Development and application of a quantitative PCR detection method to quantify Venturia oleaginea (Castagne) Rossman & Crous in asymptomatic olive (Olea europaea L.) leaves

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important to develop effective control strategies. Current knowledge is incomplete due to lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming appropriateness of the target region for developing a qPCR assay. Furthermore, High Throughput Sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive enabling the detection of about 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results, and enables detection of V. oleaginea in all olive organs, including those where OLS cannot be studied using previously available methods.