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Since 2015, a large-scale olive screening program for identifying sources of resistance to Xylella fastidiosa subsp. pauca ST53 (Xfp) was started under natural (field) and controlled conditions (greenhouse, GH) in Apulia (southern Italy). Approx. 100 genotypes including olive cultivars (from 15 different olive-growing countries) and breeding selections were planted in experimental plots located in the demarcated infected area, exposed to natural infections. The panel of tested plant material included also O. europaea subsp. cerasiformis, subsp. guanchica and var. sylvestris. Most of the experimental materials consisted of self-rooted plants propagated from the sources maintained at the World Olive Germplasm Bank and Breeding Program of IFAPA Córdoba (Spain). Sixty genotypes of the plant material under study were also evaluated in GH upon mechanical inoculations. Field trees were tested once a year and inspected for symptoms twice a year. Potted plants in the GH were monitored periodically to assess symptoms and host bacterial colonization. Under field conditions, symptoms usually began on the susceptible cultivars during the third year. Colonization was detected since the first year (incidence of 4-20%) and then progressed rapidly reaching values higher than 60% for most of the selections. For few selections, including Leccino, infections were in the range of 20%-40%. After 5 years, shoot dieback/desiccations were evident on trees belonging to all genotypes under testing, with scores ranging from 1 to 2.5 (on a scale 0-5), except only a few showing scores In the GH tests, within the 3 years of observations, symptoms of desiccations could be observed on the majority of the systemically-infected plants, with symptoms appearing at different times post-inoculation. Infection rates for the majority of the selections ranged from 50% to 89%. Overall, even if in some genotypes lower incidence of infections and longer asymptomatic periods were recorded, so far none displayed clear phenotypic traits of resistance (low prevalence, symptoms and bacterial population) similar to that observed in the resistant control (Leccino). The data collected from this large screening program showed that the occurrence of genetic traits conferring resistance to Xfp may be very limited in the olive germplasm, emphasizing the need to put in place all measures to restrain and limit its further spread in olive growing areas.

In 2020, an Interlaboratory Comparison (IC) was organized for the identification of Xylella fastidiosa (Xf) by qPCR in plant and insect samples. In the framework of this IC a test performance study (TPS) and a proficiency test (PT) involving 16 and 14 laboratories, respectively, were organized. All participating laboratories processed the samples (crude sap containing inactivated bacterial cells) from the DNA extraction step by comparing different procedures. The TPS aimed to compare the performance of different DNA extraction procedures, whereas, the PT assessed the proficiency of diagnostic laboratories carrying out Xf diagnosis. The values of accuracy, repeatability and reproducibility recorded for the three DNA extraction procedures ranged from 98.52% to 99.30% when testing plant samples, and from 94.07% to 98.75% for insect samples, with a the detection limit of approx. 10^2 CFU/ml. Overall, the data gathered confirmed the robustness of the procedures previously validated (CTAB, DNeasy mericon Food kit - Qiagen), and allowed to extend the panel of DNA extraction procedures currently available for processing the wide range of plant matrices to be checked for Xf (i.e Maxwell® RSC PureFood GMO and Authentication Kit-Promega). Both commercial kits tested, combined with automatized platforms, yielded better performance than the manual CTAB-based procedure. All laboratories participating to the PT proved to be highly proficient (values close to 100% for all performance criteria), regardless the procedure of extraction, the qPCR master mix and the amplification conditions used. However, some of the extraction methods used in few laboratories produced lower yield and quality of the total DNA recovered, thus producing higher values of quantitation cycle in qPCR. In conclusion, the TPS provided useful information on additional extraction procedures that can be used to efficiently detect the pathogen in different plant and insect matrices. While the PT provided hints on the efficiency of the diagnostic laboratories operating in 10 different EU/non-EU Countries.

