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The early detection of Xylella fastidiosa (Xf) infections is critical to the management of this dangerous plan pathogen across the world. Recent studies with remote sensing (RS) sensors at different scales have shown that Xf-infected olive trees have distinct spectral features in the visible and infrared regions (VNIR). However, further work is needed to integrate remote sensing in the management of plant disease epidemics. Here, we research how the spectral changes picked up by different sets of RS plant traits (i.e., pigments, structural or leaf protein content), can help capture the spatial dynamics of Xf spread. We coupled a spatial spread model with the probability of Xf-infection predicted by a RS-driven support vector machine (RS-SVM) model. Furthermore, we analyzed which RS plant traits contribute most to the output of the prediction models. For that, in almond orchards affected by Xf (n = 1426 trees), we conducted a field campaign simultaneously with an airborne campaign to collect high-resolution thermal images and hyperspectral images in the visible-near-infrared (VNIR, 400–850 nm) and short-wave infrared regions (SWIR, 950–1700 nm). The best performing RS-SVM model (OA = 75%; kappa = 0.50) included as predictors leaf protein content, nitrogen indices (NIs), fluorescence and a thermal indicator (Tc), alongside pigments and structural parameters. Leaf protein content together with NIs contributed 28% to the explanatory power of the model, followed by chlorophyll (22%), structural parameters (LAI and LIDFa), and chlorophyll indicators of photosynthetic efficiency. Coupling the RS model with an epidemic spread model increased the accuracy (OA = 80%; kappa = 0.48). In the almond trees where the presence of Xf was assayed by qPCR (n = 318 trees), the combined RS-spread model yielded an OA of 71% and kappa = 0.33, which is higher than the RS-only model and visual inspections (both OA = 64–65% and kappa = 0.26–31). Our work demonstrates how combining spatial epidemiological models and remote sensing can lead to highly accurate predictions of plant disease spatial distribution., Data collection was partially supported by the European Union's Horizon 2020 research and innovation program through grant agreements POnTE (635646) and XF-ACTORS (727987). R. Calderón was supported by a post-doctoral research fellowship from the Alfonso Martin Escudero Foundation (Spain).

Trabajo desarrollado bajo la financiación del proyecto “Soil Hydrology research platform underpinning innovation to manage water scarcity in European and Chinese cropping Systems” (773903), coordinado por José Alfonso Gómez Calero, investigador del Instituto de Agricultura Sostenible (IAS)., Among the agricultural practices promoted by the Common Agricultural Policy to increase soil functions, the use of cover crops is a recommended tool to improve the sustainability of Mediterranean woody crops such as olive orchards. However, there is a broad range of cover crop typologies in relation to its implementation, control and species composition. In that sense, the influence of different plant species on soil quality indicators in olive orchards remains unknown yet. This study describes the effects of four treatments based on the implementation of different ground covers (CC-GRA: sown cover crop with gramineous, CC-MIX: sown cover crop with a mixture of species and CC-NAT: cover crop with spontaneous vegetation) and conventional tillage (TILL) on soil erosion, soil physicochemical and biological properties after 8 years of cover crop establishment. Our results demonstrated that the presence of a temporary cover crop (CC), compared to a soil under tillage (TILL), can reduce soil losses and maintain good soil physicochemical properties and modify greatly the structure and diversity of soil bacterial communities and its functioning. The presence of a homogeneous CC of gramineous (Lolium rigidum or Lolilum multiflorum) (CC-GR) for 8 years increased the functional properties of the soil as compared to TILL; although the most relevant change was a modification on the bacterial community composition that was clearly different from the rest of treatments. On the other hand, the use of a mixture of plant species (CC-MIX) as a CC for only two years although did not modify greatly the structure and diversity of soil bacterial communities compared to the TILL soil, induced significant changes on the functional properties of the soil and reverted those properties to a level similar to that of an undisturbed soil that had maintained a natural cover of spontaneous vegetation for decades (CC-NAT)., This work was supported by the EU SoilMan project (grant number 01LC1620) funded through the 2015–2016 BiodivERsA COFUND call for research proposals, with national funds through PCIN-2016-048 project by the Spanish Ministry of Economy and Competitiveness (MINECO), SHui project (GA 773903), and by projects PID2019-105793RB-I00, AGL2008-00344 from ‘Ministerio de Ciencia e Innovación’, AGL-2012–37521 from ‘Ministerio de Economia y Competitividad’ of Spain, Project P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’ Junta de Andalucía, and FEDER financial support from the European Union.

Vascular pathogens are the causal agents of main diseases threatening the health and growth of olive crops worldwide. The use of endophytic microorganisms represents a challenging and promising strategy for management of vascular diseases in olive. Although current research has been focused on analyzing the structure and diversity of the endophytic microbial communities inhabiting the olive xylem, the characterization of this ecological niche has been overlooked and to date remain unexplored, despite that the characterization of the xylem sap composition is essential to unravel the nutritional requirements of xylem-limited microorganisms. In this study, branches from plantlets and adult olive trees of cultivars ‘Picual’ and ‘Arbequina' were selected to characterize the chemical composition of olive xylem sap extracted using a Scholander pressure chamber. Metabolome and ionome analyses of xylem sap were performed by proton nuclear magnetic resonance (NMR) spectroscopy-based and by inductively coupled plasma with optical emission spectroscopy (ICP-OES), respectively. Olive xylem sap metabolites included a higher relative percentage of sugars (54.35%), followed by alcohols (28.85%), amino acids (8.01%), organic acids (7.68%) and osmolytes (1.12%). Within each of these groups, the main metabolites in the olive xylem sap were mannitol, ethanol, glutamine, acetate and trigonelline, whereas K and Cl- were the main element and inorganic anion, respectively. Metabolomic profile varied when comparing olive plant age and genotype. The levels of glucose, fructose, sucrose and mannitol, choline, B and PO43 were significantly higher in adult trees than in plantlets for both olive genotypes, whereas NO3- and Rb content showed the opposite behavior. On the other hand, levels of aspartate, phenylalanine and Na were significantly higher in ‘Picual’ than in ‘Arbequina’ whereas Fe showed the opposite behavior but only for adult trees. Non-supervised hierarchical clustering analysis separated xylem sap composition firstly according to the plant age and then by the olive cultivar. Supervised PLS-DA analysis revealed that B, ethanol, Fe, Fructose, glucose, mannitol, sucrose and Sr were the most significative compounds discriminating adult trees from plantlets, whereas asparagine, aspartate, glutamate and phenylalanine or aspartate, arginine, ethanol and Sr were the most contributory compounds in the discrimination of both olive genotypes for adult trees or plantlets, respectively. Knowledge of the chemical composition of xylem sap will lead to a better understanding of the complex nutritional requirements of olive xylem-inhabiting microorganisms, including its vascular pathogens, and would allow the design of artificial growing media to improve culturing the olive microbiome.

