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Pathogens ultra-sensitive detection is vital for early diagnosis and provision of restraining actions and/or treatments. Among plant pathogens, Xylella fastidiosa is among the most threatening as it can infect hundreds of plant species worldwide with consequences on agriculture and the environment. An electrolyte-gated transistor is here demonstrated to detect X. fastidiosa at a limit-of-quantification (LOQ) of 2 ± 1 bacteria in 0.1 mL (20 colony-forming-unit per mL). The assay is carried out with a millimeter-wide gate functionalized with Xylella-capturing antibodies directly in saps recovered from naturally infected plants. The proposed platform is benchmarked against the quantitave polymerase chain reaction (qPCR) gold standard, whose LOQ turns out to be at least one order of magnitude higher. Furthermore, the assay selectivity is proven against the Paraburkholderia phytofirmans bacterium (negative-control experiment). The proposed label-free, fast (30 min), and precise (false-negatives, false-positives below 1%) electronic assay, lays the ground for an ultra-high performing immunometric point-of-care platform potentially enabling large-scale screening of asymptomatic plants.

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.

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 the insect 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. As a perspective solution, we investigated the efficacy of the low-temperature plasma and plasma-activated water to kill bacterial cells. Experiments were conducted in vitro to test the biocidal effect of the direct application of a Surface Dielectric Barrier Discharge (SDBD) plasma on bacteria cells and Plasma Activated Water (PAW). PAW activity was tested as a possible biocidal agent that can move freely in the xylem network paving the way to test the strategy on infected plants. The results showed a high decontamination rate even for cells of XfDD embedded in biofilms grown on solid media and complete inactivation in liquid culture medium.

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.

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.

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.

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.

The importance of apple allergens has been repeatedly emphasized, and their presence has been confirmed both in pollen and in fruits. In the present study, a combination of proteomic tools have been used to build a complete allergen map of apple. The water-soluble fraction of an apple extract was precipitated using a phenol-based procedure and separated by 2-DE. Initially four previously classified allergens, Mal d 1, Mal d 2, Mal d 3 and Mal d 4, could be identified in Western blots with polyclonal rabbit antibodies directed to the four respective allergens, and subsequently matched to the bands recognized by several patient sera. Further, all four known apple allergens were localized on a 2-DE map and they were matched with spots recognized by sera of patients with different allergic patterns. Moreover, a new, putative allergen could be identified using MS. We evaluated the influence of post-translational modifications and the immunoreactivity under different analytical conditions. The comparison of different visualization methods for 2-DE gels and blots revealed that even very low concentrations of the intact epitopes are detectable by IgEs of patients, and therefore might be sufficient to trigger allergic symptoms in sensitized individuals.

Surveys for virus and virus-like diseases were carried out in commercial vineyards and nurseries in seven different Syrian provinces (Aleppo, Dara’a, As Suwayda, Al Qunaytirah, Homs, Hamah, Tartous). Samples were collected at random from 835 individual vines (735 Vitis vinifera and 100 rootstock accessions) for laboratory testing. Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), and Grapevine virus A (GVA) were the only viruses recovered by mechanical transmission to herbaceous hosts. Vein necrosis developed in c. 53% of graft-inoculated 110R indicators and vein mosaic in V. riparia inoculated with material from cv. Corna Alegra. A total of 71% of the ELISA-tested V. vinifera plants (522 out of 735) were infected by one (14.8%) or more (55.8%) viruses. GVA was the most widespread (54.7%), followed by Grapevine leafroll-associated virus 1 (GLRaV-1, 47.3%), Grapevine fleck virus (GFkV, 29.7%), and Grapevine leafroll-associated virus 3 (GLRaV-3, 23.9%). Other economically relevant viruses were scarcer, i.e. Grapevine leafroll-associated virus 2 (GLRaV-2, 9%), GFLV (0.8%) and ArMV (0.1%). The most important Syrian grapevine varieties, i.e. Hellwany, Salty, Balady, and Zeiny, had average infection rates that ranged between 44% and 91%. The highest incidence of infections was observed at Damascus (90%), whereas it ranged between 68% and 79% in the other provinces, except for Hama (36%). Rootstocks were in much better sanitary condition (25% infection). GFkV (22%) was the most common virus, whilst the presence of GLRaV-3 (3%), GLRaV-1, and GFLV (1%) was negligible. Grapevine rupestris stem pitting associated virus (GRSPaV) was detected in 72.3% of the samples by RT-PCR. A high percentage of the GRSPaV-positive vines (80%) induced vein necrosis reactions in 110R, thus confirming the recently established correlation between this virus and vein necrosis.