Surveys for Xylella fastidiosa are now mandatory in the EU Member States and genetically diverse strains, i.e. those capable of infecting different hosts, have been intercepted in the currently known EU outbreaks and/or containment areas. Given the long list of the EU susceptible host plants, currently comprising more than 50 plant species, there is an compelling need to develop robust and rapid diagnostic tests (ready-to-use and on-site tests) suitable for testing and screening different plant matrices and large numbers of samples. To this end, we have compared membrane-capture-based methods and ready-to-use kits, the latter based on isothermal nucleic acid amplification techniques. Indeed, a chloroform-free method based on the use of the Maxwell® RSC PureFood GMO and Authentication Kit (Promega) was evaluated on different plant matrices, as an alternative protocol for recovering total DNA suitable for identifying X. fastidiosa in real-time PCR (qPCR) reactions, so far the most widely used diagnostic method. In a first validation test, 90 olive field-trees were tested simultaneously using different procedures: FTA-ELISA, DTBIA, qPCR assay on membrane-captured DNA, loop-mediated isothermal amplification/LAMP on fresh sap or on stored tissue-imprinted membranes, and recombinase polymerase amplification/RPA.Performance criteria for each test were determined by comparing the results with those obtained in qPCR assays performed according to Harper et al. (2010). The results, while suggesting that different types of membranes can be used for capturing bacterial cells for a subsequent serological or molecular detection, showed that all these approaches produced lower accuracy values than LAMP, RPA and standard qPCR. The chloroform-free DNA purification kit herein tested made it possible to recover DNA templates of high quality with standardised yields, suitable for the detection of the bacterium using the qPCR protocols currently validated for X. fastidiosa.

Numerous research efforts are currently underway to identify novel biocontrol agentsfor the management of diseases associated with Xylella fastidiosa. Despite the encouraging premises, most of these studies proved unable to provide long-term effects or failed once they were examined at the field-scale. An intriguing exception was that of the rhizosphere-colonizing bacterium Paraburkholderia phytofirmans PsJN, which appears efficacious to suppress Pierce’s Disease development when inoculated in grapevine infected by Xf ‘Temecula’. Having verified that PsJN did not exhibit any antagonistic in vitro effect towards Xf ‘De Donno’, we wanted to determine if it might colonize and protect olive from OQDS progress. We tested different inoculation approaches, before concluding that pin-prick method ensures the highest success rate. Motile populations of PsJN could be consistently detected distant from the inoculation point, with a newly developed SYBRÒ ­Green qPCR assay, and their long-term viability was demonstrated by successful isolation on TSA medium. We set field trials in the Apulian area affected by OQDS to investigate prophylactic and therapeutic effects of PsJN inoculation in plants naturally exposed to Xf infection. Current observations, limited to a single season, did not evidence any statistical correlation between the population dynamics of Xf and PsJN, but still, require validation on a larger time-frame. Although we collected evidence that PsJN could multiply and move in olive xylem vessels, we also observed that its concentration decreased significantly over time, suggestive of a plant response that limits PsJN expansion. We attempted a WGS metagenomic study to further investigate if PsJN might prime a host response in turn leading to a knock-on effect on the population indices of Xf and the residential microbial communities.

Numerous attempts have been made to test the use of endophytic bacteria to control diseases caused by Xylella fastidiosa (Xf), but promising results obtained in vitro have often proved inadequate once transferred to the field. A new encouraging possibility came from the observation that the bacterium Paraburkholderia phytofirmans PsJN, widely studied for its ability to defend plants from biotic and abiotic stresses, was able to reduce the symptoms caused by Xf in grapes affected by Pierce’s disease. Our study aimed at testing the effectiveness of PsJN as a biocontrol agent in the Xf strain ‘De Donno’/olive pathosystem. Although in vitro tests showed the absence of competitive inhibitory effects of PsJN on Xf ‘De Donno’ growth or its ability to form biofilm, several trials were started both in greenhouse and in Xf-contaminated orchards. Although different approaches were tested to deliver PsJN in olives, high rates of successful isolations and positive detection, achieved with a SYBR? -Green-based qPCR assay ad hoc developed in this study, were obtained only upon needle inoculation of 1- to 2-year-old shoots, in which PsJN proved to remain viable for >500 days.Current observations in open-field trials, so far limited to a single season, have not revealed significant differences in the reduction of OQDS symptoms or Xf concentration in therapeutic treatments, between plants treated or not with PsJN, nor reduction of the new infections upon preventive applications. Despite the evidence that PsJN can colonise the xylem vessels, time course diagnostic tests clearly showed that it moves slowly away from the point of inoculation and its concentration decreases significantly over time. This absence of systemic colonisation suggests a possible induced response of the plant, which will need to be analysed in detail, to optimise treatments and trigger a cascading effect on Xf. In this direction, preliminary results of PsJN impact, on the resident microbiome diversity indices, in presence/absence of Xf, have been gathered using a WGS approach.

