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Several success stories in plant pathology indicate that the use of plant genetic resistance is the most effective, durable and environmentally/economically sustainable protection system for the coexistence with harmful organism whenever curative methods lack. In the epidemic of Xylella fastidiosa ST53 in Salento, the cultivars Leccino and FS17 showed interesting resistant traits. This important finding, even if allowed the replanting of olives in infected areas, is considered a starting point being highly desirable to find a wider range of varieties to avoid a dangerous genetic uniformity and to offer alternatives to the growers and oil producers. For this purpose in Salento, a large-scale program for testing the susceptibility of Italian and Mediterranean olive germplasm started and the first long-lasting breeding activities for the resistance/tolerance were initiated. Due to the urgency, the selection of productive plants derived from uncontrolled pollination and seed dissemination was identified as a third approach aimed to save the time of long olive juvenile period and enlarge the genetic variability to be explored looking for resistance or tolerance directly under high selective pressure of inoculum in the infected area. Furthermore, each seedling plant, derived mainly from parentals of the local cultivars, is potentially a new candidate “autochthonous” variety. After 3-years of activity in the framework of the regional project ResiXO and thousands of plants observed/tested, the activities of morphological/technological characterization, as well the pathogenicity tests by pathogen inoculation in controlled conditions started on a panel of candidate potentially resistant genotypes. Positive feedbacks are also coming from a newborn initiative called Xylor (Xylella Olive Resistance), contrived as a citizen science approach launched in September 2019 to involve the local community in the exploration of a huge territory to find plants asymptomatic or with mild symptoms in the abandoned and dying olive orchards., IT; PDF; pierfederico.lanotte@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

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021., Remote sensing efforts made as part of European initiatives via POnTE, XF-ACTORS and the JRC, as well as through regional programs, have focused, among others, on the development of algorithms for the early detection of Xylella fastidiosa (Xf)-induced symptoms. Airborne campaigns carried out between 2016 and 2019 collected high-resolution hyperspectral and thermal images from infected areas in the Apulia region (Italy), in the province of Alicante and on the island of Mallorca (Spain). The remote sensing imagery collections were performed alongside field surveys and laboratory analyses to assess the presence of Xf, and the severity and incidence of disease in olive and almond trees. Radiative transfer models and machine learning algorithms were used to quantify spectral plant traits for each individual infected tree, assessing their importance as pre visual indicators of Xf-induced stress. These studies conducted across species have demonstrated that specific spectral plant traits successfully revealed Xf induced symptoms at early stages, i.e., before visual symptoms appear. The results show that spectral plant traits contribute differently to symptom detection across host species (olive vs. almond), and that abiotic-induced stress affects the performance of the algorithms used for detecting infected trees. Together, the different European initiatives studying the use of remote sensing to support the monitoring of landscapes for Xylella fastidiosa detection lead us to conclude that the early detection of Xf-induced symptoms is feasible when high-resolution hyperspectral imagery and physically-based plant trait retrievals are used, obtaining accuracies exceeding 92% (kappa>0.8). These results are essential to enable the implementation of effective control and management of plant diseases using airborne- droneand satellite-based remote sensing technologies. Moreover, these large-scale hyperspectral and thermal imaging methods greatly contribute to the future operational monitoring of infected areas at large scales, well beyond what is possible from field surveys and laboratory analyses alone.

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.

Xylella fastidiosa is an important insect‐vectored bacterial plant pathogen with a wide host range, causing significant economic impact in the agricultural and horticultural industries. Once restricted to the Americas, severe European outbreaks have been discovered recently in Italy, Spain, France, and Portugal. The Italian outbreak, detected in Puglia in 2013, has spread over 100 km, killing millions of olive trees, and is still expanding. To date, quantified assessment of important epidemiological parameters useful for risk assessment and management, such as transmission rates, symptomless periods, and time to death in field populations, has been lacking. This is due to the emergent and novel nature of the outbreak and length of time needed to monitor the course of disease progression. To address this, we developed a Bayesian method to infer epidemiological parameters by fitting and comparing compartmental epidemiological models to short snapshots of disease progression observed in multiple field plots. We estimated that each infected tree with symptoms is able to infect around 19 trees per year (95% credible range 14–26). The symptomless stage was estimated to have low to negligible infectivity and to last an average of approximately 1.2 years (95% credible range 1.0–1.3 years). Tree desiccation was estimated to occur approximately 4.3 years (95% credible range 4.0–4.6 years) after symptom appearance. However, we were unable to estimate the infectiousness of desiccated trees from the data. Our method could be used to make early estimates of epidemiological parameters in other emerging disease outbreaks where symptom expression is slow., All authors are funded by the European Union's Horizon 2020 research and innovation programme under Grant Agreement No. 727987 – XF‐ACTORS ‘Xylella fastidiosa Active Containment Through a multidisciplinary‐Oriented Research Strategy’. Field monitoring data was supported by Grant Agreement No. 635646 – POnTE ‘Pest Organisms threaten Europe’. Support was also funded by grant agreement no. 734353 – CURE‐XF ‘Capacity Building and Raising Awareness in Europe and in Third Countries to Cope with Xylella fastidiosa’.

