Optimization of a Loop-Mediated Isothermal Amplification Assay for On-Site Detection of Fusarium fujikuroi in Rice Seed

Abstract

Fusarium fujikuroi, causing bakanae disease, is one of the most important seedborne pathogens of rice, the detection of which is paramount for seed certification and for preventing field infections. Molecular tests—qPCR and loop-mediated isothermal amplification (LAMP)—are replacing the blotter test in seed health procedures, due to higher sensitivity, specificity, fast turnaround results delivery, on-site application and the possibility of quantifying endophytic seed infections. A LAMP test, which had been previously developed with primers designed to target the elongation factor 1-α sequence of F. fujikuroi, was validated according to the international validation standard (EPPO, PM7/98) on thirty-four rice seed lines of different levels of susceptibility to the disease, thus comparing it to the blotter test and with two different DNA extraction procedures. The use of crude extracted DNA provided more sensitive results than the DNA extracted with the commercial kit Omega E.Z.N.A® Plant DNA kit. The results showed that the endophytic infection of F. fujikuroi is essential for the development of the disease in the field and that the minimum amount of the pathogen necessary for the development of the disease corresponds to 4.17 × 104 cells/µL. This study confirms the applicability of the LAMP technique on-site on rice seeds with fast and quantitative detection of the pathogen.