This article belongs to the Special Issue Role of Human Interventions in Spread of Soilborne Forest Pathogens and Methods for Mitigation., Disease outbreaks caused by introduced Phytophthora species have been increasing in British forests and woodlands in recent years. A better knowledge of the Phytophthora communities already present in the UK is of great importance when developing management and mitigation strategies for these diseases. To do this, soils were sampled in “disturbed” sites, meaning sites frequently visited by the public, with recent and new plantings or soil disturbances versus more “natural” forest and woodland sites with little disturbance or management. Phytophthora diversity was assessed using high-throughput Illumina sequencing targeting the widely accepted barcoding Internal Transcribed Spacer 1 (ITS1) region of rRNA and comparing it with the mitochondrial cytochrome c oxidase I (COI) gene. Isolation of Phytophthora was run in parallel. Nothophytophthora spp. and Phytophthora spp. were detected in 79 and 41 of the 132 locations of the 14 studied sites when using ITS or COI, respectively. A total of 20 Phytophthora amplicon sequence variants (ASVs) were assigned to known Phytophthora species from eight clades (1a, 2, 2b, 3a, 5, 6b, 7a, 8b, 8c, 8d, 10a, and 10b) and 12 ASVs from six clades (1a, 2c, 3a, 3b, 6b, 7a, 8b, 8c, and 8d) when using ITS or COI, respectively. Only at two locations were the results in agreement for ITS, COI, and isolation. Additionally, 21 and 17 unknown Phytophthora phylotypes were detected using the ITS and COI, respectively. Several Phytophthora spp. within clades 7 and 8, including very important forest pathogens such as P. austrocedri and P. ramorum, were identified and found more frequently at “disturbed” sites. Additionally, eight ASVs identified as Nothophytophthora spp. were detected representing the first report of species within this new genus in Britain. Only three species not known to be present in Britain (P. castaneae, P. capsici, and P. fallax) were detected with the ITS primers and not with COI. To confirm the presence of these or any potential new Phytophthora species, sites should be re-sampled for confirmation. Additionally, there is a need to confirm if these species are a threat to British trees and try to establish any eradication measures required to mitigate Phytophthora spread in Britain., This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635646, POnTE (Pest Organisms Threatening Europe).

Vascular pathogens are the causal agents of main diseases threatening the health and growth of olive crops worldwide. The use of endophytic microorganisms represents a challenging and promising strategy for management of vascular diseases in olive. Although current research has been focused on analyzing the structure and diversity of the endophytic microbial communities inhabiting the olive xylem, the characterization of this ecological niche has been overlooked and to date remain unexplored, despite that the characterization of the xylem sap composition is essential to unravel the nutritional requirements of xylem-limited microorganisms. In this study, branches from plantlets and adult olive trees of cultivars Picual and Arbequina were selected to characterize the chemical and microbial composition of olive xylem sap extracted using a Scholander pressure chamber. Metabolome and ionome analyses of xylem sap were performed by proton nuclear magnetic resonance (NMR) spectroscopy-based and by inductively coupled plasma with optical emission spectroscopy (ICP-OES), respectively. Olive xylem sap metabolites included a higher relative percentage of sugars (54.35%), followed by alcohols (28.85%), amino acids (8.01%), organic acids (7.68%), and osmolytes (1.12%). Within each of these groups, the main metabolites in the olive xylem sap were mannitol, ethanol, glutamine, acetic acid, and trigonelline, whereas K and Cl− were the main element and inorganic anion, respectively. Metabolomic profile varied when comparing olive plant age and genotype. The levels of glucose, fructose, sucrose and mannitol, choline, B and PO43− were significantly higher in adult trees than in plantlets for both olive genotypes, whereas NO3− and Rb content showed the opposite behavior. On the other hand, levels of aspartic acid, phenylalanine, and Na were significantly higher in ‘Picual’ than in ‘Arbequina’, whereas Fe showed the opposite behavior, but only for adult trees. Microbiome composition identified Firmicutes (67%), Proteobacteria (22%) and Actinobacteriota (11%) as the main phyla, while at the genus level Anoxybacillus (52%), Cutibacterium (7%), Massilia (6%), and Pseudomonas (3%) were the most representative. Both non-supervised hierarchical clustering analysis and supervised PLS-DA analysis differentiated xylem sap chemical and microbial composition first, according to the age of the plant and then by the olive genotype. PLS-DA analysis revealed that B, ethanol, Fe, fructose, glucose, mannitol, sucrose, and Sr, and Anoxybacillus, Cutibacterium, and Bradyrhizobium were the most significant chemical compounds and bacterial genera, respectively, in the discrimination of adult olive trees and plantlets. Knowledge of the chemical composition of xylem sap will lead to a better understanding of the complex nutritional requirements of olive xylem-inhabiting microorganisms, including vascular pathogens and their potential antagonists, and may allow the better design of artificial growing media to improve the culturing of the olive microbiome., This research was funded by projects AGL2016-75606-R (Programa Estatal de I + D Orientado a los Retos de la Sociedad from the Spanish Government and the Spanish State Research Agency and FEDER-EU) and XF-ACTORS (Xylella fastidiosa Active Containment Through a Multidisciplinary-Oriented Research Strategy; grant 727987 from the European Union’s Horizon 2020 Framework Research Programme). M.A.-M. is a recipient of a research fellowship BES-2017-082361 from the Spanish Ministry of Economy and Competitiveness.

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.

Opium poppy (Papaver somniferum L.) is an economically important pharmaceutical crop in Spain. Approximately 8,000 ha are cultivated annually in southern and central Spain. To improve yields, opium poppy cultivation is expanding to more humid or irrigated areas of Spain. In the springs of 2005 and 2007, we observed poppy plants with wilt and stem rot symptoms in irrigated, commercial opium poppy (cv. Nigrum) at Carmona and Écija, which are in Seville Province in southern Spain. Closer observations of affected plants revealed darkening and water soaking of the leaves and stem at the soil level, wilting of the lower leaves or the entire plant, and dark brown discoloration of stem vascular tissues and pith of the plant. Severely affected plants became completely rotten and collapsed. Isolations from symptomatic tissues on nutrient agar consistently yielded bacterial colonies. Pure cultures of four representative bacterial strains (two per each of affected field and year of isolation) were used in triplicate for a comparative analysis of biochemical and physiological traits in the ‘carotovora’ group of Erwinia (1) with known isolates of Pectobacterium carotovorum subsp. carotovorum, P. carotovorum subsp. atrosepticum, and Dickeya chrysanthemi. The isolates from opium poppy were gram negative, facultatively anaerobic, oxidase negative, catalase positive, grew at 37°C, and did not produce gas from D-glucose. Acid was produced from D(+)-arabinose, lactose, and D(+)-trehalose, but not from α-D-methylglucoside. In addition, the opium poppy bacterial isolates caused soft rot on potato slices within 24 h at 25°C and did not induce a hypersensitive reaction on tobacco leaves. Use of the Biolog GN microplates and the OmniLog ID 1.2 system identified the four poppy isolates as P. carotovorum (showing a 66.7% similarity with the subsp. carotovorum). Pathogenicity of poppy isolates was tested on three 6-week-old opium poppy plants (cv. Nigrum) by injecting 100 μl of a bacterial suspension containing 108 CFU/ml in the basal stem. Plants that served as controls were injected with sterile water. Plants were incubated in a growth chamber adjusted to 28°C, 90% relative humidity, and a 14-h photoperiod of fluorescent light of 360 μE·m-2·s-1. Severe symptoms of soft rot and darkening developed on stems of inoculated plants within 3 to 5 days after inoculation. No symptoms developed on control plants. Bacterial strains reisolated from inoculated plants were identified as P. carotovorum on the basis of the Biolog system, as well as biochemical and physiological characters. To our knowledge, this is the first report of P. carotovorum causing soft rot of commercial opium poppy crops in Spain and elsewhere. The presence of this bacterial pathogen to irrigated crops and humid areas may pose an important constraint on the yield of opium poppy crops in Spain. References: (1) R. S. Dickey and A. Kelman. Pages 44-59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. The American Phytopathological Society, St. Paul, MN, 1988.

