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The main purpose of Point-of-Care (POC) testing is to decrease the time between specimen collection and detection results, leading to faster intervention and treatment and reduced costs, avoiding the need of large centralized laboratories. The project FREE@POC, started in January 2020 and funded by the H2020-Future and Emerging Technologies OPEN Program, aims to develop an instrument-free molecular diagnostic platform for infectious diseases-testing (HIV, Influenza and Covid-19) in human samples and pathogens (Xylella fastidiosa) in plant tissue. The Project is coordinated by Foundation for Research & Technology – Hellas (FORTH) (Greece) and involves other 5 partners: Institute for Sustainable Plant Protection (Italy), University College London Hospital (UK), GNOSIS (Greece), Institute Pasteur (France), Africa Health Research Institute (South Africa). The overall purpose of the project is to employ a new class of molecular tools (like LAMP) for efficient and specific amplification of target nucleic acids at room temperature (RT) and in crude samples, inside disposable reaction units. This instrument-free molecular diagnostic approach will be fully quantitative thanks to a novel digital colorimetric detection method using a smartphone to capture and analyze the output. Rapid transmission of test results to the patients’ file, doctor’s office, public health officials or a phytosanitary surveillance center will also be implemented. The end-users of the POC tests validated in the project will be healthcare workers at the hospital, intensive care unit or peripheral lab; care providers/nurses at a health post or community center; patients at home or in resource limited areas; farmers, nurserymen or plant inspection service at the field or entry-points., IT; PPT; angelantonio.minafra@ipsp.cnr.it

In Apulia (southern Italy) since 2016 one of the largest monitoring campaign in EU is ongoing, with an average number of 100.000 samples tested per year in the framework of the official monitoring program for Xylella fastidiosa, in the demarcated and Xylella-free areas. More than 85% of the samples were from olive trees, the remaining from almond, oleander and other host plants. Before the promulgation of the EU Regulation 2020/1201, the diagnostic program was organized with two levels of checks. Samples were first screened in 4 regional laboratories by ELISA, with each laboratories processing 300-600 samples/day. Samples yielding positive or doubtful ELISA-results along with 3-5% of the negative samples, were re-tested by qPCR as confirmation tests and for verifying the lab performance for the serological tests. An average of 8.000 sample/year were re-tested by qPCR. Using this large dataset of samples (double tested by ELISA and qPCR) we determined the diagnostic specificity and sensitivity of the test used, and assessed the influence of other factors (period of sampling, origin of the samples). Overall, less than 0.03% of the ELISA-tested samples could not be assigned as negative/positive (i.e. undetermined) and thus re-tested by qPCR, yielding in the majority of the cases (>85%) negative qPCR-results. The diagnostic sensitivity and specificity calculated on the panel of ELISA-negative/positive samples re-tested in qPCR, approx. 26.000 samples, were 94% and 97%, respectively. These values did not vary significantly when the results were analyzed based on the period of sampling or location of the sampled trees. In conclusion, under the specific scenario characterizing the Apulian epidemics (olive predominant host plants, climatic conditions) this diagnostic workflow had allowed to screen with good diagnostic accuracy a relevant number of samples/year. Given the need to replace ELISA with qPCR tests, the capability of the laboratories to process the samples is now reduced (approx. half samples/day) and other possibilities will be consider for the implementation of the upcoming monitoring campaign (i.e testing pooled samples) to ensure testing the same amount of samples., IT; PDF; donato.boscia@ipsp.cnr.it

Inspections and controls for Xylella fastidiosa are mandatory on consignment and in place of productions for the most susceptible host plants listed in the EU Decision 2017/2352, as well as on the long list of “specified plants” when propagated in nurseries located in the infected, containment and buffer zones. The main constrains for testing samples collected from lots of plants are the large number of units to be sampled and the large amount of materials (n. of leaves or shoots/cuttings) to be processed at laboratory level. We carried out experiments by simulating composite samples containing different proportions of Xylella-infected plant tissues, to verify the diagnostic sensitivity of serological and molecular tests. Petioles recovered from infected leaves of Polygala myrtifolia, Nerium oleander and Olea europaea, and scraped xylem tissue from infected cuttings of Prunus avium were pooled at different ratio with healthy materials and processed by ELISA, LAMP and qPCR. Indeed, a protocol based on the extraction of large amount of tissues (40gr of plant material, 100-200 leaves) was also tested. The results provided preliminary important indications both for sampling and laboratory testing: (i) minimum n. of leaves to be collected from the single unit of the lot; (ii) the maximum n. of units that can be pooled and processed using either the standard extraction procedures or the protocol adapted for large tissue amount. The minimum number of the leaves to be collected from the individual plant of the sample unit, ranged from 2 to 6 according to the host species, allowing to pool up to 10 plants or more in relation to the extraction protocol used. Composite sampling is increasingly becoming an acceptable practice when a large number of samples have to be selected to satisfy sample size requirements, keeping the number of diagnostic tests affordable.