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., EFSA received a request from the European Commission to update its 2015 Pest Risk Assessment (PRA) for Xylella fastidiosa in the EU territory. The update focused on potential establishment, short and long-range spread, the length of the asymptomatic period, the impact of X. fastidiosaand an update on risk reduction options, accounting for the different subspecies and Sequence Types of X. fastidiosaduring the risk assessment where data availability allowed. Here we summarise the main conclusions of the updated PRA, the uncertainties associated with the conclusions, and recommended futuredirections for research and data collection to reduce these uncertainties. The quantitative PRA provided new insights on containing existing outbreaks and preventing further spread in the EU. For example, it was demonstrated that most of the EU has potential climatic suitability for Xylella but that the southern EU is most at risk;however,X. fastidiosasubsp. multiplexdemonstrated areas of potential establishment further north in Europe compared with other subspecies. Modelling was also used to assess data from a wide range of studies on the length of the asymptomatic period, as well as to simulate the spread of the disease and management measures aimed at eradicating or containing the pathogen. The assessment showed the importance of disease control measures, including the use of buffer zones and plant removal, but also the need to couple these measures with effective vector control, rapid implementation of management measures following disease discovery, and early detection of new positive cases. The latter issue is a particular challenge for Xylellagiven the length of the asymptomatic period, particularly for some host and subspecies combinations. In a separate mandate EFSA is developing new surveillance guidelines for Xylellato enable for more effective and targeted detection surveys for the disease.

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

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., Early detection of the new outbreaks of Xylella fastidiosaand knowledge of the disease dynamics are key elements for an effective management of new foci. These elements where explored in the Update of the Scientific Opinion on the risks to plant health posed by Xylella fastidiosain the EU territory, recentlypublished by EFSA. In this opinion,a short-range spread model was used to investigate the spatial dynamics of a new outbreak in a free area and to comparatively assess the control measures aimed at local eradication of the disease. A process-based approach was used to describe the bacterial growth in a plant in relation to the symptoms/disease progression, the population dynamics of the spittlebug and the vector-mediated transmission. The model parameters were derived from the data acquired on the spread of X. fastidiosasubsp. paucain olive groves in the Apulia region. Four epidemiological scenarios were considered combining host susceptibility and vector abundance. Four management options were considered to account for the timeline for the detection and for the implementation of control measures, efficacy of vector control and plant removal. Simulation results showed that the spread rate of the disease increases over time with anon-linear pattern depending on the scenario components. High efficacy of nymph and adult vector control and short delay in detection and implementation of control measures are key factors for the successful eradication of an outbreak in a free area. Model structure and flexibility make it possible to explore a wide range of conditions to account for different vector species, bacterial strains, vegetation components, landscape structures (homogeneous, heterogeneous but continuous, patchy) and combinations of management options. This makes the model a suitable tool to support decision making for the drafting and management of emergency plans related to new outbreaks.

