Detection of Xylella fastidiosa in Host Plants and Insect Vectors by Droplet Digital PCR

Xylella fastidiosa (Xf) is a Gram-negative plant bacterium that causes severe diseases affecting several economically important crops in many countries. To achieve early detection of the pathogen, droplet digital PCR (ddPCR)-based approach was used to detect at low concentrations different species and insect vectors. In this study, we implemented reaction conditions previously developed ddPCR assay, validated its use Xf vectors as well broader list host species. More specifically, sensitivity accuracy protocol were assessed by testing five matrices (Olea europaea, Nerium oleander, Vitis vinifera, Citrus sinensis, Prunus dulcis), for first time, vector (Philaenus spumarius), either naturally infected or artificially spiked with bacterial suspension known concentrations. The lowest detected 5 ag/µL DNA 1.00 × 102 CFU/mL cells. Both techniques showed high degree linearity, R2 values ranging from 0.9905 0.9995 0.9726 0.9977, respectively, qPCR ddPCR. Under our conditions, greater analytical than O. europea, C. N. oleander. Overall, results demonstrated assay enables absolute quantification target sequences compared can support experimental research programs official controls, particularly when doubtful inconclusive are recorded qPCR.