The alarming spread of Xylella fastidiosa in EU territory and the rising susceptibility of host plants have mandated surveys and inspections in nursery and at consignments. Thus, the urge is prompted to develop robust and rapid diagnostic tests suitable for screening different host plant species and large number of samples. To this end, an alternative automated protocol for recovering total DNA for identifying X. fastidiosa in qPCR was validated on 8 different, artificially inoculated, plant matrices and on olive field samples. The results showed high quality of DNA templates with standardized yields and elevated efficiency levels. In addition, experiments were carried out, by simulating composite samples of Xylella-infected plant tissues pooled at different ratios with healthy materials and processed by qPCR.
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The results stated that a minimum of 2 to 4 infected units can be pooled with up to 20 to 40 gr of healthy tissue and processed, generating 100% of diagnostic sensitivity using the standard extraction procedures. Finally, an approach of high-resolution melting (HRM) analysis coupled with qPCR, was validated on a large panel of isolates. This procedure proved efficient and identified genotypes in 3 main clusters associated to the most abundant and widespread strains: fastidiosa, multiplex and pauca.