D'enormes progres ont ete faits dans la recherche et le developpement sur les reactifs et les methodes serologiques pour le Plum pox virus (PPV) au cours des dernieres annees. Deux evenements ont revolutionne la detection et la caracterisation serologiques du virus : le developpement de la methode ELISA en 1977, puis l'apparition de la technologie des anticorps monoclonaux specifiques. La disponibilite des kits commerciaux a popularise le diagnostic du PPV, en rendant desormais possible le diagnostic a grande echelle a des fins phytosanitaires, pour les programmes d'eradication et la lutte contre la maladie dans les pepinieres. L'utilisation de l'anticorps monoclonal universel 5B-IVIA, utilise en DASI-ELISA, est le systeme le plus precis pour la detection du PPV en routine. De meme, l'utilisation d'anticorps monoclonaux de typage donne une caracterisation exacte des principaux types de PPV decrits: 4DG5 pour PPV-D, AL pour PPV-M, EA24 pour PPV-EA, et TUV et AC pour PPV-C. Il existe, en general, une excellente correlation entre les donnees serologiques obtenues avec les anticorps monoclonaux specifiques du PPV et les donnees obtenues par des methodes moleculaires basees sur la PCR. L'ELISA utilisant un seul ou un melange d'anticorps monoclonaux restera la methode preferee pour la detection universelle et le criblage en routine du PPV dans les annees a venir. Aujourd'hui, d'autres methodes et reactifs serologiques sont egalement recommandes dans le Protocole de diagnostic OEPP, ce qui augmente le nombre de tests fiables disponibles pour la detection du PPV. Ces developpements ont aide a lutter contre la sharka au cours des dernieres annees. La cooperation internationale dans ce domaine est cruciale pour l'amelioration et la validation des outils serologiques pour la detection et la caracterisation du PPV.

International audience; Recently obtained molecular and biological information has prompted the revision of the taxonomic structure of the family Closteroviridae. In particular, mealybug-transmitted species have been separated from the genus Closterovirus and accommodated in a new genus named Ampelovirus (from ampelos, Greek for grapevine). Thus, the family now comprises three genera. Their major properties are (i) Closterovirus: type species Beet yellows virus, genome monopartite, 15.5–19.3 kb in size, a 22–25 kDa major coat protein (CP), the gene encoding the divergent CP analogue (CPd) upstream of the CP cistron, transmission by aphids, a membership of 8 definitive and 4 tentative species; (ii) Ampelo-virus: type species Grapevine leafroll virus 3, genome monopartite 16.9–19.5 kb in size, a 35–37 kDa major CP, a CPd cistron generally located downstream of the CP gene, transmission by pseudococcid and coccid mealybugs, a membership of 6 definitive and 5 tentative species; (iii) Crinivirus: type species Lettuce infectious yellows virus, genome essentially bipartite 15.3–19 kb in size, a 28–33 kDa CP, a CPd cistron downstream of the CP gene, transmission by whiteflies (Bemisia, Trialeurodes), a membership of 7 definitive and 3 tentative species. There are five unassigned species in the family.

Previously characterized monoclonal antibodies (MAbs) to Grapevine virus A (GVA) showed a differential reactivity against intact or partially destabilized virus particles [2]. In the present study, this differential reactivity was confirmed and several peptides reacting with a panel of four different antibodies were identified by the PEPSCAN method of epitope mapping. Oligopeptide sequences comprised between coat protein residues 61 (V) and 72 (T) were recognized by all the antibodies tested. One of these peptides (VGPKASK) was also reactive when expressed on recombinant phage particles as a fusion protein with protein pVIII. The specificity of this sequence for antibody binding was also demonstrated by competitive-ELISA using one of the GVA MAbs. The results of this study suggest that GVA particles carry a highly structured epitope centered on a common peptide region of the coat protein sequence.