Opium poppy is a strategic crop for the pharmaceutical industry because it is the only source of morphine, codeine, and thebaine alkaloid drugs. Approximately 7,360 ha (average from 2001 through 2007) of opium poppy (Papaver somniferum) are grown annually in France, mainly in the Northern-East (Champagne-Ardenne) and Centre-West (Centre and Poitou-Charentes) regions of the country. This acreage accounts for nearly 5.6% of the legally cultivated opium poppies worldwide. Disease symptoms resembling those of downy mildew (2) have been observed frequently in those opium-poppy-growing areas, especially in the Charente-Maritime, Cher, Loiret, and Loir et Cher departments. Disease symptoms included chlorotic to light yellow lesions on the leaf blade, curling and thickening of affected tissues, and expanding necrotic lesions that coalesced, eventually giving rise to large necrotic areas or death of the entire leaf tissues and the plant. With wet weather or high relative humidity, sporangiophores with sporangia were produced frequently on the abaxial leaf surface and occasionally on the adaxial side. Peronospora arborescens and P. cristata have been demonstrated as causal agents of opium poppy downy mildew disease and both have been reported in Europe (1–3); however, the specific identity causal agent in commercial opium poppy crops in France has not yet been determined. Microscopic observations of affected leaves in symptomatic opium poppy leaves sampled from three commercial fields in Loiret Department revealed dichotomously branching sporangiophores bearing single sporangia and oospores of shape and measurements similar to those reported for P. arborescens and P. cristata (1,3). Sporangia dimensions of P. arborescens and P. cristata overlapped, making it difficult to differentiate between the two species based solely on morphological characters (3). A species-specific PCR assay protocol (2) that differentiated P. arborescens from P. cristata was used to diagnose the pathogen. Also, the sequence of the complete 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) 1 and 2 were determined and maximum parsimony analysis was performed with the Peronospora spp. data set described by Landa et al. (2). Both species-specific PCR and phylogenetic analyses of ITS sequences showed that P. arborescens was the only Peronospora species associated with the three samples of downy-mildew-affected leaves analyzed. Thus, DNA fragments of 545, 594, and 456 bp were amplified using total DNA extracted from the sampled leaves and P2, P3, and P6 primer pairs (2), respectively. ITS sequences of all three samples showed 100% homology (GenBank Accession No. EU295529). Phylogenetic analyses using Neighbor Joining of those sequences placed the infecting Peronospora sp. in a clade (100% support) that included all P. arborescens sequences from the GenBank database with 99.2 to 99.9% homology among sequences (2,3). To our knowledge, this is the first report and molecular evidence that P. arborescens causes downy mildew disease in commercial opium poppy crops in France. References: (1) S. M. Francis. No. 686 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1981. (2) B. B. Landa et al. Phytopathology 97:1380, 2007. (3) J. B. Scott et al. Phytopathology 93:752, 2003.

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.

We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235.

We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235., We report the complete annotated genome sequence of the plant-pathogenic bacterium Xylella fastidiosa subsp. fastidiosa strain IVIA5235. This strain was recovered from a cherry tree in Mallorca, Spain.

Until now, different molecular tests can be used to assign novel X. fastidiosa isolates to subspecies clusters, among which MLST/MLSA represents the most common method. X. fastidiosa outbreaks in EU motivated the search for accurate and faster approaches to differentiate the X. fastidiosa isolates. Because MLST/MLSA requires PCR reactions and sequencing analyses, 2 independent approaches were recently developed and implemented for rapid taxonomic assignment of uncharacterized isolates: (1) single-nucleotide primer extension (SNuPE) method that allows to differentiate all subspecies and three genotypes within X. fastidiosa subsp. pauca including the typeisolate infecting olive in Italy and (2) high-resolution melting (HRM) analysis of the amplicon recovered from the gene encoding the conserved HL protein. Both assays were validated on a larger panel of isolates and proved to clearly differentiate X. fastidiosa isolates currently known to occur in the Italian, France and Spain outbreaks. These rapid approaches could represent a useful tool for prescreening of infected samples to be further analyzed by MLST or whole genome sequencing. In addition alternative genomic regions of X. fastidiosa are going to be analyzed to implement approaches aimed to assign genotypes to a subspecies cluster, with the purpose to support a rapid identification of genotypes/subspecies at interception places or when new findings occur in a pest free area

Las principales estrategias para el control de enfermedades de plantas conciernen a: (i) la eliminación del patógeno (exclusión y erradicación); (ii) el escape a la infección; (iii) el desarrollo de resistencia genética del huésped al patógeno; y (iv) la protección de la planta contra la infección (terapia). Una vez que X. fastidiosa se ha establecido en un área su control es muy difícil o imposible. Por tanto, todos los esfuerzos deben ir dirigidos a la prevención de su introducción en nuevas áreas. En este capítulo se presentan los principios de control aplicables para el manejo de las enfermedades causadas por X. fastidiosa de acuerdo a las estrategias mencionadas de forma resumida

Xylella fastidiosa, a quarantine organism in the European Union (EU), causes diseases in a wide variety of plants such as almond, cherry, grape, citrus, elm, olive, and coffee trees and many ornamental plants. Since the detection of the bacterium in Italy (2013), where it is associated to a severe epidemic on olive trees, the pathogen has also been detected in France (2015) and Germany (2016) (EPPO 2016)., The work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 635646 POnTE (Pest Organisms Threatening Europe) and No 727987 XF-ACTORS (Xylella Fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy), and the Spanish Ministry of Agriculture and Fisheries, Food and Enviroment (MAPAMA).

Article opinion eComercio Agrario Actualidad del sector agrario

Xylella fastidiosa es una bacteria de cuarentena en la Unión Europea (UE), ya que se considera una grave amenaza para la UE y los países de la cuenca del Mediterráneo por la gran variedad de plantas huéspedes que infecta, su forma de transmisión a través de insectos vectores de diversa naturaleza y por la existencia en Europa de diversos climas que son similares a los presentes en otras partes del mundo donde la bacteria presenta un problema. El Panel de Sanidad Vegetal de la Autoridad Europea de Seguridad Alimentaria (EFSA) realizó en 2015 un completo análisis de riesgo de X. fastidiosa en la UE, cuyas principales conclusiones fueron que el riesgo estimado de la bacteria para el territorio de la UE es elevado porque tiene potencial de causar enfermedades en nuestro territorio si llega a establecerse en una región, ya que muchas de las plantas huésped conocidas de la bacteria están presentes en nuestro territorio y además las condiciones ambientales de buena parte de las regiones de la UE pueden ser favorables para el desarrollo epidémico., Parte de la información recogida en este trabajo está financiada por los proyectos N. 635646 POnTE (Pest Organisms Threatening Europa) y N. 727987 XF-ACTORS (Xy/el/a Fastidiosa Active Contai nment Through a multidisciplinary-Oriented Research Strategy) del Programa de Investigación e Innovación Horizonte 2020 de la Unión Europea.

Nowadays, there is a tendency in olive production systems to reduce tillage or keep a vegetative cover to reduce soil erosion and degradation. However, there is scarce information on the effects of different soil management systems (SMS) in soil bacterial community composition of olive groves. In this study, we have evaluated the effects of soil type and different SMS implemented to control weeds in the structure and diversity of bacterial communities of 58 soils in the two geographic areas that best represent the organic olive production systems in Spain. Bacterial community composition assessed by frequency and intensity of occurrence of terminal restriction profiles (TRFs) derived from terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S ribosomal deoxyribonucleic acid were strongly correlated with soil type/field site (Eutric/Calcaric) that differed mainly in soil particle size distribution and soil pH, followed by a strong effect of SMS, in that order. Canonical discriminant (CD) analysis of TRFs properly classified all of the olive orchard soils as belonging to their respective soil type or SMS. Furthermore, only a small set of TRFs were enough to clearly and significantly differentiate soil samples according to soil type or SMS. Those specific TRFs could be used as bioindicators to assess the effect of changes in SMS aimed to enhance soil quality in olive production systems.

