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Development of a novel and rapid loop-mediated isothermal amplification assay for specific detection of Alternaria alternata and Alternaria solani

Rosa Caiazzo: National Institute of Agricultural Botany

<div>Early blight is the most significant foliar disease of potato in the USA, Asia and Africa, and incidence has been increasing in Europe and the UK too. The main species involved in the early blight complex are <em>Alternaria solani </em>and<em> Alternaria alternata</em>. Symptoms can initially appear very similar and diagnosis at the species level is only possible with microscopic assessment of spores, which is both time-consuming and requires appropriate training. Accurate identification is necessary to select appropriate control programmes. Thus a rapid, specific diagnostic assay would provide a more efficient strategy for distinguishing between <em>Alternaria</em> species and help to optimise control strategies. In this study, we developed and evaluated two loop-mediated isothermal amplification (LAMP) assays for differential diagnosis of the two pathogens, targeting the cytochrome b (cytb) to detect <em>A. solani, </em>and the major allergen Alt a gene to detect <em>A. alternata</em>. The specificity of the assays was tested against a panel of closely-related species and other pathogens affecting potato. The detection limit of the LAMP assays was 3pg of genomic DNA per reaction for <em>A. solani, </em>and 0.3pg for <em>A. alternata</em>. The assays were optimized at 64°C and 60°C, respectively using a 30 min amplification cycle. These LAMP assays have the potential to be used for monitoring the disease in the field, providing a specific, sensitive and rapid diagnostic tool for distinguishing between <em>A. solani and A. alternata</em>.</div>