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Managing outbreaks of alien plant pests is key to preserving biodiversity and safeguarding crop production. Zoning strategies are applied by plant health authorities to tailor measures to the risk of spread in relation to distance from the outbreak epicentre and the biology of the pest. Here we synthesize information on outbreak management to evaluate the diversity and consistency of such approaches. We collected information on the zoning strategies of 121 outbreaks of 25 plant pests in the European Union (EU). According to the organism’s presence and the measures applied, five zones were distinguished: an infested zone (83% of cases), a buffer zone (76%), a clear-cutting zone (28%), an eradication zone (1%) and a containment zone (1%). Infested zones and buffer zones were adjacent non-overlapping zones, while the clear-cutting zone, eradication zone or containment zone was within either the infested zone or buffer zone. A combination of infested and buffer zones was used in 51% of recorded cases. Measures differed within different zones. Destruction of infested plants in the infested zone was done in 78% of the cases, while surveillance was always applied in the buffer zone. Regulation of an organism at EU level led to a convergence of zoning strategies applied by different member states. Regulations often prescribed the greatest widths used before regulations were issued. Further analyses are needed to explore the efficacy of different strategies including the costs of each strategy. Such analyses should combine insight from practice with bio-economic modelling.

This Scientific report provides an update of the

Xylella fastidiosa (Xf) is a harmful plant pathogenic bacterium, able to infect over 500 plant species worldwide. Successful eradication and containment strategies for harmful pathogens require large-scale monitoring techniques for the detection of infected hosts, even when they do not display visual symptoms. Although a previous study using airborne hyperspectral and thermal imagery has shown promising results for the early detection of Xf-infected olive (Olea europaea) trees, further work is needed when adopting these techniques for large scale monitoring using multispectral cameras on board airborne platforms and satellites. We used hyperspectral and thermal imagery collected during a two-year airborne campaign in a Xf-infected area in southern Italy to assess the performance of spectrally constrained machine-learning algorithms for this task. The algorithms were used to assess multispectral bandsets, selected from the original hyperspectral imagery, that were compatible with large-scale monitoring from unmanned platforms and manned aircraft. In addition, the contribution of solar–induced chlorophyll fluorescence (SIF) and the temperature-based Crop Water Stress Index (CWSI) retrieved from hyperspectral and thermal imaging, respectively, were evaluated to quantify their relative importance in the algorithms used to detect Xf infection. The detection performance using support vector machine algorithms decreased from ∼80% (kappa, κ = 0.42) when using the original full hyperspectral dataset including SIF and CWSI to ∼74% (κ = 0.36) when the optimal set of six spectral bands most sensitive to Xf infection were used in addition to the CWSI thermal indicator. When neither SIF nor CWSI were used, the detection yielded less than 70% accuracy (decreasing κ to very low performance, 0.29), revealing that tree temperature was more important than chlorophyll fluorescence for the Xf detection. This work demonstrates that large-scale Xf monitoring can be supported using airborne platforms carrying multispectral and thermal cameras with a limited number of spectral bands (e.g., six to 12 bands with 10 nm bandwidths) as long as they are carefully selected by their sensitivity to the Xf symptoms. More precisely, the blue (bands between 400 and 450 nm to derive the NPQI index) and thermal (to derive CWSI from tree temperature) were the most critical spectral regions for their sensitivity to Xf symptoms in olive., Data collection was partially supported by the European Union’s Horizon 2020 research and innovation program through grants to the POnTE (Pest Organisms threatening Europe; grant 635646 from European Union’s Horizon 2020 Framework Research Programme) and XF-ACTORS (Xylella fastidiosa Active Containment Through a Multidisciplinary-Oriented Research Strategy; grant 727987 from European Union’s Horizon 2020 Framework Research Programme) projects. A. Hornero was supported by a research fellowship DTC GEO 29 “Detection of global photosynthesis and forest health from space” from the Science Doctoral Training Centre (Swansea University, UK).