A comparative study of organic and conventional commercial olive farming systems was conducted in a wide-area of Andalusia, southern Spain to determine the effect of management practices on chemical and biological soil properties. Soils from 41 accredited organic farms, 49 conventionally managed neighboring farms, and three sites containing wild or feral forms of olive were analyzed. A polyphasic approach was used combining traditional soil physicochemical analysis, culture-dependent microbiological analyses, sole-carbon-source utilization profiles using the Biolog EcoPlate assay and enzymatic activities using the API ZYM assay. Different multivariate statistical analyses clearly demonstrated that olive orchard soils could be differentiated by farm management system. Discriminant analysis differentiated among three well defined soil groups that correlated with the farm management system (conventional, organic or wild olives). Thus, enzymatic activities and sole-carbon-source utilization profiles correctly classified 75, 77 and 100% of soil samples from orchards under organic and conventional management or wild olives, respectively. Overall, all diversity and functional indexes estimated from the Biolog EcoPlate and API ZYM assays were positively and significantly correlated with SOM, organic C and N content and the C:N ratio, and negatively and significantly correlated with clay content. In general olive orchards under organic management showed significantly higher SOM, organic C and N content, and C:N ratio, as well as higher microbial diversity as measured by catabolic capability and functional indexes of the Biolog EcoPlate and API ZYM assays as compared to conventionally managed orchards. Given the crucial importance of maintaining or increasing soil health in agricultural ecosystems, this is the first demonstration that commercial olive orchards under organic management in southern Spain are agricultural systems that may contribute to promote and conserve soil quality and health. © 2013 Elsevier B.V.

Póster presentado en el 11 th International Verticillium Symposium, celebrado en Göttingen (Alemania) del 5 al 8 de mayo de 2013., Early, specific and accurate in planta detection and quantification of Verticillium dahliae are essential to prevent the spread of Verticillium wilt in olive using certified pathogen-free planting material and development of resistance. We comparatively assessed the accuracy, specificity and efficiency of eight real-time quantitative PCR (qPCR) protocols published since 2002 for the specific detection and quantification of V. dahliae in various host plant species and in soil, using a background of DNAs extracted from olive roots, stems and leaves. Results showed that some of those protocols were not specific for V. dahliae and/or were inhibited when using backgrounds other than water. Ranking of protocols according to a weighted score system placed protocol TAQ (based on IGS rDNA target gene; Bilodeau et al . 2012) and SYBR-4 (based on β -tubulin 2 target gene; Atallah et al . 211) first in sensitivity and efficiency for the quantification of V. dahliae DNA in small amounts and different types of olive tissues (root and st em) tested. Use of TAQ and SYBR-4 protocol allowed accurate quantification of V. dahliae DNA regardless of the background DNA with a detection limit being fixed at a C T of 36 (approximately 18 fg for SYBR-4 and 15 fg for TAQ) of V. dahliae ). The amount of DNA from defoliating (D) and nondefoliating (ND) V. dahliae pathotypes was monitored in Verticillium wilt-resistant 'Frantoio' olive using the TAQ and SYBR-4 protocols. In the infection bioassay, higher amounts of D- V. dahliae DNA were measured in olive stems, whereas the aver age amount of fungal DNA in roots was higher for ND-infected plants than D-infected ones. Overall, V. dahliae DNA amount in all olive tissues tested tended to slightly decrease or remain stable by the end of the experiment (35 days). The SYBR-4 and TAQ protocols further enabled detection of V. dahliae in tissues of symptomless plants, suggesting that both techniques can be useful for implementing certification schemes of pathogen-free planting material as well as a helpful tool in breeding resistance to V. dahliae in olive., Financial support was provided by grants P10-AGR-03528 from CICE-Junta de An dalucía, Spain and AGL- 2012-37521 and AGL2008-00344 from Ministerio de Econom ía y Competitividad, Spai n, and co-financed with FEDER founds from the European Union.

Until now, different molecular tests can be used to assign novel X. fastidiosa isolates to subspecies clusters, among which MLST/MLSA represents the most common method. X. fastidiosa outbreaks in EU motivated the search for accurate and faster approaches to differentiate the X. fastidiosa isolates. Because MLST/MLSA requires PCR reactions and sequencing analyses, 2 independent approaches were recently developed and implemented for rapid taxonomic assignment of uncharacterized isolates: (1) single-nucleotide primer extension (SNuPE) method that allows to differentiate all subspecies and three genotypes within X. fastidiosa subsp. pauca including the typeisolate infecting olive in Italy and (2) high-resolution melting (HRM) analysis of the amplicon recovered from the gene encoding the conserved HL protein. Both assays were validated on a larger panel of isolates and proved to clearly differentiate X. fastidiosa isolates currently known to occur in the Italian, France and Spain outbreaks. These rapid approaches could represent a useful tool for prescreening of infected samples to be further analyzed by MLST or whole genome sequencing. In addition alternative genomic regions of X. fastidiosa are going to be analyzed to implement approaches aimed to assign genotypes to a subspecies cluster, with the purpose to support a rapid identification of genotypes/subspecies at interception places or when new findings occur in a pest free area &nbsp

Downy mildew (DM) of opium poppy (Papaver somniferum) caused by Peronospora somniferi is one of the most destructive diseases of this crop due to the systemic nature of infection as compared with local infections caused by Peronospora meconopsidis, the other downy mildew pathogen of this crop. We developed an inoculation method using Peronospora somniferi sporangia as inoculum and demonstrated for the first time that local infection of leaves by sporangia give rise to systemic infections in the plant as well as of seeds. Our results also showed that this inoculation protocol was very effective in reproducing disease symptoms and assessing the resistance response to DM in opium poppy genotypes under field conditions. More interestingly, results indicate that up to 100% of seed samples from some genotypes showing a complete (symptomless) resistant phenotype were infected by the pathogen when seeds were analyzed by a P. somniferi-specific nested-PCR protocol. This latter aspect deserves further attention while breeding opium poppy for resistance to P. somniferi.

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

The association of Fusarium redolens with wilting-like symptoms in chickpea in Lebanon, Morocco, Pakistan, and Spain is reported for the first time, together with the molecular and pathogenic characterization of isolates of the pathogen from chickpea of diverse geographic origin. Maximum parsimony analysis of sequences of the translation elongation factor 1α (TEF-1α) gene grouped all F. redolens isolates from chickpea in the same main clade. Pathogenicity assays using three chickpea cultivars and isolates from different geographic origins indicated that F. redolens is mildly virulent on chickpea. Moreover, infection of chickpea by F. redolens induces a disease syndrome similar to that caused by the yellowing pathotype of F. oxysporum f. sp. ciceris, including leaf yellowing and necrosis that develop upward from the stem base, and premature senescence of the plant. In contrast, F. redolens does not cause discoloration of the vascular tissues in chickpea but does cause brown necrotic lesions in the tap root and necrosis of lateral roots. F. redolens is not easily differentiated from F. oxysporum f. sp. ciceris using morphology-based diagnosis, and the two species cause similar symptoms on chickpea; therefore, the use of molecular protocols should help to avoid misdiagnoses of Fusarium yellows in chickpea. © 2011 The American Phytopathological Society., Financial support for this research was provided by grants AGL2004-01231 from ‘Ministerio de Educación y Ciencia’ of Spain and the European Social Fund, AGR580 from ‘Consejería de Economía, Innovación y Ciencia’, Junta de Andalucía, and an ‘Intramural Project’ to B. B. Landa from the Spanish National Research Council (CSIC).

