaf, neck, and seed samples with visual confirmation via fluorescence and lateral flow assays. This integrated approach provides valuable insights into M. oryzae diversity, pathogenicity, and a robust diagnostic tool for early pathogen detection, paving the way for targeted management strategies to mitigate rice blast disease.