Plant pathogens pose increasing threats to global food security, causing yield losses that exceed 30% in food-deficit regions. Xylella fastidiosa (Xf) represents the major transboundary plant pest and one of the world’s most damaging pathogens in terms of socioeconomic impact. Spectral screening methods are critical to detect non-visual symptoms of early infection and prevent spread. However, the subtle pathogen-induced physiological alterations that are spectrally detectable are entangled with the dynamics of abiotic stresses. Here, using airborne spectroscopy and thermal scanning of areas covering more than one million trees of different species, infections and water stress levels, we reveal the existence of divergent pathogen- and host-specific spectral pathways that can disentangle biotic-induced symptoms. We demonstrate that uncoupling this biotic–abiotic spectral dynamics diminishes the uncertainty in the Xf detection to below 6% across different hosts. Assessing these deviating pathways against another harmful vascular pathogen that produces analogous symptoms, Verticillium dahliae, the divergent routes remained pathogen- and host-specific, revealing detection accuracies exceeding 92% across pathosystems. These urgently needed hyperspectral methods advance early detection of devastating pathogens to reduce the billions in crop losses worldwide., The study was partially funded by the European Union’s Horizon 2020 Research and Innovation Programme through grant agreements POnTE (635646) and XF-ACTORS (727987), as well as by projects AGL2009-13105 from the Spanish Ministry of Education and Science, P08-AGR-03528 from the Regional Government of Andalusia and the European Social Fund, project E-RTA2017-00004-02 from ‘Programa Estatal de I + D + I Orientada a los Retos de la Sociedad’ of Spain and FEDER, Intramural Project 201840E111 from CSIC, and Project ITS2017-095 Consejeria de Medio Ambiente, Agricultura y Pesca de las Islas Baleares, Spain. The views expressed are purely those of the writers and may not in any circumstance be regarded as stating an official position of the European Commission.

The quarantine bacterium Xylella fastidiosa was first detected in Salento (Apulia, Italy) in 2013 and caused severe symptoms in olives, leading to plant death. The disease, named Olive Quick Decline Syndrome (OQDS), is caused by the strain "De Donno" ST53 of the subspecies pauca of this bacterium (XfDD), which is spread by Philaenus spumarius. The epidemic poses a serious threat to the agricultural economy and the landscape, as X. fastidiosa infects several plant species and there is yet no recognized solution. Research on OQDS is focused on finding strategies to control its spread or mitigate its symptoms. In this context, we investigated the feasibility of using low-temperature plasma and plasma-activated water to kill bacterial cells. Experiments were conducted in vitro to test the biocidal effect of a Surface Dielectric Barrier Discharge plasma on bacteria. The results showed a high decontamination rate even for cells of XfDD embedded in biofilms grown on solid media. Application to trees requires protocols and tools that can reach the bacterium in the xylem vessels. Plasma Activated Water was tested as a biocidal agent that can move freely in the xylem network. Results in the liquid culture medium showed complete inactivation of XfDD cells and paved the way to test the strategy on infected plants.

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.

The invasive plant pathogen Xylella fastidiosa currently threatens European flora through the loss of economically and culturally important host plants. This emerging vector-borne bacterium, native to the Americas, causes several important diseases in a wide range of plants including crops, ornamentals, and trees. Previously absent from Europe, and considered a quarantine pathogen, X. fastidiosa was first detected in Apulia, Italy in 2013 associated with a devastating disease of olive trees (Olive Quick Decline Syndrome, OQDS). OQDS has led to significant economic, environmental, cultural, as well as political crises. Although the biology of X. fastidiosa diseases have been studied for over a century, there is still no information on the determinants of specificity between bacterial genotypes and host plant species, which is particularly relevant today as X. fastidiosa is expanding in the naive European landscape. We analysed the genomes of 79 X . fastidiosa samples from diseased olive trees across the affected area in Italy as well as genomes of the most genetically closely related strains from Central America. We provided insights into the ecological and evolutionary emergence of this pathogen in Italy. We first showed that the outbreak in Apulia is due to a single introduction from Central America that we estimated to have occurred in 2008 [95 % HPD: 1930–2016]. By using a combination of population genomic approaches and evolutionary genomics methods, we further identified a short list of genes that could play a major role in the adaptation of X. fastidiosa to this new environment. We finally provided experimental evidence for the adaptation of the strain to this new environment.

