Real-time PCR assay for Colletotrichum acutatum sensu stricto quantification in olive fruit samples.

• Colletotrichum acutatum real-time qPCR assay. • Colletotrichum acutatum detection and quantification in olive drupes. • Colletotrichum acutatum single-copy gene: klap 1. 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 early infection stages, before symptom appearance, 16 h after inoculation (Ct values = 28.29 ± 1.1). In olive samples, the derived melting curve was specific presenting a single dissociation peak (T melting = 88.7 °C). The designed assay was effectively applied in C. acutatum detection and quantification using infected olive samples, with a LOD of 0.59 ng and a LOQ of 1.8 ng, allowing its application to orchard management. [ABSTRACT FROM AUTHOR]