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 Brullé 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.

A large-scale serological characterisation of Plum pox virus (PPV) isolates was carried out with 19 monoclonal antibodies (MAbs), including the universal MAb5B and the following strain-specific MAbs: AL (specific to PPV-M), 4DG5 (specific to PPV-D), TUV and AC (specific to PPV-C), and EA24 (specific to PPV-EA). The study involved 108 PPV isolates of different geographical origin (Albania, Bulgaria, Cyprus, Czech Republic, Egypt, France, Germany, Greece, Italy, Hungary, Moldova, Romania, Slovakia, Spain, Turkey and Yugoslavia) and hosts (almond, apricot, peach, plum and cherry). The inter- and intra-strain serological relationships of PPV isolates were evaluated by DASI-ELISA. High serological variability was detected, not only between strains, but also among isolates of the same strain. Computer-assisted analysis of serological data support the hypothesis of the existence of two distinct subclusters, denoted PPV-M1 and PPV-M2, which seem to prevail in Mediterranean and Eastern–Central European countries, respectively.

A monoclonal antibody to an Albanian isolate of plum pox potyvirus (PPV) was obtained (MAbAL), that specifically recognized strain M of this virus. The specificity of MAbAL, assessed by comparative ELISA on 130 PPV isolates of different geographical origin, 22 of which were also tested by comparative IC-PCR, gave consistent and highly reproducible results. MAbAL seems to be elicited by a stable surface determinant that makes it particularly suitable for successful use under a wide range of conditions. MAbAL is an useful addition to the panel of PPV-specific MAbs available to date.

A virus for which the name of pothos latent virus (PoLV) is proposed, was isolated by inoculation of sap from symptomless plants ofScindapsus aureus. PoLV had isometric particlesc. 30 nm in diameter, a monopartite genome consisting of a non polyadenylated, single-stranded RNA moleculec. 4,300 nucleotides in length, constitutingc. 17% of the particle weight, and a single type of coat protein subunit with aMr ofc. 40,000 Daltons. The biological properties (host range reactions) of PoLV resembled those ofTombusviridae for it infected most of the artificial hosts locally, inducing symptoms recalling those elicited by several species of the above family. Like tombus- and carmoviruses, PoLV had two subgenomic RNAs which, however, differed in size from those of both genera. The dsRNA pattern was also distinctly different. Cytopathological features recalled those of tombusviruses except for the lack of multivesicular inclusion bodies. PoLV was serologically related to, but distinct from twoCarmovirus (i.e., galinsoga mosaic and Ahlum waterborne viruses) and threeTombusvirus species (i.e. eggplant mottled crinkle, Sikte waterborne and Lato river viruses). Thus, PoLV had properties somewhat intermediate between those ofTombusvirus andCarmovirus genera but bridged the two taxa through the serological relationship with some of their species. The taxonomic position of PoLV is still undetermined. It must await the results of molecular investigations now underway.

Grapevines were surveyed for the presence of virus and virus-like diseases in the Albanian viticultural districts of Shkoder, Lesh, Kruje, Durres, Tirana, Elbasan, Lushnje and Vlora. Symptoms of grapevine degeneration, leafroll and rugose wood were observed in all areas surveyed, whereas fleck was found in volunteer plants of Vitis rupestris at Elbasan, and enation disease in a few vines near Durres. Viruses identified were grapevine fanleaf nepovirus, grapevine fleck virus, grapevine virus A, and grapevine leafroll-associated closteroviruses I and III. ELISA tests showed that 83.5% of 530 Vitis vinifera vines and 46% of 24 American rootstocks individually checked were infected by one or more viruses. The presence of Xiphinema index, the major vector of grapevine fanleaf nepovirus, was recorded from vineyards affected by yellow mosaic.

