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The spittlebugs Philaenus spumarius and Neophilaenus campestris (Hemiptera: Aphrophoridae) are xylem-feeder insects that have been identified as vectors of Xylella fastidiosa in Europe. In the present study, we aim to identify volatile organic compounds (VOCs) that may act as semiochemicals for these species. Using the dynamic headspace technique, we collected VOCs from Olea europaea L. and Polygala myrtifolia L. , highly susceptible plant species to X. fastidiosa, Pinus halepensis Mill., a common plant where N. campestris is found during summer, and from host plant species that are used as cover crops or exist as natural vegetation in olive orchards, such as Cistus creticus L. , Medicago sativa L., Cynodon dactylon (L.) Pers. , Lolium arundinaceum (Schreb.) Darbysh., Apium graveolens L. and Petroselinum crispum (Mill.) Fuss. We tested the response of female antennae on those blends with Gas Chromatography-Electroantennographic Detection (GC-EAD). The chemical profile of C. creticus and P. halepensis was rich in terpenes, alcohols, aldehydes, and esters. In the O. europaea profile, the main compounds were terpenes. As for P. myrtifolia, the volatile profile consisted mostly of alkanes and their substitutes. In the volatile profile of M. sativa, C. dactylon and L. arundinaceum, common volatile compounds were detected. Petroselinum crispum and Apium graveolens chemical profiles were dominated by terpenes. Several compounds elicited a consistent response to the female antennae of both species. In total, 65 compounds elicited consistent EAD responses for P. spumarius and 16 compounds for N. campestris. α –pinene was found in all tested plants and elicited consistent EAD responses of P. spumarius in five plants. In addition, antennae of P. spumarius females responded to camphor, limonene, 4-methyl octane and sabinene. These compounds were found in the volatile profile of at least 5 out of 8 examined plant species. Behavioral bioassays using Y-tube olfactometry were performed on volatiles that elicited antennal responses during electrophysiological studies. Among the compounds tested in behavioral studies, namely (−)- α- pinene, (+)- α -pinene, sabinene, (−)- S -limonene and (1 R)-(+)-camphor, only the last one elicited a significant attraction response by P. spumarius females. The results achieved shed light on the VOCs from selected host plant species of X. fastidiosa that are perceived by two important insect vectors and a non-host plant, P. crispum. The identification of semiochemicals for manipulating spittlebugs' behavior contribute to the development of efficient monitoring tools for X. fastidiosa vectors. Electrophysical responses of Philaenus spumarius and Neophilaenus campestris adults , vectors of Xylella fastidiosa, were recorded on volatiles collected from 9 different plants. Olfactometer bioassays showed significant attraction to camphor for P. spumarius females. [Display omitted] • VOCs from significant plants in the epidemiology of X. fastidiosa were identified. • Electrophysiological responses of spittlebugs were recorded by GC-EAD. • Several compounds elicited EAD responses for P. spumarius and N. campestris. • Behavioral experiments with electrophysiological active compounds were conducted. • P. spumarius females significantly chose the camphor odor in a Y-tube olfactometer. [ABSTRACT FROM AUTHOR]

We developed two real-time detection assays, TaqMan real-time PCR and LAMP, using primers and probe designed based on a sequence annotated to code for a Haemagglutinin-related protein (Hg) of Xylella fastidiosa (Xf), a gene uniquely present in the Italian olive (De Donno of olive) and American mulberry strains, for specific detection of the target Xf strains. These assays were validated with DNA samples extracted from Xf -infected plant samples and from two species of insect vectors (Philaenus spumarius , Ps ; and Neophilaenus campestris , Nc). Both techniques were proven to be highly sensitive (100 fg of Xf -genomic DNA) and specific to the Italian De Donno and American mulberry strains of Xf. When our LAMP was utilized in a duplex manner by combining with previously published universal primers and probe for detection of all Xf -subspecies and strains, the duplex LAMP showed high versatility in the simultaneous detection and differentiation of the Italian De Donno and American mulberry stains form other subspecies/strains. Furthermore, the Hg gene-specific LAMP primers and TaqMan probe were exploited to develop a new approach; henceforth referred to as the F luorescence of T aqMan P robe upon Dequenching - L oop mediated Isothermal Amp lification (FTP-LAMP). In the FTP-LAMP, the Xf - Hg specific fluorophore-quenched probe was added to a standard LAMP reaction and fluoresces only when bound to its target, allowing for a sequence-specific detection of the Xf -Italian De Donno and American mulberry strains in a LAMP context. Our FTP-LAMP assay showed to be highly sensitive detecting down to 100 fg genomic DNA of Xf, when tested on Xf -genomic DNA extracted from infected plants, DAS-ELISA-crude saps and insect vectors. Furthermore, the assay showed high specificity (98.7% vs 89% for LAMP) when applied on DNA templates from insect vectors. With the addition of an extra target sequence-specific probe acting as a direct Xf -specific dye, the FTP-LAMP has gained more specificity and reduced one of the main problems of the LAMP assay (false positives) when used for detecting of Xf in insect vectors. To the best of our knowledge, this study reports the development of the first LAMP assay and the first novel FTP-LAMP method for specific detection of the Italian De Donno and the American mulberry strains of Xf. Together with the Xf universal LAMP primers in a duplex approach, the FTP-LAMP could represent a useful tool not only for the specific detection of the olive-associated strain in Italy, but also to differentiate the De Donno strain from other strains of Xf already reported in Italy and Europe (Germany, France, Spain and Portugal). • Development of TaqMan real-time PCR, LAMP and FTP-LAMP assays for Xylella fastidiosa • Specific detection and differentiation of CoDiRO and mulberry strains • Detection of Xylella fastidiosa in plants and insect vectors [ABSTRACT FROM AUTHOR]