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The bacterium Xylella fastidiosa (Xf) is a plant pathogen that can block the flow of water and nutrients through the xylem. Xf symptoms may be confounded with generic water stress responses. Here, we assessed changes in biochemical, biophysical and photosynthetic traits, inferred using biophysical models, in Xf-affected almond orchards under rainfed and irrigated conditions on the Island of Majorca (Balearic Islands, Spain). Recent research has demonstrated the early detection of Xf-infections by monitoring spectral changes associated with pigments, canopy structural traits, fluorescence emission and transpiration. Nevertheless, there is still a need to make further progress in monitoring physiological processes (e.g., photosynthesis rate) to be able to efficiently detect when Xf-infection causes subtle spectral changes in photosynthesis. This paper explores the ability of parsimonious machine learning (ML) algorithms to detect Xf-infected trees operationally, when considering a proxy of photosynthetic capacity, namely the maximum carboxylation rate (Vcmax), along with carbon-based constituents (CBC, including lignin), and leaf biochemical traits and tree-crown temperature (Tc) as an indicator of transpiration rates. The ML framework proposed here reduced the uncertainties associated with the extraction of reflectance spectra and temperature from individual tree crowns using high-resolution hyperspectral and thermal images. We showed that the relative importance of Vcmax and leaf biochemical constituents (e.g., CBC) in the ML model for the detection of Xf at early stages of development were intrinsically associated with the water and nutritional conditions of almond trees. Overall, the functional traits that were most consistently altered by Xf-infection were Vcmax, pigments, CBC, and Tc, and, particularly in rainfed-trees, anthocyanins, and Tc. The parsimonious ML model for Xf detection yielded accuracies exceeding 90% (kappa = 0.80). This study brings progress in the development of an operational ML framework for the detection of Xf outbreaks based on plant traits related to photosynthetic capacity, plant biochemistry and structural decay parameters., This research was supported by grant: ITS2017-095: Design and Implementation of control strategies for Xylella fastidiosa, Project 5. Government of the Balearic Islands, Spain. Data collection was partially supported by the European Union's Horizon 2020 research and innovation program through gran agreement XF-ACTORS (727987).

Trabajo presentado en el Simposium Científico Técnico Expoliva, celebrado online del 22 al 25 de septiembre de 2021., Los patógenos vasculares son los agentes causales de las principales enfermedades que amenazan la salud y la producción olivarera en todo el mundo. El uso de microorganismos endofíticos representa una estrategia desafiante y prometedora para el manejo de las enfermedades vasculares en el olivo. Aunque la investigación actual se ha centrado en analizar la estructura y diversidad de las comunidades microbianas que habitan el xilema del olivo, la caracterización de este nicho ecológico ha sido pasada por alto hasta la fecha. En este trabajo, se seleccionaron plantones jóvenes y olivos adultos de los genotipos 'Picual' y 'Arbequina' para caracterizar la composición química de la savia del xilema, así como su diversidad microbiana con el objetivo de mejorar la comprensión de los requisitos nutricionales de los microorganismos que habitan el xilema, incluyendo sus patógenos vasculares, para permitir el diseño de medios de crecimiento artificiales que ayuden a mejorar el cultivo del microbioma del olivo.

