A NEW REAL-TIME PCR METHOD DEVELOPED FOR THE DETECTION OF THE MAIN VIRUSES PRESENT IN OLIVE ORCHARDS

Abstract

Sensitive detection of viruses in olive orchards is actually of main importance since these pathogenic agents cannot be treated, their dissemination is quite easy, and they can have negative effects on olive oil quality. Traditionally, techniques based on serology have been used widely for the detection of olive viruses, but more recently, molecular biology-based methods that include the highly laborious dsRNA analysis, have started to be performed. The low viral titres in olive tissues is the major constrain of the techniques and do not always allow the successful, accurate and reproducible detection. Phytosanitary certification programs depend on a reliable and sensitive detection of these viruses. In plant virology, real-time PCR (qPCR) is increasingly being used to improve the sensitivity and accuracy while maintaining reliability. The work describes the development and application of new SYBRGreen qPCR assays for the detection of the Olive leaf yellowing-associated virus, Olive latent virus-1, Tobacco necrosis virus-D and Olive mild mosaic virus, the main viruses that affect the olive trees. The main goal is to increase the accuracy of detection of olive viruses and, consequently, to improve their control. The work involves i) design of specific primers for each target virus; ii) studies on the sensitivity of the technique and primers specificity; iii) validation of the technique with plant material from different orchards. This work enables for the first time a reliable, sensitive and reproducible estimation of virus accumulation in infected olive trees that will allow gaining new insights in virus biology essential for disease control.