Agrobacterium rhizogenes-mediated transformation was applied toVitis spp. andNicotiana spp. infected by different grapevine phloem-limited viruses (grapevine fleck virus, grapevine virus A, grapevine virus B) to obtain root cultures for virus purification. All plant species were successfully transformed, and several clones were established in liquid culture. Transformed grapevine roots contained as much virus as non transformed roots and more than leaves, as assessed by ELISA and thin sectioning. Likewise, transformed roots ofNicotiana benthamiana Domin. contained in average more GVA than leaves, especially those at the base and the top of the plant, whereas withNicotiana occidentalis wheel., GVB was apparently less concentrated than in leaves.Nicotiana root grew faster than those ofVitis. All viruses multiplied and persisted in root cultures, which were successfully used for purification. Virus yields were the same (GFkV and GVB) or higher (GVB) than those reported in the literature. Grapevine roots may prove useful for culturing and purifying other non-mechanically transmissible grapevine viruses.

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A virus with highly flexuous filamentous particles c. 800 nm long, showing distinct transverse striations was isolated with high frequency (60%) by inoculation of Nicotiana occidentalis with sap from grapevine accessions indexing positive for corky bark. The virus, for which the name grapevine virus B (GVB) is proposed, has an ssRNA genome with mol. wt. of c. 2.5 x 10(6) Da (c. 7600 nt) and coat protein subunits with mol. wt. of c 23,000 Da. GVB has a very restricted herbaceous host range and was experimentally transmitted by the mealybug Pseudococcus ficus. The physicochemical and ultrastructural properties of GVB resemble those of closteroviruses. However, it is serologically unrelated to other grapevine closteroviruses including grapevine virus A, with which it shares some biological and physicochemical properties.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

Grapevines of the Maltese islands were surveyed for the presence of virus diseases. Symptoms of fanleaf degeneration, leafroll, rugose wood and fleck were observed in the main grape-growing areas. The occurrence of these diseases was experimentally confirmed by graft transmission to woody indicators, which also revealed the presence of vein necrosis, a latent virus-like disorder. Viruses identified were grapevine fanleaf nepovirus, grapevine fleck virus, grapevine A closterovirus, grapevine leafroll-associated closteroviruses I and III. ELISA tests showed that 86% of 322 vines individually checked were infected by one or more viruses.

A virus with isometric particles c. 30 nm in diameter and angular contour was isolated by inoculation of sap from a Tunisian grapevine with mild mottling and leaf deformation. The virus sedimented in sucrose density gradients as three components: T (empty shells), M (particles containing a molecule of ssRNA with an apparent size of 5,800 nucleotides, constituting 35% of the particle weight) and B (particles containing a molecule of ssRNA with apparent size of 6,800 nucleotides, constituting 41% of the particle weight). Virus particles had buoyant densities of 1.31 (T), 1.45 (M), and 1.49 g/cm3 (B) in cesium chloride equilibrium gradients. The coat protein subunits consisted of a single polypeptide with mol. wt. of c. 59,000 daltons. An antiserum was produced with a titer of 1:256, which did not react with healthy plant antigens. Cells of artificially infected herbaceous hosts showed cytoplasmic vesiculate-vacuolate inclusion bodies, virus-containing tubules, mostly associated with plasmodesmata and/or cell wall protrusions, and crystalline aggregates of virus particles and empty capsids. The physicochemical and ultrastructural properties of this virus resemble very much those of nepoviruses. However, it was serologically unrelated to 19 different members of the group, including all those reported to infect grapevines. Therefore, the virus is possibly a hitherto unreported nepovirus for which the name of grapevine Tunisian ringspot virus (GTRV) is proposed.

The Cucumber mosaic virus (CMV) is an isodiametric plant virus with an extremely wide host range, present worldwide. CMV chimeric particles (R9-CMV), engineered to express a 27-aa synthetic peptide derived from Hepatitis C virus (HCV), were demonstrated to be stable under simulated gastric and intestinal conditions. Then the possibility of inducing a humoral immune response in rabbits fed with R9-CMV infected lettuce plants was demonstrated, suggesting that this system could function as a confirming tool of a bioreactor for the production of a stable edible vaccine against HCV.