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

Trabajo presentado en la 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021., Xylella fastidiosa (Xf) causes plant diseases that lead to massive economic losses in agricultural crops, making it one of the pathogens of greatest concern to agriculture nowadays. Detecting Xf at early stages of infection is crucial to prevent and manage outbreaks of this vector-borne bacterium. Recent remote sensing (RS) studies at different scales have shown that Xf-infected olive trees have distinct spectral features in the visible and infrared regions (VNIR). However, RS-based forecasting of Xf outbreaks requires tools that account for their spatiotemporal dynamics. Here, we show how coupling a spatial Xf-spread model with the probability of Xf-infection predicted by an RS-driven modeling algorithm based on a Support Vector Machine (RS-SVM) helps detecting the spatial Xf distribution in a landscape. To optimize such model, we investigated which RS plant traits (i.e., pigments, structural or leaf protein content) derived from high-resolution hyperspectral imagery and biophysical modelling are most responsive to Xf infection and damage. For that, we combined a field campaign in almond orchards in Alicante province (Spain) affected by Xf (n=1,426 trees), with an airborne campaign over the same area to acquire high-resolution thermal and hyperspectral images in the visible-near-infrared (400-850 nm) and short-wave infrared regions (SWIR, 950-1700 nm). We found that coupling the epidemic spread model and the RS-based model increased accuracy by around 5% (OA = 80%, kappa = 0.48 and AUC = 0.81); compared to the best performing RS-SVM model (OA = 75%; kappa = 0.50) that included as predictors leaf protein content, nitrogen indices (NIs), fluorescence and a thermal indicator, alongside pigments and structural parameters. The parameters with the greatest explanatory power of the RS model were leaf protein content together with NI (28%), followed by chlorophyll (22%), structural parameters (LAI and LIDFa), and chlorophyll indicators of photosynthetic efficiency. In the subset of almond trees where the presence of Xf was tested by qPCR (n=318 tress), the combined RS-spread model yielded the best performance (OA of 71% and kappa = 0.33). Conversely, the best-performing RS-SVM model and visual inspections produced OA and kappa values of 65% and 0.31, respectively. This study shows for the first time the potential of combining spatial epidemiological models and remote sensing to monitor Xf-disease distribution in almond trees.

Trabajo presentado en la 2nd European conference on Xylella fastidiosa (how research can support solutions), celebrada en Ajaccio el 29 y 30 de octubre de 2019., An outbreak of Xylella fastidiosa (Xf) subsp. multiplex ST6 was first identified in June 2017 in Alicante province (Valencian Community, Spain) affecting mainly almond trees, but also other species including ornamentals, cultivated Prunus spp. and landscape plants. The current demarcated area (DA) in Alicante covers 134,581 hectares, and affects more than 70 municipalities. Genetic analysis may provide evidence of biological, ecological and host-range diversity among Xf strains of the same subspecies and STs and help to elucidate the pathway of introduction and spread in an affected area. Pairwise comparisons of the chromosomal genomes of two ST6-sequenced strains isolated from almond trees in Alicante, ESVL and IVIA5901, showed an average nucleotide identity higher than 99.9%. Interestingly, the two strains differ for the presence of the plasmids pXF64-Hb_ESVL and pUCLA-ESVL detected only in the ESVL strain. The aim of this study was to determine the incidence and distribution of plasmid-bearing strains of Xf subsp. multiplex ST6 in Alicante. PCR tests were performed on 20 strains isolated from different hosts, and on approximately 100 DNA samples from infected almond trees collected in eight municipalities within the DA of Alicante. PCR results on the cultured isolates showed the occurrence of ST6-strains harbouring the two plasmids, or only the plasmid pUCLA-ESVL or none of them. Interestingly, in some of the DNA samples from infected almonds, only the plasmid pXF64-Hb_ESVL could be detected. More specifically, 4% of ST6-infected samples harboured the plasmid pXF64-Hb_ESVL, 12% only pUCLA-ESVL, 16% both plasmids, and 44% neither of the two plasmids. In five of the eight municipalities, we found samples harbouring the two plasmids, and only in the samples from the municipality of Benifato the PCR tests failed to amplify the targeted plasmids. Future work will increase the number of samples to cover the entire DA to better understand and relate the presence of plasmids to the epidemiology of this disease in combination with the use of multilocus sequence typing and genome sequencing of more Xf subsp. multiplex ST6 strains., The present work was presented in the framework of the Joint Annual Meeting of the EU Horizon 2020 Projects POnTE ‘Pest Organisms Threatening Europe' (GA 635646) and XF-ACTORS ‘Xylella fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy' (GA 727987).