Olive Quick Decline Syndrome (OQDS), caused by the vector transmitted bacterium Xylella fastidiosa (Xf), is the most devastating olive disease in south Apulia. Studies in the Pierce’s Disease grapevine pathosystem indicate that the biofilm-embedded bacteria engulf the xylem conduits thus eliciting the formation of tylose-like structures and gums aiming to isolate the pathogen. We attempted to evaluate the presence of vascular occlusions in diverse Xf-infected olive cultivars and to assess their nature by fluorescence and transmission electron microscopy (TEM). The observations were performed on healthy and Xf-infected twigs of the Cellina di Nardò, Leccino and FS17 cultivars. The percentages of completely occluded vessels were visually determined by fluorescence microscopy on hand made cross-sections of one-year-old olive twigs from artificially pin-pricked infected and mock-inoculated plants grown under controlled conditions. Occlusions were negligible in healthy plants, while significantly higher in Xf-infected plants reaching the highest value (9.65%) in the susceptible cultivar Cellina di Nardò, while these were 6.81% and 1.33% in the Leccino and FS17, respectively. In addition, the distribution of occluded vessels was not uniform among individual twigs of the same cultivars, ranging from 1% to 34% in Cellina di Nardò, 0.044% to 14% in Leccino and 1.09% to 1.53% in FS17. Tissue portions in which occluded vessels were found by fluorescence microscopy, were embedded in resin to be observed by TEM. Thin sections observations showed that the olive response to the Xf infection does not include the production of tylose-like structures. Conversely, the obstructions we seen by fluorescence microscopy are due to bacterial cell aggregates embedded in a hard matrix. Indeed, as observed by the IAWA (International Association of Wood Anatomists) bulletins for taxonomic identification of hardwood species, olive, differently from grapevine, has very small, half-bordered vessel ray and vessel-parenchyma pits which make unlikely the tyloses development., IT; PPT; vitomontilon@virgilio.it

Strains of Xylella fastidiosa subsp. pauca characterized by a specific genotype, the so called sequence type "ST53", have been associated with a severe disease named Olive Quick Decline Syndrome (OQDS). Despite the relevant research efforts devoted to control the disease caused by X. fastidiosa, so far there are no therapeutic means able to cure the infected host plants. As such, the aim of this study was the identification of antagonistic bacteria potentially deployable as bio-control agents against X. fastidiosa. To this end, two approaches were used, i.e. the evaluation of the antagonistic activity of: i) endophytic bacteria isolated from olive trees located in an infected area but showing mild or no symptoms, and ii) Bacillus strains, as they are already known as bio-control agents. Characterization of endophytic bacterial isolates revealed that the majority belonged to different species of the genera Sphingomonas, Methylobacterium, Micrococcus and Curtobacterium. However, when they were tested in vitro against X. fastidiosa ST53 none of them showed antagonistic activity. On the contrary, when strains belonging to different species of the genus Bacillus were included in these tests, remarkable antagonistic activities were recorded. Some B. velezensis strains also produced culture filtrates with inhibitory activity against X. fastidiosa ST53. Taking also into account that two of these B. velezensis strains (namely strains D747 and QST713) are already registered and commercially available as bio-control agents, our results pave the way for further studies aimed at the development of a sustainable bio-control strategy of the OQDS.