The proper identification and quantification of F. oxysporum populations inhabiting soil and plant rhizosphere niches are of importance for soil microbial ecology and plant pathology. In this study, we report the improvement of a PCR protocol for the specific identification of the F. oxysporum species complex and its conversion into a real-time qPCR assay for the quantification up to 1. pg of the fungus DNA in soil and different plant tissues. The amplification efficiency, sensibility and reproducibility of qPCR assays were not influenced by presence of non-target DNA from either plant or soil. The applicability of the newly developed qPCR protocol for F. oxysporum population studies was demonstrated using the technique for quantifying the fungus in different complex environmental samples. The use of the qPCR protocol allowed to accurately quantify up to 25. pg of F. oxysporum/g of naturally infested field soil, as well as to identify significant differences in the amount of F. oxysporum DNA in roots of different chickpea cultivars grown in a field soil infested with diverse pathogenic and nonpathogenic F. oxysporum populations. This qPCR protocol may be especially important for studies on soil microbial ecology and plant pathology since it provides a new opportunity for analyzing F. oxysporum populations and their interactions with the soil microflora, environment and plant host genotypes. © 2010 Elsevier B.V., Financial support for this research was provided by grants AGL2004-01231 from ‘Ministerio de Educación y Ciencia’ of Spain and the European Social Fund, AGR580 from ‘Consejería de Innovación Ciencia y Empresa’, Junta de Andalucía, and an ‘Intramural Project’ to B. B. Landa from the Spanish National Research Council (CSIC).

Montes-Borrego, M., Munoz Ledesma, F. J., Jimenez-Diaz, R. M., and Landa, B. B. 2009. A nested-polymerase chain reaction protocol for detection and population biology studies of Peronospora arborescens, the downy mildew pathogen of opium poppy, using herbarium specimens and asymptomatic, fresh plant tissues. Phytopathology 99:73-81. A sensitive nested-polymerase chain reaction (PCR) protocol was developed using either of two primer pairs that improves the in planta detection of Peronospora arborescens DNA. The new protocol represented an increase in sensitivity of 100- to 1,000-fold of detection of the oomycete in opium poppy tissue compared with the detection limit of single PCR using the same primer pairs. The new protocol allowed amplification of 5 to 0.5 fg of Peronospora arborescens DNA mixed with Papaver somniferum DNA. The protocol proved useful for amplifying Peronospora arborescens DNA from 96-year-old herbarium specimens of Papaver spp. and to demonstrate that asymptomatic, systemic infections by Peronospora arborescens can occur in wild Papaver spp. as well as in cultivated opium poppy. Also, the increase in sensitivity of the protocol made possible the detection of seedborne Peronospora arborescens in commercial opium poppy seed stocks in Spain with a high frequency, which poses a threat for pathogen spread. Direct sequencing of purified amplicons allowed alignment of a Peronospora arborescens internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequence up to 730-bp long when combining the sequences obtained with the two primer sets. Maximum parsimony analysis of amplified Peronospora arborescens ITS rDNA sequences from specimens of Papaver dubium, P. hybridum, P. rhoeas, and P. somniferum from different countries indicated for the first time that a degree of host specificity may exist within populations of Peronospora arborescens. The reported protocol will be useful for epidemiological and biogeographical studies of downy mildew diseases as well as to unravel misclassification of Peronospora arborescens and Peronospora cristata, the reported causal agents of the opium poppy downy mildew disease.

This study uses an ¿omics¿ approach to evaluate the bacterial biodiversity changes during fermentation process of natural green cracked Aloreña de Málaga table olives, from raw material to fermented fruit. For this purpose, two industries separated by almost 20 km in Guadalhorce Valley (Málaga, Spain) were analysed for obtaining both brines and fruit samples at different moments of fermentation (0, 7, 30 and 120 days). Physicochemical and microbial counts during fermentation showed the typical evolution of this type of processes, apparently dominated by yeasts. However, high-throughput barcoded pyrosequencing analysis of V2¿V3 hypervariable region of the bacterial 16S rRNA gene showed at 97% identity the presence of 131 bacterial genera included in 357 operational taxonomic units, not detected by the conventional approach. The bacterial biodiversity was clearly higher in the olives at the moment of reception in the industry and during the first days of fermentation, while decreased considerably as elapse the fermentation process. The presence of Enterobacteriaceae and Lactobacillaceae species was scarce during the four months of study. On the contrary, the most important genus at the end of fermentation was Celerinatantimonas in both brine (95.3% of frequency) and fruit (89.4%) samples, while the presence of well-known spoilage microorganisms (Pseudomonas and Propionibacterium) and halophilic bacteria (Modestobacter, Rhodovibrio, Salinibacter) was also common during the course of fermentation. Among the most important bacterial pathogens related to food, only Staphylococcus genus was found at lowfrequencies (b0.02% of total sequences). Results showthe need of this type of studies to enhance our knowledge of the microbiology of table olive fermentations. It is also necessary to determine the role played by these species not previously detected in table olives on the quality and safety of this fermented vegetable., The research leading to these results has received funding from Junta de Andalucía Government through the PrediAlo project (AGR-7755: www.predialo.science.com.es) and FEDER European funds. FNAL wishes to express thanks to the Spanish government for his RyC postdoctoral research contract while VRG would like to thank ceiA3, Spanish Government, Bank of Santander, IG-CSIC and ‘Aloreña de Málaga’ Olive Manufacturing Association for her pre-doctoral fellowship.

Haro, Carmen et al., [Context] Gut microbiota, which acts collectively as a fully integrated organ in the host metabolism, can be shaped by long-term dietary interventions after a specific diet., [Objective] The aim was to study the changes in microbiota after 1 year's consumption of a Mediterranean diet (Med diet) or a low-fat, high-complex carbohydrate diet (LFHCC diet) in an obese population., [Design] Participants were randomized to receive the Med diet (35% fat, 22% monounsaturated) and the LFHCC diet (28% fat, 12% monounsaturated)., [Setting and Participants] The study was conducted in 20 obese patients (men) within the Coronary Diet Intervention With Olive Oil and Cardiovascular Prevention (CORDIOPREV) study, an ongoing prospective, randomized, opened, controlled trial in patients with coronary heart disease., [Main Outcome Measure] We evaluated the bacterial composition and its relationship with the whole fecal and plasma metabolome., [Results] The LFHCC diet increased the Prevotella and decreased the Roseburia genera, whereas the Med diet decreased the Prevotella and increased the Roseburia and Oscillospira genera (P = .028, .002, and .016, respectively). The abundance of Parabacteroides distasonis (P = .025) and Faecalibacterium prausnitzii (P = .020) increased after long-term consumption of the Med diet and the LFHCC diet, respectively. The changes in the abundance of 7 of 572 metabolites found in feces, including mainly amino acid, peptide, and sphingolipid metabolism, could be linked to the changes in the gut microbiota., [Conclusions] Our results suggest that long-term consumption of the Med and LFHCC diets exerts a protective effect on the development of type 2 diabetes by different specific changes in the gut microbiota, increasing the abundance of the Roseburia genus and F. prausnitzii, respectively., The CORDIOPREV study is supported by the Fundación Patrimonio Comunal Olivarero, Junta de Andalucía (Consejería de Salud, Consejería de Agricultura y Pesca, Consejería de Innovación, and Ciencia y Empresa), Diputaciones de Jaén y Córdoba, Centro de Excelencia en Investigación sobre Aceite de Oliva y Salud and Ministerio de Medio Ambiente, Medio Rural y Marino, and Gobierno de España. In addition, it was partly supported by research grants from the Ministerio de Ciencia e Innovación (AGL2009–122270 to J.L.-M., FIS PI10/01041 to P.P.-M., and PI10/02412 to F.P.-J.), Ministerio de Economia y Competitividad (AGL2012/39615, PIE14/00005, and PIE14/00031 to J.L.-M.; PI13/00619 to F.P.-J.), Consejería de Economía, Innovación y Ciencia, Proyectos de Investigación de Excelencia, Junta de Andalucía (AGR922 to F.P.-J.), Consejería de Salud, Junta de Andalucía (PI0193/09 to J.L.-M., PI-0252/09 to J.D.-L., and PI-0058/10 to P.P.-M.), Consejería de Innovación Ciencia y Empresa (CVI-7450 to J.L.-M.), and Fondo Europeo de Desarrollo Regional. A.C. is supported by an Instituto de Salud Carlos III research contract (Programa Miguel-Servet CP14/00114). J.F.A.-D. is supported by an Instituto de Salud Carlos III research contract (Programa Rio-Hortega).