Additional file 1. Major CTV contigs with position on the mapping genome and coverage depth. The first BlastN hits are reported with the % of nucleotide identity and a relative strain attribution is presented.

Trabajo presentado en la 2nd European conference on Xylella fastidiosa (how research can support solutions), celebrada en Ajaccio el 29 y 30 de octubre de 2019., The epidemic spread of Xylella fastidiosain Apulia (southern Italy) with the continuous expansions of the border of the infected area, incited several investigations into the genetics and genomics of the strains associated with the infections. These studies were aimed to infer genetic correlations with known strains of the bacterium as well as among the initially discovered Apulian outbreaks and those that emergedfrom time to time. MLST and full genome sequencing provided clear evidence that a single genotype, denoted ST53, of the subspecies paucawas causing the epidemics on olives and other hosts. In this work, we tested a panel of SSR markers in the attempt to disclose information on the genetic diversity and evolution of the bacterial population, even if,given the reduced spatial and temporal scales of this recent epidemic, a relatively low level of variation was expected. Five SSR markers selected among those previously reported in the literature proved to be polymorphic for the ST53-isolates from Apulia, that even if yielding a small number of alleles (from 2 to 5), when combined provide a good resolution, distinguishing several genotypes. Additionally, the design of a new set of 12 SSR markers for fine-scale genotyping of the Apulian isolates, yielded higher number of allelic variation, paving the way to perform micro-evolutionary and epidemiological studies. Indeed, the successful use of these markers on DNA plant samples, will allow a large-scale study,taking advantage of a six-year dataset of plant DNA collected and stored in the frameworkof the official monitoring programmestarted in the region at the beginning of the epidemics in 2013 and covering the entire demarcated area in Apulia that currently exceeds 200,000 ha., This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N. 635646 ‘Pest Organisms Threatening Europe POnTE’.

Report of theTest Performance Study: Molecular detection of Xylella fastidiosa through quantitative real time assays- Implementation of the Proficiency test EU-XF- PT-2017-02.This interlaboratory validation followed the proficiency test (PT) EU-XF- PT-2017-02 carried out in early 2017, and aimed to assess the performance and the accuracy of different qPCR assays to detect Xylella fastidiosa in host plants. In the framework of this test performance study (TPS), five different qPCR assays were compared using a common panel of DNA templates. DNA extracts were those previously recovered in the framework of the PT EU-XF- PT-2017-02, from olive tissues spiked with bacterial suspension of X. fastidiosa subsp. pauca strain De Donno, at known concentrations (CFU/ml). More specifically, the DNA extracts used in this TPS were recovered from artificially contaminated olive sap, processed using either the DNeasy® mericon™ Food kit (Qiagen) or the CTAB-based extraction protocol.&nbsp

Presentation on the results of the international Proficiency Test for the diagnosis of Xylella fastidiosa organised in the framework of the projects: POnTE, XF-ACTORS and PROMODE

Proficiency test (PT) is a way in which the performance and competence of laboratories are evaluated and assessed. In proficiency testing, standardized samples are prepared with known status regarding thepresence of the target pathogen(s). These are sent out to participating laboratories that analyze them usingtheir own methods, equipment and reagents, and send results back to the Organizer(s). The Organizer(s)analyzes the results and provides a report detailing all participants’ results in confidential manner togetherwith actual sample status.The current proficiency test aimed to evaluate the performance (efficiency and accuracy) of laboratoriesinvolved in the diagnosis of Xylella fastidiosa by using molecular and serological methods. Thisinterlaboratory comparison is part of the research activities on the implementation of surveillance andmonitoring program for X. fastidiosa, within the framework of the following ongoing European projects: - Euphresco project (2015-F-146) “Harmonized protocol for monitoring and detection of Xylellafastidiosa in its host plants and its vectors” - H2020 “POnTE – Pest Organisms Threatening Europe (635646)” - H2020 “XF-ACTORS - Xylella fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy (727987)”.&nbsp

