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Olive anthracnose is caused by fungal species within the Colletotrichum acutatum, C. gloeosporioides and C. boninense complexes. Anthracnose causes severe pre- and post-harvest olive drupe fall. This study aimed to design a species-specific qPCR assay, based on klap1 gene, suitable for C. acutatum s.s. quantification in cv. Galega Vulgar fruit samples. The developed qPCR assay presented a detection limit of 10.14 fg/reaction, and a linear cycle threshold of R2 = 0.996. C. acutatum inoculum was detected in pulverized olive fruits, and in