A large program of mechanical inoculations and vector-mediated transmission experiments was launched in the past 2 years, and continuously implemented, in the attempt to disclose the role of the olive-infecting strain of X. fastidiosa in the olive quick decline disease (OQDS) and to ascertain the crop species under threat due to the expansion of the severe epidemic in southern Italy. Artificial inoculations were performed using the selected olive strain “De Donno” on different olive and grape cvs, stone fruit and citrus species, and ornamentals. These plant species, but including a larger number of olive cultivars, were also tested through field experiments set in the demarcated infected area, by exposing Xylella-free plants to the natural inoculum pressure and/or by caging naturally infected Philaenus spumarius. Comparison of the bacterial infection rates recovered upon mechanical inoculations and vector transmission (field experiments), showed perfect agreement regarding the susceptibility of the different host species: (i) high rates of systemically infected plants were obtained for olives, with some differences in relation to the cultivars, oleanders and Poligala myrtifolia; (ii) very low host colonization occurred for cherry and almond plants; (iii) no bacterial movement and host colonization could be detected in grapes, citrus, apricot, peach and plum. The pathogenicity of this strain has been also demonstrated; severe symptoms were detected on the most susceptible hosts. Acknowledgment This work was supported by the European Food Safety Authority (EFSA) in the framework of the “Pilot project on X. fastidiosa to reduce risk assessment uncertainties” (NP.EFSA.ALPHA.2014.07) and by the European Union’s Horizon 2020 Research and Innovation Programme, under grant agreement No. 635646, POnTE (Pest Organisms Threatening Europe).

Osservazioni e rilievi di campo integrati da un programma di campionamenti e saggi diagnostici di laboratorio confermano in alcune delle varietà oggetto di studio una minore incidenza delle infezioni di Xylella fastidiosa, a cui sono associate sintomatologie di deperimento e manifestazioni di disseccamento più lievi e attenuate rispetto a quanto osservato in cultivar definite altamente suscettibili, ove la presenza di infezioni di X. fatidiosa compromette la sopravvivenza stessa delle piante. I risultati, oltre a confermare i fenomeni di resistenza già osservati in precedenti studi sulla cultivar Leccino, individuano nella selezione FS-17®, un ulteriore e potenziale fonte di resistenza al batterio. Al contrario, si rafforzano le evidenze sull’elevata suscettibilità delle cultivar Cellina di Nardo e Ogliarola salentina. Si presenta infine una rapida sintesi delle diverse attività in corso sulla ricerca di fonti di resistenza.

A devastating outbreak of a strain of Xylella fastidiosa is ravaging the olive trees of southern Puglia, Italy (Saponari et al., 2015). The bacterium, discovered in 2013, causes the Olive Quick Decline Syndrome (OQDS), a destructive disease characterized by apical and marginal leaf scorching, extensive branch and twig dieback, followed by death of the trees (Martelli et al., 2016). Genome studies (Giampetruzzi et al., 2015; Loconsole et al., 2016;) have shown that the olive infecting strain of X. fastidiosa belongs to the subspecies pauca, sequence type 53 (ST53), whereas epidemiological investigations and transmission tests proved that the xylem-feeding meadow froghopper Philaenus spumarius is the vector of the bacterium (Saponari et al., 2014).

