Potential of LAMP assay as a tool for ecological monitoring and on-site detection of Bipolaris oryzae in rice

Abstract

One of the most significant rice seed-borne pathogens, Bipolaris oryzae causes serious yield losses worldwide every year. Traditional seed health testing involves incubation for up to a week, but molecular diagnosis using PCR-based assays requires sophisticated equipment and is not amenable to on-site testing. Loop-mediated isothermal amplification (LAMP) is ideal for field-level diagnosis since it offers quick, high-specific amplification of target template sequences at a single temperature. The exon 2 of the Glycosyl Transferase Family 90 protein of B. oryzae was targeted for designing the LAMP primers. The LAMP assay proposed was incredibly sensitive, detecting the B. oryzaetemplate's presence at concentrations as low as 10 fg at 60 °C in 30 min. The specificity of the test was assessed using genomic DNA retrieved from 22 different phytopathogens. The assay was employed to biologically monitor the existence of this pathogen by testing it on ten weed species in rice fields infected with B. oryzae. The pathogen was detected in 8 weed species except Brachypodium distachyon and Phalaris minor. The assay was used to detect the presence of B. oryzae in seeds from 13 different rice varieties. Seed samples from two cultivars tested negative while the remaining cultivars were confirmed to harbor the pathogen. Using this novel assay, B. oryzae can be detected rapidly in rice seeds and farm soil. The proposed LAMP assay has the potential to be employed in seed health laboratories, plant quarantine stations and on-site detection of B. oryzae in seeds and plants.