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.
Highlights
Rice (Oryza sativa L.) is a staple food consumed worldwide, with a cultivated area of 162 million ha and a production of 755 million tons
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
A loop-mediated isothermal amplification (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, comparing it to the blotter test and with two different DNA extraction procedures
Summary
Rice (Oryza sativa L.) is a staple food consumed worldwide, with a cultivated area of 162 million ha and a production of 755 million tons. In Italy, rice production is currently 1,498,133 tons, an area of 220,027 ha, concentrated in the northern regions [2] that apply technologically advanced rice cultivation systems [3]. The most frequent symptoms of the disease are yellowing and excessive elongation of the affected seedlings [9,10] caused by the production of gibberellic acid by the pathogen [11], which has led to the Japanese name bakanae, meaning “foolish seedling”. If the level of seed borne inoculum is high, the probability of causing infection in the field increases, but several factors can influence the infection cycle, such as weather conditions, cropping practices, resistance or susceptibility of the cultivar, the virulence of the strain of the pathogen and the efficiency of the inoculum [13]. The disease became more relevant to rice seed companies, increasing the need of specific diagnostic tools, as well as the development of effective management strategies [16]
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