Development of multiplex PCR protocols for simultaneous detection of Alternaria padwickii, Bipolaris oryzae, Pyricularia oryzae and Ustilaginoidea virens in rice seed

Abstract

Rice, a staple crop sustaining over half of the global population, is widely cultivated and traded as crop as well as exchanged globally in the form of germplasm. This crop is particularly susceptible to seed-borne fungal pathogens such as Alternaria padwickii, Bipolaris oryzae, Pyricularia oryzae and Ustilaginoidea virens, which pose significant threats to seed health, conservation, and transboundary movement. To address these risks, we developed robust two multiplex PCR protocols capable of simultaneously detecting A. padwickii, B. oryzae and P. oryzae and A. padwickii, P. oryzae and U. virens using specific primer sets: ApEF-1F&1R designed from translation elongation factor 1 (EF 1) region of A. padwickii, BoSP7-F&R designed from ATCC 44560 unplaced genomic scaffold_136 of B. oryzae, PoM-1F&1R designed from hydrophobin-like protein (MPG1) gene region of P. oryzae and UvP-1F&1R designed from internal transcribed spacer (ITS) region of U. virens. These primers amplified distinct target sequences of 175, 325, 520, and 240 bp, respectively, with an optimal annealing temperature of 62°C. Sensitivity test revealed that the primers detect only target pathogens at concentration of DNA as low as 0.1 ng μl⁻¹ of each pathogen, demonstrating high specificity. These findings aim to provide a robust tool for ensuring seed health testing, potentially improving disease management, reducing post-harvest losses, and supporting disease-free long-term conservation, seed certification, and expediting quick quarantine processing of rice seed.