Genetic studies showed that the genotype of the olive-infecting strain of X. fastidiosa (Xf- De Donno) belongs to the sequence type ST53 within subspecies pauca, already reported to occur in Costa Rica. The analysis of single nucleotide polymorphisms (SNPs) and the study of the pan-genome of 27 available whole genomes were performed to determine the phylogenetic placement of Xf-De Donno. Maximum-parsimony and maximum likelihood trees constructed using the SNPs and the pangenome data distinguished the subsp. fastidiosa, multiplex, pauca, sandyi, and morus and groups the Italian and three Costa Rican ST53 isolates in a compact clade that diverges from the South American pauca isolates. These findings were supported by the characterization of a conjugative plasmid shared by the four ST53 isolates and by the identification of a gene encoding a putative histidine kinase-like ATPase, which is not present in isolates from the subsp. multiplex, sandyi and pauca, but was detected in the four ST53 and ST21 isolates of the subspecies fastidiosa from Costa Rica. These data support the common and recent origin of the ST53 isolates. The complete and annotated genome sequence of the strain De Donno of X. fastidiosa was obtained by a combined Illumina and PacBio sequencing. This strain, recovered from an olive tree affected by Olive Quick Decline Syndrome (OQDS), when mechanically inoculated in different olive cultivars, caused symptoms identical to those observed in contaminated olive groves.

We report here the complete and annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. pauca strain De Donno. This strain was recovered from an olive tree severely affected by olive quick decline syndrome (OQDS), a devastating olive disease associated with X. fastidiosa infections in susceptible olive cultivars.

While different sources of natural resistance to Xylella fastidiosa (Xf) have been described in grapevines and citrus, lack of consolidated information exists on the wide panel of cultivars characterizing the vast olive germplasm. Preliminary observations on few cultivars, support the evidence that differential cultivar responses to Xf infections may exist. To explore the response of a larger panel of cultivars, in April 2015, an experimental olive plot, located within the Xf-heavily affected olive groves, was established in the Apulia Region (Italy). Twenty-four trees for each of the ten different cultivars were planted in randomized blocks. Each tree was caged with 15-20 specimens of Philaenus spumarius collected from the neighboring infected olive groves. Upon removing the cages, the trees are then continuously exposed to the natural vector populations occurring in the area. Nine and 12-months after planting, the trees were sampled, tested for Xf and inspected for symptoms. The first data confirmed the infectivity of the vector populations occurring in the Apulian contaminated area and the Xf susceptibility of the olive cultivars tested. Almost 50% of the trees tested positive, with an infection incidence ranging from 25% (Leccino) to 78% (Koroneiki). Symptoms of shoot dieback started to appear 1-year after planting, limitedly on few replicates of Cellina di Nardò. In April 2016, the number of cultivars has been increased up to 30. Periodical surveys for symptoms and quantitative analyses to monitor the differential bacterial titer and expression of target genes involved in the host response, are underway.

To assess the presence of the xylem-limited bacterium Xylella fastidiosa subsp. pauca strain CoDiRO in olive trees, a specific sampling method was evaluated. Symptomatic and symptomless plants were randomly selected in four olive orchards located in the province of Lecce (Southern Italy). The crown of each plant was subdivided into a lower and an upper portion; four samples were collected from each layer in the main four cardinal directions. A total of eight samples per plant, composed of one- or two-year-old asymptomatic twigs, were collected next to branches showing leafscorch symptoms. In this preliminary study, the null hypothesis was tested. i.e. there is no difference between the lower and the upper portions of the tree canopy and across the four cardinal directions. Samples (472), collected from 60 plants belonging to 11 different olive cultivars, were tested by qPCR. Out of 236 samples taken from the upper and lower parts of the canopy only 38.1% of lower samples, in contrast to 56.8% taken from the upper crown layer, were positive to the bacterium,. The McNemar test determined that there is a statistically significant difference in the proportion of positive samples between the upper and lower crown (p < 0.001). The Cochran’s Q test was performed to evaluate differences in the four cardinal directions. The null hypothesis suggesting there is no difference across cardinal directions was confirmed (p = 0.097). Based on these preliminary results, it appears that sampling should be directed to the upper part of the canopy. However, further studies are needed to improve the efficiency of the sampling technique.

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.