Olive quick decline syndrome (OQDS) is a devastating disease of olive trees in the Salento region, Italy. This disease is caused by the bacterium Xylella fastidiosa, which is widespread in the outbreak area, however, the &ldquo, Leccino&rdquo, variety of olives has proven to be resistant with fewer symptoms and lower bacterial populations than the &ldquo, Ogliarola salentina&rdquo, variety. We completed an empirical study to determine the mineral and trace element contents (viz, ionome) of leaves from infected trees comparing the two varieties, to develop hypotheses related to the resistance of Leccino trees to X. fastidiosa infection. All samples from both cultivars tested were infected by X. fastidiosa, even if leaves were asymptomatic at the time of collection, due to the high disease pressure in the outbreak area and the long incubation period of this disease. Leaves were binned for the analysis by variety, field location, and infected symptomatic and infected asymptomatic status by visual inspection. The ionome of leaf samples was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) and compared with each other. These analyses showed that Leccino variety consistently contained higher manganese (Mn) levels compared with Ogliarola salentina, and these levels were higher in both infected asymptomatic and infected symptomatic leaves. Infected asymptomatic and infected symptomatic leaves within a host genotype also showed differences in the ionome, particularly a higher concentration of calcium (Ca) and Mn levels in the Leccino cultivar, and sodium (Na) in both varieties. We hypothesize that the ionome differences in the two varieties contribute to protection against disease caused by X. fastidiosa infection.

Plant pathogens cause significant losses to agricultural yields and increasingly threaten food security1, ecosystem integrity and societies in general2–5. Xylella fastidiosa is one of the most dangerous plant bacteria worldwide, causing several diseases with profound impacts on agriculture and the environment6. Primarily occurring in the Americas, its recent discovery in Asia and Europe demonstrates that X. fastidiosa’s geographic range has broadened considerably, positioning it as a reemerging global threat that has caused socioeconomic and cultural damage7,8. X. fastidiosa can infect more than 350 plant species worldwide9, and early detection is critical for its eradication8. In this article, we show that changes in plant functional traits retrieved from airborne imaging spectroscopy and thermography can reveal X. fastidiosa infection in olive trees before symptoms are visible. We obtained accuracies of disease detection, confirmed by quantitative polymerase chain reaction, exceeding 80% when high-resolution fluorescence quantified by three-dimensional simulations and thermal stress indicators were coupled with photosynthetic traits sensitive to rapid pigment dynamics and degradation. Moreover, we found that the visually asymptomatic trees originally scored as affected by spectral plant-trait alterations, developed X. fastidiosa symptoms at almost double the rate of the asymptomatic trees classified as not affected by remote sensing. We demonstrate that spectral plant-trait alterations caused by X. fastidiosa infection are detectable previsually at the landscape scale, a critical requirement to help eradicate some of the most devastating plant diseases worldwide.

Xylella fastidiosa Wells et al. 1987 (family Xanthomonadaceae) is a Gram-negative, non-motile, non-sporing bacterium endowed with a very peculiar biological and epidemiological behaviour: it colonizes the xylem vessels of the hosts and is transmitted from plant to plant by xylem fluid-feeding insects (leafhoppers of the family Cicadellidae). Because of its polyphagy, when X. fastidiosa enters a new environment with congenial climatic conditions, it becomes rapidly entrenched in the territory. This is what has presumably taken place in Salento, an area where, a few years ago, a destructive disease of olive (Olea europaea L.) developed, denoted olive quick decline syndrome (OQDS), together with a leaf scorch condition of oleander, cherry and almond. Information stemming from studies carried out by the Bari team has laid the basis for implementing a strategy for OQDS containment, based on: (i) accurate surveillance of the territory north of the infected area; (ii) elimination of inoculum sources in newly developed infection foci through the uprooting of infected hosts and the surrounding healthy plants; and (iii) control of vectors by mechanical weeding in late winter/spring to kill juvenile forms, followed by insecticide treatments against the adults when, in late spring, they move from weeds to olives.