Trabajo presentado en la 2nd European conference on Xylella fastidiosa (how research can support solutions), celebrada en Ajaccio el 29 y 30 de octubre de 2019., A first case of infection caused by Xylella fastidiosasubsp. multiplexST6 was detected in an olive tree in the municipality of Villarejo de Salvanés, in the southeast of the Community of Madrid (Spain) in April 2018. This constituted the first detection of X. fastidiosain this crop on the Iberian Peninsula, and the second concerning other crops after the detection of the pathogen in almondtrees in Alicante, in the Valencian Community (Spain), where X. fastidiosasubsp. multiplexST6 was also identified, although until now it has not been detected causing infection in olive trees in that region.The Community of Madrid accounts for 27,000 hectares of olive trees, most of them in Villarejo de Salvanés. This area has a continental climate, with below zero minimum winter temperatures. After the official declaration of the outbreak, the actions established in the European Decision 2017/2352, theContingency Plan against X. fastidiosaof the Spanish Ministry of Agriculture, Fisheries and Food, and that of the Community of Madrid were immediately applied to eradicate the bacterium and prevent its spread.Among these measures, around 2,000 samples from olive trees and other host plants of the demarcated area (DA), 300 insect vectors, and olive trees from other municipalities were analysed. All of them gave a negative result by real-time PCR official protocols. As a direct consequence of the application of the eradication measures, the results obtained so far would indicate that this detection was a unique event and the bacterium would be neither disseminated nor established in the EU, although surveillance measures within the DA must continue as established in the Action Plan of the Community of Madrid.

Trabajo presentado en la European Conference on Xylella 2017 (Finding answers to a global problem), celebrada en Palma de Mallorca del 13 al 15 de noviembre de 2017., The spatial dynamics of Olive Quick Decline Syndrome (OQDS) caus ed by X. fastidiosa subsp. pauca were determined in 20 olive plots in a selected olive growing area within the infected zone of the Lecce province, Puglia by assessing disease incidence (DI) and severity (DS) (0-5 rating scale) in June, 2016. Eight of the 20 plots with a wide range of initial DI and DS values were selected to assess the spatial and temporal dynamics of the OQDS and were evaluated in October 2016 and February 2017. Analyses are in progress and include the use of the Spatial Analysis by Distance Indices (SADIE) (Perry et al., 1999) to quantify the spatial pattern of OQDS. Spatial association between time periods was determined by the SADIE association index (Winder et al., 2001). Spatial pattern of symptomatic trees was estimated as regular (22.2% of plots), non-aggregated (33.3% of plots) or aggregated (44.4% of plots). Overall, both, DI and DS increased in the second and third assessments as compared to the initial score in June 2016 by 28.7±4.2% and 2.02±5.1% in DI, respectively; and by 0.53 ±0.27 and 0.04±0.06 in DS, respectively. Spatial pattern was characterized by the occurrence of several clusters of diseased trees. Increasing clustering over time was indicated by stronger values of the clustering index and the increase in patch cluster size. Spatial association was also found in the clustering of diseased trees between evaluation periods., This work has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N. 635646 “Pest Organisms Threatening Europe POnTE”. Research supported by EU grant 635646 POnTE.

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Trabajo presentado en la European Conference on Xylella 2017 (Finding answers to a global problem), celebrada en Palma de Mallorca del 13 al 15 de noviembre de 2017., With the goal of understanding the nutritional requirements of xylem-limited microbiome, the chemical composition of xylem sap extracted from two Olea europaea var. europaea cultivars (Picual and Arbequina) was evaluated. Selected plants were established on their own feet or grafted onto rootstocks of Olea europaea var. sylvestris. Xylem sap of olives branches was collected using a external 45-cm external Scholander Super Chamber. Metabolome analysis was performed by proton nuclear magnetic resonance (NMR) spectroscopy-based study and the ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy (ICP-OES). In total 26 metabolites were identified by NMR spectroscopy, including amino acids (alanine, arginine, aspartate, glutamate, glutamine, isoleucine, leucine, methionine, proline, threonine, tyrosine, and valine), sugars (glucose, fructose, mannitol, myo-inositol, and sucrose), organic acids (formic, fumaric and succinic acid), alcohols (ethanol and methanol), and other molecules. On the other hand ICP-OES allowed the quantification of 14 elements and five inorganic anions. Xylem sap analysis will lead to a better understanding of the biology and complex nutritional requirements of olive xylem-inhabiting microorganisms, including Xylella fastidiosa, and to help designing artificial growing media to improve culturing of the olive microbiome., Study supported by Projects 727987 XF-ACTORS (EU-H2020) & AGL2016-75606-R (MEIC Spain and FEDER-EU).