While different sources of natural resistance to X. fastidiosa have been described in grapevines and citrus, lack of solid information exists on possible sources of resistance/tolerance in the cultivars that characterize the wide olive germplasm. Preliminary field observations and laboratory analyses of a few cultivars, have shown that differential responses to X. fastidiosainfections exist. To confirm these preliminary findings, a large panel of olive cultivars is being specifically investigated. Currently, the screening procedure relies on field observations looking for symptomless subjects (trees of known cultivars/volunteer seedlings), mechanical inoculations, qualitative and quantitative diagnostic assays (ELISA & qPCR) and, in selected cases, comparative transcriptomic profiling. Field experiments include the planting of the target cultivars/selections in an infected area under high inoculum pressure. All the plots are located in the Apulia Region (Italy) in the demarcated infected area, surrounded by X. fastidiosa heavily affected olive groves. A first experimental plot was established in April 2015 with 10 different cultivars, which was extended in 2016 to 49 cultivars, and will be further enlarged in 2017 with the addition of 40 new accessions. Other plots, comprising newly planted or grafted cultivars (over 260 cvs) have been also established, bringing to over 300 the total number of accessions under evaluation. Cvs Leccino and FS-17®, both expressing interesting traits of resistance, have already been identified.

While different sources of natural resistance to X. fastidiosa have been described ingrapevines and citrus, lack of solid information exists on possible sources of resistance/tolerance inthe cultivars that characterize the wide olive germplasm. Preliminary field observations and laboratoryanalyses of a few cultivars, have shown that differential responses to X. fastidiosa infections exist. To confirm these preliminary findings, a large panel of olive cultivars is being specifically investigated.Currently, the screening procedure relies on field observations looking for symptomless subjects (treesof known cultivars/volunteer seedlings), mechanical inoculations, qualitative and quantitativediagnostic assays (ELISA & qPCR) and, in selected cases, comparative transcriptomic profiling. Fieldexperiments include the planting of the target cultivars/selections in an infected area under highinoculum pressure. All the plots are located in the Apulia Region (Italy) in the demarcated infectedarea, surrounded by X. fastidiosa heavily affected olive groves. A first experimental plot wasestablished in April 2015 with 10 different cultivars, which was extended in 2016 to 49 cultivars, andwill be further enlarged in 2017 with the addition of 40 new accessions. Other plots, comprising newlyplanted or grafted cultivars (over 260 cvs) have been also established, bringing to over 300 the totalnumber of accessions under evaluation. Cvs Leccino and FS-17®, both expressing interesting traits ofresistance, have already been identified.

A novel virus has been identified by next-generation sequencing (NGS) in privet (Ligustrum japonicum L.) affected by a graft-transmissible disease characterized by leaf blotch symptoms resembling infectious variegation, a virus-like privet disease with an unclear aetiology. This virus, which has been tentatively named 'privet leaf blotch-associated virus' (PrLBaV), was absent in non-symptomatic privet plants, as revealed by NGS and reverse transcription-polymerase chain reaction (RT-PCR). Molecular characterization of PrLBaV showed that it has a segmented genome composed of two positive single-stranded RNAs, one of which (RNA1) is monocistronic and codes for the viral replicase, whereas the other (RNA2) contains two open reading frames (ORFs), ORF2a and ORF2b, coding for the putative movement (p38) and coat (p30) proteins, respectively. ORF2b is very probably expressed through a subgenomic RNA starting with six nucleotides (AUAUCU) that closely resemble those found in the 5?-terminal end of genomic RNA1 and RNA2 (AUAUUU and AUAUAU, respectively). The molecular signatures identified in the PrLBaV RNAs and proteins resemble those of Raspberry bushy dwarf virus (RBDV), currently the only member of the genus Idaeovirus. These data, together with phylogenetic analyses, are consistent with the proposal of considering PrLBaV as a representative of the second species in the genus Idaeovirus. Transient expression of a recombinant PrLBaV p38 fused to green fluorescent protein in leaves of Nicotiana benthamiana, coupled with confocal laser scanning microscopy assays, showed that it localizes at cell plasmodesmata, strongly supporting its involvement in viral movement/trafficking and providing the first functional characterization of an idaeovirus encoded protein.