Olive quick decline syndrome (OQDS) is a devastating disease of olive trees in the Salento region, Italy. This disease is caused by the bacterium Xylella fastidiosa, which is widespread in the outbreak area; however, the “Leccino” variety of olives has proven to be resistant with fewer symptoms and lower bacterial populations than the “Ogliarola salentina” variety. We completed an empirical study to determine the mineral and trace element contents (viz; ionome) of leaves from infected trees comparing the two varieties, to develop hypotheses related to the resistance of Leccino trees to X. fastidiosa infection. All samples from both cultivars tested were infected by X. fastidiosa, even if leaves were asymptomatic at the time of collection, due to the high disease pressure in the outbreak area and the long incubation period of this disease. Leaves were binned for the analysis by variety, field location, and infected symptomatic and infected asymptomatic status by visual inspection. The ionome of leaf samples was determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) and compared with each other. These analyses showed that Leccino variety consistently contained higher manganese (Mn) levels compared with Ogliarola salentina, and these levels were higher in both infected asymptomatic and infected symptomatic leaves. Infected asymptomatic and infected symptomatic leaves within a host genotype also showed differences in the ionome, particularly a higher concentration of calcium (Ca) and Mn levels in the Leccino cultivar, and sodium (Na) in both varieties. We hypothesize that the ionome differences in the two varieties contribute to protection against disease caused by X. fastidiosa infection.

An outbreak of

An outbreak of Xylella fastidiosa subsp. multiplex sequence type ST6 was discovered in 2017 in mainland Spain affecting almond trees. Two cultured almond strains, “ESVL” and “IVIA5901,” were subjected to high throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated they belong to the subspecies multiplex, and pairwise comparisons of the chromosomal genomes showed an average nucleotide identity higher than 99%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The availability of these draft genomes contribute to extend the European genomic sequence dataset, a first step toward setting new research to elucidate the pathway of introduction and spread of the numerous strains of this subspecies so far detected in Europe., This work was funded by European Union’s Horizon 2020 Framework Research Programme Projects XF-ACTORS (Xylella fastidiosa Active Containment Through a Multidisciplinary-Oriented Research Strategy grant 727987) and MSCA-RISE-2016 CURE-XF (Capacity Building and Raising Awareness in Europe and in Third Countries to Cope with Xylella fastidiosa); COST Action CA16107 EuroXanth, supported by European Cooperation in Science and Technology; and Project Desarrollo de estrategias de erradicación, contención y control de en España: Diagnóstico, estructura genética y gama de huéspedes project E-RTA2017-00004-C06-02 from Programa Estatal de I+D+I Orientada a los Retos de la Sociedad of the Spanish Government. This work used the Vincent J. Coates Genomics Sequencing Laboratory at University of California, Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.

An outbreak of Xylella fastidiosa was discovered in late 2018 in northern Italy affecting several plant species. Multilocus sequence typing analyses detected the presence of strains clustering in X. fastidiosa subsp. multiplex and harboring a hitherto uncharacterized sequence type, ST87. Three cultured strains (TOS4, TOS5, and TOS14) were subjected to high-throughput sequencing and the draft genomes assembled. Phylogenetic analysis conclusively indicated that they belong to the subspecies multiplex. The genetic information generated for these newly discovered strains further supports the evidence that sequence types are associated with the emergence of X. fastidiosa in Europe, posing major challenges for predicting the main threatened European and Mediterranean crops and plant species.

The vector-borne bacterial pathogenXylella fastidiosais widely distributed in the Americas; in the last decade it has emerged as a serious threat for agricultural crops, natural environment and landscape in Europe. Following the first EU outbreak in 2013 in southern Italy, associated with a severe disease in olive trees, annual mandatory surveys are now in place in the Member States, leading to the discovery of bacterial outbreaks in different countries. Among the latest findings, an outbreak has been reported in the Italian region of Tuscany, with infections identified in seven different plant species. In this work, we report the isolation and the genetic characterization of isolates associated with this newly discovered outbreak. Multilocus sequence typing approach revealed the occurrence of isolates harbouring a new sequence type, denoted ST87, genetically related to strains of subsp.multiplex, but different from the genotypes of this subspecies previously characterized in Europe. Five cultured strains were successfully recovered from four of the seven host plants, an important achievement for advancing the studies on genomics and pathogenicity of theseisolates and thus assess their potential threat for European agriculture.