17 páginas, 4 tablas, 4 figuras., The microbial ecology of the nitrogen cycle in agricultural soils is an issue of major interest. We hypothesized a major effect by farm management systems (mineral versus organic fertilizers) and a minor influence of soil texture and plant variety on the composition and abundance of microbial nitrifiers. We explored changes in composition (16S rRNA gene) of ammonia-oxidizing archaea (AOA), bacteria (AOB), and nitrite-oxidizing bacteria (NOB), and in abundance of AOA and AOB (qPCR of amoA genes) in the rhizosphere of 96 olive orchards differing in climatic conditions, agricultural practices, soil properties, and olive variety. Majority of archaea were 1.1b thaumarchaeota (soil crenarchaeotic group, SCG) closely related to the AOA genus Nitrososphaera. Most AOB (97%) were identical to Nitrosospira tenuis and most NOB (76%) were closely related to Nitrospira sp. Common factors shaping nitrifiers assemblage composition were pH, soil texture, and olive variety. AOB abundance was positively correlated with altitude, pH, and clay content, whereas AOA abundances showed significant relationships with organic nitrogen content and exchangeable K. The abundances of AOA differed significantly among soil textures and olive varieties, and those of AOB among soil management systems and olive varieties. Overall, we observed minor effects by orchard management system, soil cover crop practices, plantation age, or soil organic matter content, and major influence of soil texture, pH, and olive tree variety., This research was supported by grants P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’-Junta de Andalucía, ‘Interprofesional del Aceite de Oliva Español-Fundación CITOLIVA’ and AGL-2012-37521 from the Spanish Office of Science (MINECO) to BBL,DARKNESS CGL2012-32747 (MINECO) to EOC, and Fondo Europeo de Desarrollo Regional (FEDER).

Two haplotypes of Xylella fastidiosa subsp. pauca (Xfp) that correlated with their host of origin were identified in a collection of 90 isolates infecting citrus and coffee plants in Brazil, based on a single-nucleotide polymorphism in the gyrB sequence. A new single-nucleotide primer extension (SNuPE) protocol was designed for rapid identification of Xfp according to the host source. The protocol proved to be robust for the prediction of the Xfp host source in blind tests using DNA from cultures of the bacterium, infected plants, and insect vectors allowed to feed on Xfp-infected citrus plants. AMOVA and STRUCTURE analyses of microsatellite data separated most Xfp populations on the basis of their host source, indicating that they were genetically distinct. The combined use of the SNaPshot protocol and three previously developed multilocus SSR markers showed that two haplotypes and distinct isolates of Xfp infect citrus and coffee in Brazil and that multiple, genetically different isolates can be present in a single orchard or infect a single tree. This combined approach will be very useful in studies of the epidemiology of Xfp-induced diseases, host specificity of bacterial genotypes, the occurrence of Xfp host jumping, vector feeding habits, etc., in economically important cultivated plants or weed host reservoirs of Xfp in Brazil and elsewhere.

Based on sequence data from ITS rDNA, cox1 and cox2, six Peronospora species are recognised as phylogenetically distinct on various Papaver species. The host ranges of the four already described species P. arborescens, P. argemones, P. cristata and P. meconopsidis are clarified. Based on sequence data and morphology, two new species, P. apula and P. somniferi, are described from Papaver apulum and P. somniferum, respectively. The second Peronospora species parasitizing Papaver somniferum, that was only recently recorded as Peronospora cristata from Tasmania, is shown to represent a distinct taxon, P. meconopsidis, originally described from Meconopsis cambrica. It is shown that P. meconopsidis on Papaver somniferum is also present and widespread in Europe and Asia, but has been overlooked due to confusion with P. somniferi and due to less prominent, localized disease symptoms. Oospores are reported for the first time for P. meconopsidis from Asian collections on Papaver somniferum. Morphological descriptions, illustrations and a key are provided for all described Peronospora species on Papaver. cox1 and cox2 sequence data are confirmed as equally good barcoding loci for reliable Peronospora species identification, whereas ITS rDNA does sometimes not resolve species boundaries. Molecular phylogenetic data reveal high host specificity of Peronospora on Papaver, which has the important phytopathological implication that wild Papaver spp. cannot play any role as primary inoculum source for downy mildew epidemics in cultivated opium poppy crops. © 2014 Voglmayr et al.

Downy mildew (DM) caused by the biotrophic obligate oomycete Peronospora arborescens (Berk.) is one of the most economically limiting diseases of opium poppy (Papaver somniferum L.) worldwide. The first symptoms appear as small chlorotic leaf lesions, which can evolve to curled and thickened tissues that become deformed and necrotic as the disease develops. The present study explored the use of high-resolution thermal and multi-spectral imagery as an indicator of DM infection. Work was conducted in two opium poppy field plots artificially infected by P. arborescens. Airborne thermal and multi-spectral imagery were acquired at 200 mm resolution on three dates in spring of 2009 using an unmanned aerial vehicle (UAV). Leaf reflectance and transmittance spectra of DM asymptomatic and symptomatic opium poppy leaves were measured using an integrating sphere. Simulation work was conducted with the coupled PROSPECT + SAILH radiative transfer model to assess the effects of the variability found in an opium poppy plot developing a DM epidemic on the normalized difference vegetation index (NDVI) and the green/red index (R550/R670) calculated from the multi-spectral imagery. The airborne flights enabled DM detection by using image-derived canopy temperature (Tc) normalized by air temperature (Tc − Ta) and the green/red index (R550/R670). Tmin for each grid unit was calculated to estimate pure-vegetation temperature removing background and soil effects. Tmin − Ta and R550/R670 were assessed as a function of aggregated NDVI clusters to compare asymptomatic and symptomatic plants normalized by similar growth levels. Results demonstrated that Tc − Ta and the R550/R670 index were related to physiological stress caused by DM infection. In addition, Tmin − Ta was found to decrease as the NDVI increased and symptomatic plants reached significantly higher (P, Financial support for this research was provided by the Spanish Ministry of Education and Science through projects AGL2009-13105, AGL2012-40053-C03-01 and PET2006_0444, the Regional Government of Andalusia through project P10-AGR-6497 and the European Union through the European Regional Development Fund (ERDF). R. Calderón was supported by research fellowship BES-2010-035511 from the Spanish Ministry of Education and Science.