Xylella fastidiosa is a plant pathogenic bacterium, recently introduced in Europe that is causing a decline in olive trees in the South of Italy. Preliminary genetic studies using Multi Locus Sequence Typing (MLST) analysis revealed that the bacterial genotype recovered from the infected olive trees belongs to the sequence type “ST53” within subspecies pauca. This genotype has also been reported to occur in Costa Rica, and more recently in France. Extensive MLST analyses coupled with next generation sequencing (NGS) approaches have been conducted to assess the population genomics of the isolates occurring in the epidemic area of the Salento peninsula. All the different tested susceptible hosts and all the olive trees sampled in the most recently reported outbreaks show to harbor the same sequence type ST53. NGS using complementary approaches based on Illumina and PacBio technologies was used to reconstruct the genome of the selected reference isolate “De Donno”, being the first sequenced olive strain. Indeed, the draft genomes of two additional olive isolates were obtained both from the pure culture and total plant DNA. The sequence dataset of these novel reconstructed genomes was inferred with the recently sequenced draft genomes of the Costa Rican ST53 genotypes to assess their phylogenetic relatedness. Comparative analysis based on single nucleotide polymorphisms and the study of the pan-genome of these whole genome sequences along with those currently available for X. fastidiosa, confirmed that within the subsp. pauca the Italian and Costa Rican ST53 isolates formed a distinct phylotype in a clade divergent from the South American pauca isolates. The clustering and distinctiveness of the ST53 isolates support the hypothesis of their common origin, and the limited genetic diversity among these isolates suggests this is an emerging clade.

The implementation and validation of diagnostic tests included serological and molecular approaches with the aim of pursuing sensitive and reliable identification of the bacterium in a wide range of plant species, including symptomatic and asymptomatic hosts. Serological assays focused on ELISA and DTBIA procedures. The newly developed polyclonal antisera, raised against the CoDiRO strain, was processed as ELISA and DTBIA reagent, thus evaluated for testing a wide range of plant matrices. Indeed, an X. fastidiosa MopB recombinant protein was developed for being used as standardized reference positive control in the diagnostic kits, avoiding the manipulation and the use of bacterial suspension preparations. In addition, experiments have been made to determine the best procedure for preparing and preserving non-infectious samples (with inactivated bacterium) for diagnostic purposes, including the preparation of the samples to be used for interlaboratory validation and performance test studies. Specifically, freeze-drying and dehydration protocols were used to prepare experimental samples that can be safely manipulated by different diagnostic laboratories without specific quarantine requirements. For molecular assays, a commercial kit (DNeasy mericon food kit, Qiagen) was successfully adapted for the automatized purification of high-quality DNA from a wide range of host plants. The qPCR assay was implemented by including the simultaneous detection of a plant DNA internal control (multiplex qPCR). A sensitive bacterial detection was also obtained through the development of a real-time LAMP assay based on crude plant sap. All the currently available methods were then tested and compared to a panel of asymptomatic infected plants (olive, oleander, cherry). All approaches were able to identify at a different level the presence of the bacterium, with qPCR assays being the most sensitive tests detecting up to 102 CFU/ml.