In spring 2017, phytoplasma suspected symptoms were reported on 25% of 15-year-old almond plants, cultivars Filippo Ceo and Genco grafted onto GF677, in a commercial orchard (20 ha) located at Grottaglie, Apulia (southeast Italy). Among the symptoms, development of many axillary buds with small and yellowish leaves, and witches' brooms developing from the trunk, were the most frequent, followed by leaf rosetting, proliferation of slender shoots, tree decline, and dieback.

Most diseases caused by Xylella fastidiosa have been reported from North and South America. However, since 2013, widespread X. fastidiosainfections have been reported in Apulia region of southern Italy and were associated with a devastating olive disease called olive quick decline syndrome (OQDS). Localized epidemic incidence called for urgent emergency measures and surveillance programs which resulted in detection of this bacterium elsewhere in the EU, namely, Corsica and mainland France, Balearic Islands and mainland Spain. From investigations carried out, different scenarios have emerged with regard to spread and suggested that several independent introductions occured in Europe in the past years. This was indirectly confirmed by numerous interceptions of infected consignments during intensified inspections at ports of entry. The EU outbreaks have been associated with a long list of susceptible species, including several hitherto undescribed hosts and associated to new X. fastidiosa strains. The rapid spread of X. fastidiosa has highlighted the serious threat the bacterium poses to the EU and Mediterranean agricultural industries. The current epidemic in southern Italy is an example of the pathogen’s destructive impact. Harmonized and coordinated EU research programs are underway to gain knowledge on the complex interactions of this pathogen under the EU conditions with the goal being to improve tools for early detection and control.

Xylella fastidiosa (Xf) is a xylem-limited bacterial plant pathogen that is responsible for Olive Quick Decline Syndrome (OQDS), a devastating disease reported in Salento (Apulia, Italy). Field observations show that olive cv. ‘Leccino’ show milder symptoms when compared to the highly susceptible cv. ‘Ogliarola’. A prior transcriptome analysis of ‘Leccino’ and ‘Ogliarola’ cultivars in response to infection by X. fastidiosa ‘De Donno’ strain has revealed that a Calcium-Dependent Protein Kinase (CDPK1) gene is upregulated in ‘Ogliarola’ infected leaves. Moreover Ca accumulation in leaves have been associated with symptomatic tobacco, blueberry, grapes and pecan plants infected with Xf. Calcium is a critical second messenger and its cellular distribution can trigger diverse physiological processes including stress response and plant defence. Based on these observations we pursued a study of the ionome and CDPK1 gene expression profiles of symptomatic and asymptomatic infected leaves of cv. ‘Leccino’ and ‘Ogliarola’ from three orchards. Comparison between the two cultivars reveals changes in ionome and CDPK1 gene expression. ‘Leccino’ symptomatic leaves had significant (p < 0.01) higher Ca concentration as compared to asymptomatic leaves, while differences for the susceptible cv. ‘Ogliarola’ were non-significant (p > 0.2). In addition, sodium levels were higher in symptomatic leaves of both varieties. qRT-PCR confirmed that CDPK1 expression in ‘Ogliarola’ is significantly increased relatively to ‘Leccino’ trees grown in the same field and the increase is higher in symptomatic leaves. The impact of these changes on disease progression will be discussed., The present work was done in the framework of an STM granted by the EU COST Action CA16107 and with the financial and scientific support of the EU H2020 research projects POnTE (GA 635646).

The olive quick decline syndrome (OQDS) is a disease that appeared suddenly a few years ago in the province of Lecce (Salento peninsula, southeastern Italy). Among the factors that may be involved in its aetiology, the most relevant is Xylella fastidiosa, a quarantine pathogen of American origin, whose presence in Italy represents its first confirmed record in the European Union. X. fastidiosa is a Gram-negative bacterium that invades the xylem of a wide range of hosts, from which it is acquired by xylem-feeding insect vectors and transferred to other plants. The bacterium multiplies within the plant vessels and occludes them, thus impairing water uptake. Besides olive, the Salentian strain of X. fastidiosa infects in nature a number of woody (almond, cherry) and shrubby (oleander, broom, Acacia saligna, Polygala myrtifolia, Westringia fruticosa, Rosmarinus officinalis, Rhamnus elaternus, Myrtus communis) hosts, with no evidence for grapevines or citrus being hosts. The bacterium was isolated in culture and identified as a genotype of X. fastidiosa subsp. pauca, molecularly identical to an isolate from Costa Rica. Philaenus spumarius (meadow spittlebug), a froghopper quite common in the Salento area where it thrives on olive, was identified as the main vector. Disease eradication and sanitation of infected olives are unfeasible. However, strategies are being enacted for restraining the spread of pathogen and vector(s) within the boundaries of the currently infected zone.