Background: In the last years, many olive plantations in southern Spain have been mediated by the use of self-rooted planting stocks, which have incorporated commercial AMF during the nursery period to facilitate their establishment. However, this was practised without enough knowledge on the effect of cropping practices and environment on the biodiversity of AMF in olive orchards in Spain. Methodology/Principal Findings: Two culture-independent molecular methods were used to study the AMF communities associated with olive in a wide-region analysis in southern Spain including 96 olive locations. The use of T-RFLP and pyrosequencing analysis of rDNA sequences provided the first evidence of an effect of agronomic and climatic characteristics, and soil physicochemical properties on AMF community composition associated with olive. Thus, the factors most strongly associated to AMF distribution varied according to the technique but included among the studied agronomic characteristics the cultivar genotype and age of plantation and the irrigation regimen but not the orchard management system or presence of a cover crop to prevent soil erosion. Soil physicochemical properties and climatic characteristics most strongly associated to the AMF community composition included pH, textural components and nutrient contents of soil, and average evapotranspiration, rainfall and minimum temperature of the sampled locations. Pyrosequencing analysis revealed 33 AMF OTUs belonging to five families, with Archaeospora spp., Diversispora spp. and Paraglomus spp., being first records in olive. Interestingly, two of the most frequent OTUs included a diverse group of Claroideoglomeraceae and Glomeraceae sequences, not assigned to any known AMF species commonly used as inoculants in olive during nursery propagation. Conclusions/Significance: Our data suggests that AMF can exert higher host specificity in olive than previously thought, which may have important implications for redirecting the olive nursery process in the future as well as to take into consideration the specific soils and environments where the mycorrhized olive trees will be established. © 2014 Montes-Borrego et al., This research was supported by grants from Projects AGL2008-00344/AGR from ‘Ministerio de Ciencia e Innovación’, Project P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’ of Junta de Andalucía, Project AGL-2012-37521 from ‘Ministerio de Economía y Competitividad’ of Spain, and Fondo Europeo de desarrollo regional (FEDER) >Una manera de hacer Europa> from the European Union. The grant 219262 ArimNET_ERANET FP7 2012–2015 Project PESTOLIVE from Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), also provided partial financial support. M. Montes-Borrego enjoyed a contract from ‘Consejería de Economía, Innovación y Ciencia’ of Junta de Andalucía, Spain.

Beauveria bassiana strain 04/01-Tip obtained from larvae of the opium poppy stem gall Iraella luteipes endophytically colonizes opium poppy plants and protect it against this pest. Development of a specific, rapid and sensitive technique that allows accurately determining the process and factors leading to the establishment of this strain in opium poppy plants would be essential to achieve its efficient control in a large field scale. For that purpose in the present study, species-specific primers that can be used in conventional or quantitative PCR protocols were developed for specifically identification and detection of B. bassiana in plant tissues. The combination of the designed BB.fw/BB.rv primer set with the universal ITS1-F/ITS4 primer set in a two-step nested-PCR approach, has allowed the amplification of up to 10. fg of B. bassiana. This represented an increase in sensitivity of 10000- and 1000-fold of detection than when using the BB.fw/BB.rv primers in a single or single-tube semi-nested PCR approaches, respectively. The BB.fw and BB.rv primer set were subsequently optimized to be used in real time quantitative PCR assays and allowed to accurately quantify B. bassiana DNA in different plant DNA backgrounds (leaves and seeds) without losing accuracy and efficiency. The qPCR protocol was used to monitor the endophytic colonization of opium poppy leaves by. B. bassiana after inoculation with the strain EABb 04/01-Tip, detecting as low as 26. fg of target DNA in leaves and a decrease in fungal biomass over time. PCR quantification data were supported in parallel with CLMS by the monitoring of spatial and temporal patterns of leaf and stem colonization using a GFP-tagged transformant of the B. bassiana EABb 04/01-Tip strain, which enabled to demonstrate that B. bassiana effectively colonizes aerial tissues of opium poppy plants mainly through intercellular spaces and even leaf trichomes. A decline in endophytic colonization was also observed by the last sampling times, i.e. from 10 to 15. days after inoculation, although fungal structures still remained present in the leaf tissues. These newly developed molecular protocols should facilitate the detection, quantification and monitoring of endophytic B. bassiana strains in different tissues and host plants and would help to unravel the factors and process governing the specific endophytic association between opium poppy and strain EABb 04/01-Tip providing key insights to formulate a sustainable strategy for I. luteipes management in the host. © 2013 Elsevier Inc., This research was supported by project AGR-7681 from the Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía, Spain and by ALCALIBER I+D+i S.L.U.

Management of soilborne fungal plant pathogens in sustainable cropping systems should be based on strategies that integrate all available control measures. The use of PGPR is recognized as one of the most promising methods for controlling soilborne fungal plant pathogens. However, we are currently far away of being able to understand and exploit the full potential of PGPR as an effective disease management strategy at field scale. Both, abiotic and biotic factors modulate metabolic processes of plants and microorganisms within their respective biological limits, and consequently may modify interactions among them in a given pathosystem. It is well documented that incidence and severity of plant diseases are modulated by the soil environment and that the efficacy of some PGPR biocontrol agents decrease as conditions become more favorable for disease development. Soil biotic and abiotic factors may influence biological control mediated by PGPR by either (1) predisposing pathogens to microbial antagonism, (2) regulating the growth or production of metabolites by specific antagonists, or (3) modulating disease development and consequently the level of disease suppression achieved. To complicate more the scenario, there is global observational evidence that many natural biological systems are being affected by regional climate changes. Many environmental factors associated to climate change, particularly temperature increase, may influence the mechanisms of biocontrol mediated by PGPR, but there is little knowledge of how those specific factors may affects the interactions among soilborne fungal pathogens and PGPR. Research on the effects of climate change on plant diseases is limited, especially on biocontrol. Currently, the use of climate risk mapping tools may facilitate the geographic assessment of potential pathogen and PGPR distribution and how those factors may interact under the different scenarios of climatic change and may help in designing new strategies for the efficient use and improved performance of well-performing PGPR as effective biocontrol agents of soilborne fungal plant pathogens.

Classical and molecular methods were used to study the nematode communities associated with rhizosphere soil and roots of a collection of 16 olive cultivars from a world olive germplasm bank in Mengibar (Jaen province, southern Spain). Classical nematological analysis, including soil nematode extraction, species counting and morphological identification showed that 24 taxa belonging to 9 genera (including Aphelenchoides, Criconemoides, Ditylenchus, Filenchus, Helicotylenchus, Merlinius, Paratylenchus, Tylenchus, and Xiphinema) and 8 families (including Anguinidae, Aphelenchidae, Belonolaimidae, Criconematidae, Hoplolaimidae, Longidoridae, Tylenchidae and Tylenchulidae) of plant-parasitic nematodes were present, with one species (Helicotylenchus digonicus) being prevalent in all samples. The low values of the plant-parasitic nematode index (PPI) indicated a high disturbance of the field soil probably due to application of herbicides and fertilizers. Cluster analysis of population densities of the various nematode species, nematode trophic groups, and ecological indices grouped most olive cultivars into three main clusters indicating that olive genotypes differ in the nematode communities in their rhizosphere soil. The use of T-RFLP analysis discriminated to a higher extent the nematode communities present in the rhizosphere soil from the different olive cultivars as compared to the morphological-based analysis. This study provides the first evidence of an effect of the olive genotype on nematode community composition by combining classical morphological and molecular approaches. © 2011 Elsevier Ltd., Authors gratefully acknowledge Projects AGL2008-00344/AGR and HA2008-0014 from ‘Ministerio de Ciencia e Innovación’, Project P10-AGR-5908 from ‘Consejería de Economía, Innovación y Ciencia’ of Junta de Andalucía and FEDER financial support from the European Union.