Xylella fastidiosa es una bacteria de cuarentena en la Unión Europea (UE) (su introducción está explícitamente prohibida por no estar presente en la 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, así como por su forma de transmisión a través de insectos vectores de diversa naturaleza, y por la existencia en Europa de condiciones climáticas que favorecerían su desarrollo epidémico. Además, la bacteria posee un enorme potencial patogénico ya que infecta y causa enfermedad grave a diferentes cultivos agrícolas de gran importancia económica, incluyendo al almendro, cítricos, melocotón, olivo y vid, así como a una gran variedad de árboles y plantas no cultivadas, como arces, adelfas, y diferentes especies silvestres y forestales típicas del bosque mediterráneo, tales como quercus, olmos, jara y retama, entre otros. Esto hace que a esta bacteria se la considere uno de los organismos fitopatógenos de más riesgo para la Unión Europea.

Xylella fastidiosa es una bacteria de cuarentena en la Unión Europea (bacteria incluida en la Lista A1 EPPO desde 1981), 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, así como por su forma de transmisión a través de insectos vectores de diversa naturaleza, y por la existencia en Europa de condiciones climáticas que favorecerían su desarrollo epidémico. Además, la bacteria posee un enorme potencial patogénico ya que infecta y causa enfermedad severa en diferentes cultivos agrícolas de gran importancia económica, incluyendo el almendro, cítricos, melocotón, olivo y vid, así como una gran variedad de árboles y plantas no cultivadas como arces, adelfas, y diferentes especies silvestres y forestales típicas del bosque mediterráneo como Quercus, olmos, jara, y retama, entre otros. Esto hace que a esta bacteria se la considere uno de los organismos fitopatógenos de más riesgo para la Unión Europea.

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., Regional Government of Andalusia (project P10-AGR-6497) to MMB; European Union through the European Regional Development Fund (ERDF).

Trabajo presentado en el 15th Congress of the Mediterranean Phytopathological Union (Plant health sistaining Mediterranean ecosystems), celebrado en Córdoba (España) del 20 al 23 de junio de 2017.-- Olmo, D. et al., Xylella fastidiosa is a quarantine organism in the European Union (EU), that was first detected in Europe in Italy in 2013 where it is associated to a severe epidemic on olive trees., This study was supported by funding from the European Union’s Horizon 2020 research and innovation programme, under grant agreements No. 635646 POnTE (Pest Organisms Threatening Europe) and No. 727987 XF-ACTORS (Xylella Fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy).

Trabajo presentado en la European Conference on Xylella 2017 (Finding answers to a global problem), celebrada en Palma de Mallorca del 13 al 15 de noviembre de 2017., A remote sensing campaign carried out in Puglia in summer 2016 collected hyperspectral and thermal images of ca 200,000 olive trees at sub-meter resolution. In the 1200 ha study area within the X. fastidiosa infected zone, 3500 trees were simultaneously evaluated in the field for severity of X. fastidiosa symptoms. The hyperspectral sensor used in this experiment acquired data in the visible and near-infrared spectral region (400-885 nm) with 260 bands of 6.5 nm FWHM at 1.85 nm/pixel and 12-bit radiometric resolution. The sensor was radiometrically calibrated in the laboratory, and images atmospherically corrected to obtain surface reflectance using total incoming irradiance measured in the field. The high resolution hyperspectral and thermal imagery acquired over the orchards allowed the delineation of individual trees using object-based crown detection algorithms. Crown temperature and hyperspectral indices were calculated for each tree to classify disease severity levels using different machine learning algorithms, including linear discriminant analysis, support vector machines and neural networks. The success and applicability of these early detection methods to other areas will be discussed in the context of a new airborne campaign planned in July 2017 over X. fastidiosa infected zones in Mallorca. In this new airborne campaign the impact of X. fastidiosa symptoms on crown reflectance will also be evaluated in the 800-1700 nm spectral region., This work was supported by JRC and has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N. 635646 “Pest Organisms Threatening Europe POnTE” and grant agreement N. 727987 “Xylella fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy XF-ACTORS”.