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.

First, third, and fifth authors: Università degli Studi di Bari Aldo Moro, Dipartimento di Scienze del Suolo della Pianta e degli Alimenti, via Amendola 165/A, Bari, Italy; second, fourth, seventh, and tenth authors: Consiglio Nazionale delle Ricerche, Istituto per la Protezione Sostenibile delle Piante, Bari, via Amendola 122/D, Bari, Italy; sixth author: Department of Environmental Science, Policy and Management, University of California, Berkeley, CA, 94720-3114; eighth author: Institute for Sustainable Agriculture, Consejo Superior de Investigaciones Científicas, 14004 Córdoba, Spain; and ninth author: Centro de Investigación en Enfermedades Tropicales (CIET), Facultad de Microbiología, Universidad de Costa Rica, 2060 San José, Costa Rica., The work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 635646: POnTE (Pest Organisms Threatening Europe).

Discovery of Xylella fastidiosa from olive trees with "Olive quick decline syndrome" in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brulle were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy.

Accurate and early detection of Xylella fastidiosa (Xf) is a major challenge due to the wide range of host plants (different matrices/ tissues, rate of host colonization) and the occurrence of symptomless bacterial infections. The recent establishment of this exotic plant pathogenic bacterium in the EU territory and the large panel of EU susceptible host plants increased the need for rapid diagnostic tools suitable for processing large number of samples and from different sources. Although, several approaches are currently available for the detection of Xf in the host plants and vectors, there is a need for harmonized protocols and user-friendly diagnostic tests. In this study, we compared the sensitivity and the reliability of a selected panel of currently available protocols (ELISA, PCR, qPCR), in comparison with novel approaches based on automatized diagnostic platform and on DTBIA and LAMP-based assays. The overall results showed that: (i) although resulting in different diagnostic sensitivity all the approaches tested were able to detect the bacterium in samples from symptomless plants; (ii) Real-time LAMP assay based using crude plant sap can represent a rapid and reliable screening test; (iii) Real-time quantitative PCR assays had the higher diagnostic and analytical sensitivity; (iv) the use of automatized platform allowed to prepare PCR-templates with high and standardized quality for highly reliable diagnostic results; (v) DTBIA had the lowest diagnostic sensitivity, yet representing a useful approach when movement of Xf infected materials is limited due to the phytosanitary regulations.

© 2016, Koninklijke Nederlandse Planteziektenkundige Vereniging. After the first confirmed outbreak of Xylella fastidiosa in the European Union (EU), associated with an olive disease denoted olive quick decline syndrome, mandatory surveys are now carried out in the member States and inspections increased at EU entry points such as ports. Such activities led to the interception of X. fastidiosa-infected coffee plants in consignments originating from Central America. Similarly, the geographic expansion of the olive decline epidemic area of the Apulia region (southern Italy) prompted investigations to identify new host plants. Here we report the interception of three novel bacterial sequence types in Italy, based on multi-locus sequence typing, that cluster with different X. fastidiosa subspecies, illustrating the risk of the introduction of additional pathogen genetic diversity into Europe. In the epidemic area of Apulia, new foci as well as host plant species positive with X. fastidiosa, including cherry, myrtleleaf and rosemary, were found to be all infected with the same sequence type of this bacterium (ST53, or CoDiRO strain). This work highlights the limited knowledge of X. fastidiosa phylogenetic and phenotypic diversity, the risk of novel X. fastidiosa introductions via contaminated plant material, and corroborates other studies indicating that the Apulia epidemic emerged from a single introduction of this pathogen into the region.