Bacteria have evolved mechanisms that allow them to grow and survive in highly competitive environments like soil and the rhizosphere. Using classical microbiological, physiological, and genetic analyses, we isolated and identified for the first time Duganella spp. associated with the rhizosphere of woody plants in Mediterranean environments that are able to produce violacein, a blue-purple secondary metabolite of considerable biotechnological interest. Based on physiological and biochemical characterization and phylogenetic analysis of different genes including 16S rRNA, gyrB, and vioA (implicated in the synthesis of violacein), the seven Duganella spp. strains isolated and studied were differentiated according to their host of origin (wild versus cultivated olives) and potentially might belong to new species. All the Duganella spp. strains produced violacein in vitro, with natural production levels significantly higher than that previously reported for other violacein-producing bacteria without optimizing growing conditions. The important biological, medical, and industrial applications of violacein make these bacteria good candidates for their biotechnological exploitation because low violacein yields are considered as one of the main limitations of using wild-type strains for extensive exploitation and pigment production. Independent of violacein production, purple-pigmented strains from olives showed proteolytic and lipolytic activities and a weak siderophore production. No in vitro inhibitory activity was demonstrated for bacteria or crude violacein filtrates against plant-pathogenic Gram-negative bacteria and fungi, but they did inhibit Gram-positive bacteria. © 2011 Springer Science+Business Media, LLC., This research was supported by grants AGL2008-00344 and HA2008-0014 from ‘Ministerio de Ciencia e Innovación’ of Spain and the European Social Fund and PI2007-40I012 ‘Intramural Project’ to B. B. Landa from the Spanish National Research Council (CSIC).

In this study, we developed a reliable, quick, and accurate quantitative polymerase chain reaction (qPCR) assay based on the MIQE (Minimum Information for publication of Quantitative Real-Time PCR Experiments) guidelines for the quantification of Peronospora arborescens in infected downy mildew-symptomless opium poppy (Papaver somniferum) tissues and commercial seed stocks. The protocol was highly reproducible and allowed accurate quantification of pathogen DNA up to 10 fg in different plant DNA backgrounds without losing specificity and efficiency. Moreover, to further overcome difficulties conferred by the strict biotrophy of this pathogen, we developed dilution series of DNA extracted from a plasmid with the target pathogen DNA as a cloned insert. This facilitated the demonstration of the robustness of the protocol in different laboratories with different qPCR equipment and reagents, which may help in its use on a broad scale. Finally, we validated the usefulness of the qPCR protocol for quarantine purposes and downy mildew resistance screening by quantifying P. arborescens in complex, naturally infested opium poppy samples. Thus, a pathogen biomass of 0.0003 to 0.007%o or of 0.110 to 5,557 ppm was quantified in symptomless capsules in commercial seed stocks, or in stem samples from symptomless opium poppy plants systemically infected by the pathogen, respectively. © 2011 The American Phytopathological Society., Financial support for this research was provided by grants PET2006_0444 from ‘Ministerio de Educación y Ciencia’ of Spain and the European Social Fund, and from an ‘Intramural Project’ to B. B. Landa from the Spanish Council for Research (CSIC) and from ALCALIBER I+D+i, S.L.

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infestedwith several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.© 2011 The American Phytopathological Society., Financial support for this research was provided by grants AGL2004- 01231 from ‘Ministerio de Educación y Ciencia’ of Spain and the European Social Fund, AGR580 from ‘Consejería de Economía, Innovación y Ciencia’, Junta de Andalucía, and an ‘Intramural Project’ to B. B. Landa from the Spanish National Research Council (CSIC).

Wild olive trees, namely oleaster, are considered the ancestor of cultivated olive and a unexplored source of genetic variability that might contain important traits of agronomic and biotechnological interest. The longevity and genetic diversity of oleasters may have favoured selection of specific and well adapted rhizosphere microbial populations that can constitute unique reservoirs of microbial antagonists of Verticillium dahliae, the main soilborne fungal pathogen of olive worldwide. The objective of this present study was to determine the structure and diversity of bacterial communities in the rhizosphere and endosphere of oleaster from 11 havens in Cádiz and Córdoba provinces of Andalusia, southern Spain. To carry out the study we used a multiphasic approach. First, the occurrence and diversity of rhizosphere bacteria was monitored by a cultivation-independent-approach, using fluorescent terminal restriction fragment length polymorphism (FT-RFLP) analyses of amplified 16S rDNA sequences. FT-RFLP patterns revealed a high heterogeneity in the composition of the sampled rhizosphere bacterial communities and suggested the existence of plant genotype-site-specific communities, with each oleaster haven being a unique reservoir of bacterial diversity. Secondly, to investigate the antagonistic potential of these root-associated bacterial populations, a total of 675 bacterial isolates obtained from oleaster rhizosphere and endosphere were screened by dual testing for inhibition of in vitro growth of the highly virulent, olive defoliating pathotype of V. dahliae. Out of 675 tested bacterial isolates, 94 (14%) showed a strong antagonistic activity against a defoliating V. dahliae pathotype. Of the antagonistic bacteria, a slightly lower proportion (12.9% of total bacteria) were inhabitant of the oleaster rhizosphere compared to that in the endosphere (16.5%). The biotechnological potential of those isolates was assessed by in vitro production of different hydrolytic enzymes, indole-1.3-acetic acid (IAA), siderophores, and antimicrobial compounds. Overall, most of bacterial antagonists (58.5 to 78.3%) showed proteolytic, lipolytic, and chitinolytic activity, and produced IAA and siderophores. Finally, analysis of the 16S rDNA gene sequence indicated that most of the 94 bacterial antagonists belong to genera Bacillus (56.4%), Pseudomonas (27.7%), and Paenibacillus (7.4%). Overall, the rhizosphere and endosphere of wild olives were proved as a good reservoir of bacteria antagonists against V. dahliae. Several of those bacteria showing high and broad antagonism potential may therefore be considered for further analyses as promising biocontrol agents against V. dahliae in olive. © 2011 Springer Science+Business Media B.V., This research was supported by grants AGL2008-00344 and HA2008-0014 from ‘Ministerio de Ciencia e Innovación’ of Spain and the European Social Fund.

Severe downy mildew diseases of opium poppy (Papaver somniferum) can be caused by Peronospora arborescens and P. cristata, but differentiating between the two pathogens is difficult because they share morphological features and a similar host range. In Spain, where severe epidemics of downy mildew of opium poppy have occurred recently, the pathogen was identified as P. arborescens on the basis of morphological traits. In this current study, sequence homology and phylogenetic analyses of the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) were carried out with DNA from P. arborescens and P. cristata from diverse geographic origins, which suggested that only P. arborescens occurs in cultivated Papaver somniferum in Spain. Moreover, analyses of the rDNA ITS region from 27 samples of downy-mildew-affected tissues from all opium-poppy-growing regions in Spain showed that genetic diversity exists within P. arborescens populations in Spain and that these are phylogenetically distinct from P. cristata. P. cristata instead shares a more recent, common ancestor with a range of Peronospora species that includes those found on host plants that are not members of the Papaveraceae. Species-specific primers and a PCR assay protocol were developed that differentiated P. arborescens and P. cristata and proved useful for the detection of P. arborescens in symptomatic and asymptomatic opium poppy plant parts. Use of these primers demonstrated that P. arborescens can be transmitted in seeds and that commercial seed stocks collected from crops with high incidence of the disease were frequently infected. Field experiments conducted in microplots free from P. arborescens using seed stocks harvested from infected capsules further demonstrated that transmission from seedborne P. arborescens to opium poppy plants can occur. Therefore, the specific-PCR detection protocol developed in this study can be of use for epidemiological studies and diagnosing the pathogen in commercial seed stocks; thus facilitating the sanitary control of the disease and avoidance of the pathogen distribution in seeds.