Grapevine phloem-limited isometric virus (GPLIV) is the name proposed for a non mechanically-transmissible virus found in Italian and Tunisian grapevines. In density gradient centrifugation purified virus preparations sedimented as two components: T, made up of empty protein shells, and B, composed of intact nucleoprotein particles. B particles had a buoyant density of 1.45 g/cm3 at equilibrium in CsCl and contained 35% RNA consisting of a single molecule with an apparent size of 7.4 kb. The coat protein consisted of a single species with a mol.wt of 28,000 daltons. Purified virus preparations did not infect herbaceous hosts by manual inoculation. A specific antiserum with a titre of 1: 64 raised in rabbits, was used for identification of, GPLIV in field-grown Tunisian grapevines and in leafroll-affected Italian vines before and after heat treatment. Although heat treatment eliminated the virus from the majority of the plants, leafroll symptoms persisted in several GPLIV-free vines, indicating that there is no clear-cut relationship between GPLIV and this disease. ZusammenfassungEinige Eigenschaften eJnes im Phloem begrenzten, nicht mechanisch iibertragbaren Weinstockvirus Der Name grapevine phloem-limited isometric virus [GPLIV) wird fur ein nicht mechanisch ubertragbares, in Italien und Tunesien vorkommendes Virus vorgeschlagen, Nach Dichtegradiemenzentnfugation konnten zwei Komponenten aus gereinigten Viruspraparaten festgestellt werden: T, die aus leeren Proteinhullen bestand, und B, die ganze Nucieoproteinpartikein enthielt. Die B-Komponenten hatten eine schwimmende Dichte von 1,45 g/cm3 im Gleichgewicht mit CsCl und emhielten 35 % RNS, die aus eincm einzelnen Molekul mit einer scheitibaren Grose von 7,4 kb bestand. Das Hullprotein setzte sich aus einer Art mit einem Mokkulargewicht von 28 000 Dalton zusammen. Gereinigte Viruspraparaie waren nicht in der Lage, Wirtspflanzen nach einer kiinsthchen Inokulation zu lnfizieren. Ein aus Kaninchen gewonnenes, spezifisches Antiserum, mit einem Titer von 1: 64, wurde bei der Feststellung vom CPLIV in Weinstocken aus Feldern in Tunesien und in Reben in Italien. die geroiite Blatter aufwiesen, vor und nach einer Hitzebehandlung angewandt. Obwohl Nach einer Hitzebehandlung das Virus in fast alien Pflanzen nicht mehr nachzuweisen war, wurden die gerollten Blattersymptome bei einigen GPLIV-freien Reben nicht aufgehoben. Dies deutet darauf hin, das es keinen eindeutigen Zusammenhang zwischen dem GPLIV und dieser Krankheit gibt.

A vaccine against Hepatitis C virus (HCV) is urgently needed due to the unsatisfactory clinical response to current therapies. We evaluated the immunological properties of a chimeric Cucumber mosaic virus (CMV), a plant virus engineered to express on its surface a synthetic peptide derived from many HVR1 sequences of the HCV envelope protein E2 (R9 mimotope). Evidence was obtained that the chimeric R9-CMV elicits a specific humoral response in rabbits. Furthermore, in patients with chronic HCV infection, purified preparations of R9-CMV down-modulated the lymphocyte surface density of CD3 and CD8, and induced a significant release of interferon (IFN)-gamma, interleukin (IL)-12 p70 and IL-15 by lymphomonocyte cultures. Finally, an R9 mimotope-specific CD8 T-cell response, as assessed by intracellular IFN-gamma production, was achieved in the majority of the patients studied. Our results open up new prospects for the development of effective vaccines against HCV infection. Moreover, the wide edible host range of CMV makes the production of an edible vaccine conceivable.

An RT-PCR/nested PCR technique was developed for the simultaneous detection and typing of plum pox virus (PPV) and its major types--Dideron (D), Marcus (M), El-Amar (EA) and Cherry (C). Degenerated oligonucleotides were synthesized for the general detection of PPV, flanking the coding sequence for the N-terminal portion of the coat protein (CP), within which strain-specific differences were identified. On the basis of these characteristic differences, degenerated primer pairs were designed to differentiate between the four major subgroups of the virus in nested PCR reactions. The validity of the technique was tested on viral strains and cloned cDNAs overlapping the CP region. High specificity was observed with no detectable cross-reactions. The results of general PPV detection with the new primers and those of the PCR-based detection of the 3' non-coding region of the viral genome correlated with complete coincidence. The PCR typing results correlated well with those of the RsaI-RFLP and serological typing and revealed a surprisingly high incidence of PPV-D in Hungary.