Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus.

Sofia Bertacca,Daniela Trippa,Maria Isabel Font San Ambrosio,Antonio Giovino,Slavica Matic,Stefano Panno,Annalisa Marchese,Ana Alfaro,Andrea Giovanni Caruso,Emanuela Noris,Salvatore Davino

doi:10.3390/plants11050660

Abstract

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material.

Highlights

The olive tree (Olea europaea L.) belonging to the Oleaceae family is the most widely cultivated species of the Olea genus
We aimed to investigate the presence of Olea europaea geminivirus (OEGV) in Sicily and to develop a rapid detection protocol based on the loop-mediated isothermal amplification (LAMP) methodology
To ascertain the presence of OEGV in Sicily, a total of 80 samples representing 10 different cvs collected from two olive production sites in the Agrigento province were analyzed by end-point PCR

Summary

Introduction

The olive tree (Olea europaea L.) belonging to the Oleaceae family is the most widely cultivated species of the Olea genus. In Sicily, the olive crop has been cultivated since ancient times, and it is characterized by many ancient landraces/cultivars of high organoleptic quality [6]; its germplasm is distinguished by a wide genetic diversity, possibly related to its past domestication and spread and to some reproductive biological peculiarities such as self-incompatibility [7]. The current tendency in olive tree cultivation is moving towards the use of local cvs for high quality oil production (such as DOP—protected designation of origin), which is typical of specific geographic areas. For this reason, the local administration currently supports studies and activities aimed at the characterization and recovery of local and ancient cvs, in order to establish germplasm collections that limit genetic erosion [9]. No genes encoding AC4/C4 were found on the OEGV genome

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