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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.

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.

This research was carried out in order to evaluate the presence and distribution of viral infections causing severe disease in eggplant plants collected from different districts in Bekaa valley, Lebanon. Most infected plants showed virus-like symptoms consisting predominantly of leaf blotch, mottling chlorotic and ring spots; leaf twisting and plant dwarf were also observed in the visited fields. Symptomatic and asymptomatic plants were collected and screened by ELISA test for the presence of several different pathogenic viruses potentially present in the area. Results showed that potato virus Y (PVY) was the most prevalent virus found by ELISA (detected in the 15.3% of the tested plants), followed by eggplant mottled dwarf virus (EMDV, 2.9%) and cucumber mosaic virus (CMV, 1.2%), while tomato spotted wilt virus (TSWV), alfalfa mosaic virus (AMV) and pepper mottle virus (PepMoV) were not detected. Biological indexing of symptomatic ELISA-negative plants, followed by electron microscopy, indicated the presence of virus-like particles of the genus Potexvirus, which was subsequently confirmed as potato virus X (PVX) by RT-PCR and Sanger sequencing. PVX was found in 35.3% of the tested plants, all sampled in the northern Bekaa area. In a phylogenetic analysis, the partial coat protein gene sequence of a selected Lebanese isolate, PVX-AK1, clustered together with other PVX isolates from Asia. Furthermore, the 124-aa sequence of PVX-AK1 shared 100% identity with PVX-UK3, an isolate which is known as avirulent in potato genotypes carrying either Nx or Rx resistance genes. This work revealed a picture of the previously uninvestigated phytosanitary status of eggplant crops in an important horticultural area of Lebanon.