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

The quarantine bacterium Xylella fastidiosa was first detected in Salento (Apulia, Italy) in 2013 and caused severe symptoms in olives, leading to plant death. The disease, named Olive Quick Decline Syndrome (OQDS), is caused by the strain “De Donno” ST53 of the subspecies pauca of this bacterium (XfDD), which is spread by the insect Philaenus spumarius. The epidemic poses a serious threat to the agricultural economy and the landscape, as X. fastidiosa infects several plant species and there is yet no recognized solution. Research on OQDS is focused on finding strategies to control its spread or mitigate its symptoms. As a perspective solution, we investigated the efficacy of the low-temperature plasma and plasma-activated water to kill bacterial cells. Experiments were conducted in vitro to test the biocidal effect of the direct application of a Surface Dielectric Barrier Discharge (SDBD) plasma on bacteria cells and Plasma Activated Water (PAW). PAW activity was tested as a possible biocidal agent that can move freely in the xylem network paving the way to test the strategy on infected plants. The results showed a high decontamination rate even for cells of XfDD embedded in biofilms grown on solid media and complete inactivation in liquid culture medium.

Preventive measures for infectious diseases caused by the harmful plant pathogenic bacterium Xylella fastidiosa include inspections and diagnostic tests on imported consignments of plants and in nurseries. Currently, mandatory checks on plant propagating materials are enforced in Europe (EU regulation 2021/1201) for the most susceptible species found in the European outbreaks, and prior to move propagating materials of the “specified plants” from nurseries located in the so-called “demarcated areas”. These requirements imply sampling and laboratory manipulation of a large number of samples, nevertheless plants to be sampled are often small size potted plants. While statistically based methods for inspections and sampling are available, namely the International Standards for Phytosanitary Measures n. 31, validated laboratory procedures to test large volumes of plant materials are lacking. In this work, we optimized two distinct protocols to detect X. fastidiosa in pooled plant materials collected from lots of plants for planting. The first protocol was designed to test in pool few samples (up to 8), the second to process through a single diagnostic test plant material from a high number of samples (up to 225). Accuracy of the newly developed protocols was assessed by pooling at different ratio tissues collected from healthy and infected Polygala myrtifolia, Nerium oleander, Olea europaea, Lavandula stoechas and Prunus avium. Moreover, tests included pools of plantlets of Brassicaceae and Solanaceae artificially inoculated with stem portions of infected periwinkle. Using both protocols, high diagnostic sensitivity values were generated using serological and molecular tests, with qPCR consistently yielding the highest performance values, regardless the host species tested.