Xylella fastidiosa subsp. pauca strain CoDiRO was identified as associated with the “Olive Quick Decline Syndrome”, a devastating disease first observed in October 2013 in the southeastern Apulia. At least 350 plant species belonging to 75 families are reported as hosts of X. fastidiosa. These provide a source of inoculum for the vectors (xylem sap-feeding leafhoppers), thus playing a major epidemiological role and facilitating the entrenchment of the pathogen in the affected area. To investigate the CoDiRO strain host range in Salento, monthly samplings of the native flora of two heavily infected olive groves and of the side of adjacent roads were conducted from January 2014 onwards. One of the groves was grass-covered, whereas periodic tillage was performed in the other. Overall, more than 200 species of 50 families were sampled, observed for the presence of symptoms, photographed and identified. In the spring, Philaenus spumarius the main vector of the Salentinian X. fastidiosa strain was abundantly present on the herbaceous flora and shrubs at all sites. All samples, in pools of no less that 3 to 5 plants, were tested by DAS-ELISA and uncertain/positive results were verified by conventional and real time PCR. Bacterial isolates were obtained in axenic culture from some positive species. In a two-year survey, only Euphorbia terracina proved to be Xylella-positive among the herbaceous hosts, whereas some shrubs and subshrubs i.e. Asparagus acutifolius, Cistus creticus, Myrtus communis, Phillyrea latifolia, Rhamnus alaternus and Rosmarinus officinalis were infected. These results provide as strong indication that, rather than weeds, are the perennial shrubs that play a major role in the epidemiology of the X. fastidiosa in this area.

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.

To assess the presence of the xylem-limited bacterium Xylella fastidiosa subsp. pauca strain CoDiRO in olive trees, a specific sampling method was evaluated. Symptomatic and symptomless plants were randomly selected in four olive orchards located in the province of Lecce (Southern Italy). The crown of each plant was subdivided into a lower and an upper portion; four samples were collected from each layer in the main four cardinal directions. A total of eight samples per plant, composed of one- or two-year-old asymptomatic twigs, were collected next to branches showing leafscorch symptoms. In this preliminary study, the null hypothesis was tested. i.e. there is no difference between the lower and the upper portions of the tree canopy and across the four cardinal directions. Samples (472), collected from 60 plants belonging to 11 different olive cultivars, were tested by qPCR. Out of 236 samples taken from the upper and lower parts of the canopy only 38.1% of lower samples, in contrast to 56.8% taken from the upper crown layer, were positive to the bacterium. The McNemar test determined that there is a statistically significant difference in the proportion of positive samples between the upper and lower crown (p < 0.001). The Cochran’s Q test was performed to evaluate differences in the four cardinal directions. The null hypothesis suggesting there is no difference across cardinal directions was confirmed (p = 0.097). Based on these preliminary results, it appears that sampling should be directed to the upper part of the canopy. However, further studies are needed to improve the efficiency of the sampling technique.

Mapping of transcripts from the â olive xylem transcriptomeâ to a set of Core Eukaryotic Genes (CEGs). Percentages of mapped transcripts toward the four CEGs groups are reported. (PPTX 46Â kb)

A survey on viruses infecting olive trees was performed in Croatian Istria. Major olive viruses were detected using molecular (RT-PCR), biological (bioassay on indicator plants) and serological (DAS-ELISA) diagnostic techniques. Specifically, fifteen olive varieties from a total of 62 olive trees have been tested for the presence of eight viruses. Cherry leaf roll virus (CLRV), Strawberry latent ring spot virus (SLRSV) and Olive latent virus-1 (OLV-1) were detected by one-step RT-PCR in ten autochthonous and five introduced olive varieties. The most frequent found was CLRV, detected in 11.3% of tested trees both in autochthonous and in introduced varieties, while OLV-1 and SLRSV were detected only in autochthonous varieties and in lower incidence. In order to confirm the results obtained by one-step RT-PCR, inocula prepared from the flowers were used for inoculation of herbaceous indicator plants. Symptoms on herbaceous hosts were monitored and CLRV and SLRSV were detected by DAS-ELISA, confirming the results of molecular test. The most successful mechanical transmission was on Chenopodium quinoa, while the most evident symptoms were observed on Cucumis sativus L. cv. Sunčani potok plants.

Length distribution of transcripts of the combined â olive xylem transcriptomeâ . (PPTX 255Â kb)

Graphic plots showing the range of Log2 fold change values as they appear in the RNASeq quantitation using SeqMonk. Per probe normalisation was applied to the graph to obtain a wider dynamic range. This has the effect of emphasising the changes between conditions whilst ignoring the magnitude of the differences. a) Log2 fold change of candidate transcripts of housekeeping genes; b) Log2 fold change of candidate transcripts of target genes. (PPTX 3708Â kb)