The bacterial pathogen Xylella fastidiosa (Xf) is characterized by a wide plant host range and insect vectors, and on the basis of phylogenetic studies it was subdivided into different subspecies. Results from strain typing, phylogenetic analyses, and other data comparisons have shown that phylogenetic clusters exhibit host-based genetic relationships. Until now, different molecular tests can be used for the differentiation of Xf isolates, among which MLST/MLSA represents the most common method to determine classification and phylogenetic placement of novel isolates. Xf outbreaks in EU motivated the search for accurate and faster approaches for detection and identification of the bacterium in different plant matrices. Because MLST/MLSA requires several PCR reactions and sequencing analyses, we have developed two independent approaches for rapid taxonomic assignment of uncharacterized isolates: (1) single-nucleotide primer extension (SNuPE) method for the multiplex amplification of six Xylella DNA sequences (targeting all subspecies and three genotypes within Xf subsp. pauca including the type-isolate infecting olive in Italy); (2) high-resolution melting (HRM) analysis of the amplicon recovered from the gene encoding the conserved HL protein. Both assays proved to clearly differentiate Xf isolates currently known to occur in the Italian and France outbreaks. Indeed, validation on a larger panel of isolates covering the different subspecies consistently allowed to rapidly differentiate the isolates in different clusters. In conclusion, these approaches could represent a useful tool for pre-screening and selection of infected samples to be further analyzed by MLST or whole genome sequencing. Future project activities will be focalized on the implementation of both protocols on a larger number of isolates and new variants of Xf as they will appear.

The recent Xylella fastidiosa subsp. pauca (Xfp) outbreak in olive (Olea europaea) groves in southern Italy is causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations disclosed that Xfp-infected plants of cv. Leccino show milder symptoms, than the more widely grown and highly susceptible cv. Ogliarola salentina. To ascertain whether these field observations underlie a tolerant condition of cv. Leccino, which could be exploited for lessening the economic impact of the disease on the local olive industry, transcriptional changes occurring in plants of the two cultivars affected by Xfp were investigated. A global transcriptome profiling comparing susceptible (Ogliarola salentina) and tolerant (Leccino) olive cultivars, infected or not by Xfp, was done on messenger RNA (mRNAs) extracted from xylem tissues. The study revealed that 659 and 447 genes were differentially regulated in cvs Leccino and Ogliarola upon Xfp infection, respectively, whereas 512 genes were altered when the transcriptome of both infected cultivars was compared. Analysis of these differentially expressed genes (DEGs) shows that the presence of Xfp is perceived by the plants of both cultivars, in which it triggers a differential response strongly involving the cell wall. Up-regulation of genes encoding receptor-like kinases (RLK) and receptor-like proteins (RLP) is the predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina. Moreover, both cultivars react with a strong re-modelling of cell wall proteins. These data suggest that Xfp elicits a different transcriptome response in the two cultivars, which determines a lower pathogen concentration in cv. Leccino and indicates that this cultivar may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. These findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aiming at investigating molecular basis and pathways modulating its different defense response.

The CoDiRO strain of Xylella fastidiosa subsp. pauca (Xfp) is ravaging olive (Olea europaea) groves in southern Italy, causing a destructive disease denoted Olive Quick Decline Syndrome (OQDS). Field observations show that the Xfp-infected plants of the cv. Ogliarola salentina develop more severe symptoms than that of cv. Leccino. A global transcriptome profiling comparing the two olive cultivars, infected or not by Xfp, was performed to ascertain whether a tolerant condition of cv. Leccino exists, which could be exploited for lessening the economic impact of the disease on the local olive industry. The study revealed that 659 and 447 genes were differentially regulated upon Xfp infection, in cvs Leccino and Ogliarola salentina, respectively, whereas 512 genes resulted altered between the two infected cultivars. The analysis showed that plants of both cultivars perceive the presence of Xfp, mainly involving cell wall-associated proteins. The predominant response of cv. Leccino, which is missing in cv. Ogliarola salentina, consists on the up-regulation of genes encoding receptor-like kinases and receptor-like proteins. This different transcriptome response determines a lower pathogen concentration in the cv. Leccino, suggesting that it may harbor genetic constituents and/or regulatory elements which counteract Xfp infection. These findings suggest that cv. Leccino is endowed with an intrinsic tolerance to Xfp, which makes it eligible for further studies aimed at investigating molecular pathways underlying its different defense response.