Detection by Sensitive Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification of Olive Leaf Yellowing Associated Virus and Its Incidence in Italy and Spain

Abstract

Olive trees (Olea europea L.) are constantly threatened by many viruses, such as the olive leaf yellowing-associated virus (OLYaV), that belong to the Olivavirus genus, family Closteroviridae. In this work, the OLYaV incidence in different regions of Italy and Spain, which represent the two most important European areas for olive production, was evaluated through the development of a real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for reliable and sensitive OLYaV detection. The specificity and accuracy of the developed real-time RT-LAMP assay were determined; the assay showed that potential cross-reactivity with other viruses belonging to the Closteroviridae family was excluded. The LAMP assay detected OLYaV with a higher sensitivity than conventional end-point RT-PCR, detecting a total of 1.34 × 10−2 genome copies. A total of 80 and 120 plants of different olive cultivars from Spain (Comunitat Valenciana, Andalusia) and Italy (Sicily, Calabria, Apulia, Lazio, and Umbria) regions were tested, respectively. The percentage of infected plants was 46.25% and 30% for Spain and Italy, respectively, while the most susceptible cultivars were “Serrana Espadán” and “Villalonga” from Comunitat Valenciana and Andalusia regions (Spain) and “Ogliarola barese” from Apulia region (Italy). In addition, the survey demonstrated that the real-time RT-LAMP showed good sensitivity for OLYaV-positive sample detection, especially on asymptomatic olive trees. For this reason, the developed assay could be very suitable for phytopathological laboratories as a reliable and efficient method for a rapid and sensitive routine test on olive samples.