Development of high-throughput real-time PCR assays for the Colletotrichum acutatum detection on infected olive fruits and olive oils

Filipe Azevedo-Nogueira, Sónia Gomes, Teresa Carvalho, and Paula Martins-Lopes

The detection of latent Colletotrichum spp infection in olive drupes is crucial, to avoid contamination in the olive oil production chain. In order to detect the presence of C. acutatum in complex olive matrices a real-time PCR assay was developed, using olive drupe and oil samples from C. acutatum susceptible and tolerant olive cultivars (Galega Vulgar, Cobrançosa and Picual) with different infection levels. A C. acutatum specific sequence, belonging to the Internal Transcribed Spacers region, was used to design the real-time PCR detection assay, resulting in an 490 bp amplicon with a consistent melting temperature (T_m = 87.8 °C). The assay allowed a rapid and high-sensitive C. acutatum detection mean, being able to detect the infection in a latent phase, for the first time, in olive drupes, 16 hai, and in olive oils containing 20% of infected olives. This novel method can be used to monitor C. acutatum presence in olive orchards.