Detection of Avirulence Gene AvrPi9 in Magnaporthe oryzae, a Rice Blast Fungus, Using a Combination of RPA and CRISPR-Cas12a Techniques

Rice blast disease is one of the most devastating diseases rice production worldwide, which causes by an ascomycete fungus, Magnaporthe oryzae. The virulence fungus determined avirulence genes (Avr genes). Therefore, identification Avr important for resistance variety improvement. are currently identified using pathogenicity assay with near-isogenic lines (NILs) or PCR amplification and gene sequencing, both time-consuming labor-intensive methods. This study aims to develop a simple method AvrPi9 as model. A recombinase polymerase (RPA) technique was carried out amplify incubating genomic DNA gene-specific primers at 37°C 20 min. Cas12a-based detection performed 5 fluorescence signal visualized naked eye under LED transilluminator. found that can be amplified detected RPA method. AvrPi9_crRNA2 has higher efficiency than AvrPi9_crRNA1. sensitivity 3.8 ng target AvrPi9_crRNA1 1.9 AvrPi9_crRNA2. Cas12a combination newer in plants several advantages over traditional It considered easier use more efficient terms time labor, making it potentially useful tool plant breeders pathologists.