The draft genome sequence of Xylella fastidiosa CO33 isolate, retrieved from symptomatic leaves of coffee plant intercepted in northern Italy, is reported. The CO33 genome size is 2,681,926 bp with a GC content of 51.7%. Isolate CO33, cultured from a coffee plant intercepted in northern Italy, represents a novel multilocus sequence typing profile, ST72 (G. Loconsole, personal communication). Isolates genetically related to CO33 were found in several coffee plants imported in October 2014 from Costa Rica through the Netherlands (European Food Safety Authority [EFSA] 2015).

Sharka disease, caused by the Plum Pox Virus (PPV), is endangering apricot industry in many countries worldwide. Both PPV-D (Dideron) and PPV-M (Marcus) strains are able to determine severe crop losses, with the latter strain being the most dangerous. We selected 'Lito', among the cultivars described in literature as resistant, and crossed to the susceptible accession 'B081604311' to produce a double pseudo-test cross progeny. A population of 118 individuals was phenotyped and genotyped to identify molecular markers linked to the genetic determinant/s of Sharka resistance. Both PPV D and M strains were inoculated in seedling replicates and symptoms visually scored for three years. ELISA and PCR assays were also done. A detailed SSR-based molecular map was developed and used for QTL analysis. A major QTL for both PPV-M and PPV-D strains was found at the top of the linkage group 1 of 'Lito', in the same region where a QTL of resistance to Sharka was found in 'Stark Early Orange', the resistant parent of 'Lito', by Lambert and coworkers (2007). To pave the way for the positional cloning of the DNA sequence, the 'Lito' × 'BO81604311' progeny has been enlarged and further 241 seedlings were genotyped with old and new SSR primers that allowed saturating the QTL region in the LG1. Phenotyping with both PPV strains is under way. A random shearing 10X BAC library prepared with inserts >100 kb is being screened with markers surrounding the QTL. Positive BACs will be physically mapped to produce a contig of the genomic region including the main QTL. Finally, as a first step towards genetic transformation of apricot for resistance to PPV, constructs for either CP overexpression or post-transcriptional silencing were used, and transgenic callus lines were recovered from Agrobacterium-inoculated mature tissues, highly recalcitrant to shoot regeneration.

Mal d 2, a thaumatin-like protein from apple was previously described to react to almost 75% of the apple allergic patient sera. Based on the molecular structure of this protein, the present study focused on the conformational stability of Mal d 2 in relation to in vitro IgE-binding under different physico-chemical conditions and proteolysis. The structural integrity of Mal d 2 was monitored using SDS–PAGE, Western blotting using polyclonal antibodies and human sera, fluorescence spectrometry and circular dichroism. Results confirmed the stability of Mal d 2. However, Mal d 2 was reactive to human serum IgEs mainly after reduction of disulphide bridges fixing the α-helical domain II. Contrary to previous assumptions, the current findings suggest that the allergenic epitopes of Mal d 2 are hidden inside the protein structure and none of the rigorous conditions applied in industrial juice processing or digestive proteolysis enhance or reduce the binding to IgE molecules.

Little cherry, an economically important disease of cherry is caused by at least two different viruses. One of these is Little cherry virus 1 (LChV-1) for the detection of which no efficient serological tools are available, so that diagnosis is based on molecular methods. In this study, different immunization strategies for producing antibodies against the viral coat protein of LChV-1 were tried, using either purified virus preparations, or bacterially expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene in vivo. Effective induction of specific antibodies to LChV-1 CP was obtained using DNA intramuscular immunization followed by a single boost with the recombinant protein. The entire coat protein sequence was cloned in a mammalian expression vector and, after being coated by an amphiphilic non-toxic reagent was delivered into rabbit. A protein boost increased the specific immune response against the virus protein. The sensitivity of this antiserum is lower if compared with that of antisera raised conventionally against other viruses, thus it requires improvements for use for diagnostic purposes.