Optimizing molecular assays to support early detection of Xylella fastidiosa in host plants

Efficient sampling approaches combined with reliable, fast testing procedures are crucial factors in surveillance for early detection of Xylella fastidiosa (Xf). In the frame of the European project ‘XF-ACTORS’, work was performed on developing effective sampling methods to support reliable detection of Xf, particularly in asymptomatic plants. The objectives of the work were to: (a) evaluate the efficiency of two methods for DNA extraction from plant material (1 g), employing two commercial kits, one silica-column-based and one magnetic-bead-based, in automated DNA-extraction devices. (b) test the performance of both DNA extraction methods in pooled (10 g) plant samples. c) evaluate the sensitivity of an isothermal DNA amplification and detection system applied on crude plant material preparation without DNA purification, using a portable fluorometer (AmpliFire, Douglas Scientific). The plant material was derived from cultivated host species of high economic importance and wide distribution in Greece: Olea europaea, Prunus cerasifera, Ficus carica, and various ornamentals with a major role in Xf epidemiology: Lavandula sp., Nerium sp., Pelargonium sp., Pistacia sp., Juniperus sp. and Tagetes sp. It was spiked with different levels of inactivated Xf cells. The DNA extracts were assessed for their quality (Α260/Α280), quantity (ng/μl) and performance in qPCR protocols recommended in EPPO/ISPM diagnostic standards. In the isothermal DNA amplification and detection system experiment, the plant material was derived from: Polygala myrtifolia, a species used in this project as an indicator host for monitoring the presence of Xf insect-vectors, and Myrtus microphylla, a species exhibiting strong inhibition of amplification in conventional PCR. The results obtained contribute to the improvement of sampling and molecular testing of plants in Xf surveillance programs, supporting reliable early detection of this pathogen, as far as the effectiveness of the selected DNA extraction method is concerned, especially when pooled samples are tested.