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New methods for testing rice seed: LAMP assays for the detection of Fusarium fujikuroi and Magnaporthe oryzae

Sara Franco Ortega: Agroinnova - University of Torino

<div><em>Fusarium fujikuroi </em>and<em> Magnaporthe oryzae</em> are the causal agents of bakanae and rice blast, respectively. The estimated worldwide losses caused by both pathogens can reach up to 30% of the total production. The identification of both fungi on rice seed is a prerequisite for the pathogen-free certification and essential for the control of the pathogens. ISTA currently recomends methods for the detection of rice pathogens, based on the plating of 400 seeds and morphological identification of cultures growing on the seeds 10-days post-plating. This method may result in misidentifications due to the high number of shared characteristics among closely related species and the co-growth of multiple organisms. The LAMP assays developed in this study can overcome the drawback caused by culturing-methods. The LAMP assays were designed from the elongation factor 1-alpha and calmodulin genes for <em>F. fujikuroi </em>and<em> M. oryzae</em>, respectively. Both assays were validated according to the international EPPO standard (PM7/98) in terms of specificity, sensitivity, reproducibility and repeatibility. The results showed a limit of detection of 100-999 fg DNA for <em>F. fujikuroi</em> and 10-99 pg for <em>M. oryzae.</em> Five infected rice seed lots were used to compare the traditional culturing method with the LAMP method using a commercial DNA extraction kit. The results demonstrated the reliability of the LAMP methods for the surveillance of <em>F. fujikuroi </em>and <em>M. oryzae </em>in seed testing laboratories.</div>

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