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., Detecting and monitoring the spatial and temporal dynamics of the symptoms and the severity of the damage caused by Xylella fastidiosa(Xf) is a key priority to prevent its expansion. This study evaluates the use of Sentinel-2 imagery together with a radiative transfer (RT) approach to monitor epidemics caused by Xf in olive trees. A time series of Sentinel-2a imagery collected over two years was used to describe the temporal dynamics of Xf-infected olive orchards located in the region of Apulia (southern Italy). Airborne hyperspectral acquisitions were used for validation along with field visual surveys carried out for more than three thousand trees with different disease incidence (DI) and severity (DS) levels. A careful evaluation of the sensitivity of Sentinel-2 imagery to canopy alterations produced by a progressive Xf infection in olive orchards has been accomplished based on model simulations and field observations.Our results demonstrate that the assessment of Xf infection monitoring based on Sentinel-2 data requires the use of self-corrected vegetation indices (VIs) and RT modelling. Among the tested VIs, those that minimise the atmospheric and background effects such as ARVI, ATSAVI and OSAVI performed better than traditional vegetation indices usedas a quantitative proxy measure of the fractional cover (FC) of green and healthy vegetation such as NDVI, RDVI or MSR. Model simulations and field observations showed that the background effects have a significant impact on the temporal variation of DI levels detected with Sentinel-2a imagery. The use of 3-D RT modelling improved the DI estimates by 25% when accounting for the background effects, and by 32% when its heterogeneity was also considered. Therefore, the methodology proposed using a 3-D RT and Sentinel-2 data can provide useful spatiotemporal indicators to track the damage caused by Xfinfections across large areas.

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.

Trabajo presentado en la European Conference on Xylella 2017 (Finding answers to a global problem), celebrada en Palma de Mallorca del 13 al 15 de noviembre de 2017., The spatial dynamics of Olive Quick Decline Syndrome (OQDS) caus ed by X. fastidiosa subsp. pauca were determined in 20 olive plots in a selected olive growing area within the infected zone of the Lecce province, Puglia by assessing disease incidence (DI) and severity (DS) (0-5 rating scale) in June, 2016. Eight of the 20 plots with a wide range of initial DI and DS values were selected to assess the spatial and temporal dynamics of the OQDS and were evaluated in October 2016 and February 2017. Analyses are in progress and include the use of the Spatial Analysis by Distance Indices (SADIE) (Perry et al., 1999) to quantify the spatial pattern of OQDS. Spatial association between time periods was determined by the SADIE association index (Winder et al., 2001). Spatial pattern of symptomatic trees was estimated as regular (22.2% of plots), non-aggregated (33.3% of plots) or aggregated (44.4% of plots). Overall, both, DI and DS increased in the second and third assessments as compared to the initial score in June 2016 by 28.7±4.2% and 2.02±5.1% in DI, respectively; and by 0.53 ±0.27 and 0.04±0.06 in DS, respectively. Spatial pattern was characterized by the occurrence of several clusters of diseased trees. Increasing clustering over time was indicated by stronger values of the clustering index and the increase in patch cluster size. Spatial association was also found in the clustering of diseased trees between evaluation periods., 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”. Research supported by EU grant 635646 POnTE.

The identification in 2013 of an outbreak of Xylella fastidiosa (Xf) in olive groves in the Salento peninsula (southern Italy) resulted in a plant health emergency of unprecedented proportions for the EU. Infected olive trees show extensive desiccation of the canopy and severe quick decline symptoms. In the outbreak area, the bacterium was found to be efficiently spread by the meadow spittlebug Philaenus spumarius, abundant on the olive canopies during the dry season. The initial demarcated foci rapidly expanded over the past 4 years, establishing a new demarcation line distant 80 km from the first reported outbreak; while few species were found infected in 2013 the currently known susceptible hosts reached the number of ca. 30 different plant species. Phytosanitary measures to combat the spread and mitigate the impact of the bacterial infections, included restrictions for the new plantations, for the movement of propagating materials and removal of infected trees. The severe damage suffered by the infected olive trees combined with the imposed phytosanitary restrictions determined severe economic and social impacts in the local community, raising major concerns against the application of the containment measures and determining the failure to implement timely, effective and coordinated preventive measures. Due to the novelty of the Xylella‐associated disease in olives and in general the fact that Xf is conquering new geographical area, like the EU territories, the EU Commission mobilized dedicated resources to build EU research actions to fulfil research gaps for this emerging pathogen threatening the entire EU territory. Between 2015 and 2016, two relevant research projects in the framework of the H2020 programs have been funded: the project "Pest Organisms Threatening Europe" (POnTE) and the project "Xylella Fastidiosa Active Containment Through a multidisciplinary‐Oriented Research Strategy" (XF‐ACTORS) the latter targeting exclusively Xf. From the intense research activity developed in the past three years some major results have been already achieved, providing data on the genetic and biological properties of the population of the bacterium, the range of hosts, the identification and biology of the vector, the identification of olive cultivars with promising traits of resistance.