Trabajo presentado en el 1st Congreso Ibérico de Livicultura, las V Jornadas Nacionales del Grupo de Olivicultura de la Sociedad Española de Ciencias Hortícolas (SECH) y el VII Simpósio Nacional de Olivicultura de la Associação Portuguesa de Horticultura (APH), celebrado en Badajoz del 13 al 15 de abril de 2016., Xylella fastidiosa es una bacteria Gram-negativa, habitante del xilema, de muy lento crecimiento in vitro que se transmite de forma no específica por varias especies de insectos que se alimentan del xilema incluidos en Cicadélidos (Hemiptera: Cicadellidae: Cicadellinae) y Cercópidos (Hemiptera: Cercopidea). Esta bacteria causa grandes pérdidas económicas y es el agente causal de la enfermedad Pierce de la vid PD, Vitis vinifera; el enanismo del melocotonero, Prunus persica; la clorosis variegada de los cítricos (CVC) en Citrus spp., entre otras. Las epidemias de X. fastidiosa han dado lugar a pérdidas severas en cítricos en Sudamérica y vid en Norteamérica desde hace décadas. Hasta hace poco, X. fastidiosa solo se había detectado infectando olivo en California, EEUU y en las regiones de La Rioja y Córdoba en Argentina. Sin embargo, en octubre de 2013, una cepa de X. fastidiosa ha sido asociada con un decaimiento rápido de olivo (OQDS) que está causando devastación en la provincia de Lecce, Apulia, Italia, donde está afectando actualmente a más de 10.000 has. La monitorización exhaustiva de todo el territorio de los estados miembros de la UE siguiendo la normativa de la Decisión (EU) 2015/789 ha demostrado también la presencia de X. fastidiosa en Francia en la isla de Córcega y diversas localidades de la Costa Azul (región de los Alpes Marítimos). En esta charla se dará una visión general de los principales avances científicos que se han producido en relación a las cepas de X. fastidiosa interceptadas o presentes en Europa, haciendo un especial énfasis en la variante genética que afecta a olivo. Se presentará el estado actual de la epidemia en olivo en la región de Apulia, las opciones de contención y control de la enfermedad, y los posibles riesgos existentes para la sostenibilidad del olivar. Finalmente se presentarán los objetivos científicos que se van a abordar en el Proyecto de Investigación POnTE: Pest Organisms Threathening Europe financiado por la UE en su programa H2020 que pretende contribuir a diseñar una estrategia europea para la gestión integrada de la enfermedad, con el objetivo de desarrollar las medidas de prevención, detección, contención y erradicación más adecuadas desde el punto de vista técnico, económico y medioambiental.

Trabajo presentado en el XVIII Congreso de la Sociedad Española de Fitopotología, celebrado en Palencia del 20 al 23 de septiembre de 2016., Xylella fastidiosa (Xf) es una bacteria Gram-negativa de cuarentena en la Unión Europea, que crece restringida en el xilema de las plantas y presenta a menudo variantes patogénicas específicas (subspecies) de ciertos huéspedes. Xf causa importantes enfermedades y pérdidas de rendimientos en diferentes cultivos y plantas forestales y ornamentales, llegando a infectar más de 340 especies vegetales. La asignación genotípica de Xf a nivel de subespecie es necesaria para permitir realizar inferencias acerca de la biología de los aislados y/o su procedencia en el momento que se realiza alguna interceptación de esta bacteria en un país donde no está presente. En este estudio se ha desarrollado un nuevo protocolo basado en la minisecuención, que consiste en una reacción de extensión de un cebador en un único nucleótido (SNuPE), utilizando cuatro cebadores simultáneamente, y que permite la identificación de todas las subespecies de Xf (subsp. fastidiosa, multiples, sandyi y pauca) y de tres genotipos de Xf subsp. pauca, incluyendo la cepa CoDiRO causante de la enfermedad de decaimiento rápido del olivo en el sur de Italia. El protocolo resultó robusto para la predicción de todas las subespecies y genotipos de Xf en pruebas ciegas realizadas utilizando ADN extraído de: i) cultivos puros de Xf, ii) diferentes plantas infectadas por Xf de forma artificial o natural, y iii) insectos vectores portadores de Xf. Este protocolo SNuPE múltiple puede ser muy útil como una primera metodología de análisis rápida y robusta para la asignación taxonómica de cepas de Xf a nivel de subespecie o genotipo (Xf subsp. pauca), en estudios epidemiológicos de campo o para fines de cuarentena cuando se intercepte material infectado por la bacteria., Investigación financiada por el Proyecto POnTE H2020-SFS-2014-2015 Sustainable Food Security call (Topic SFS-03a-2014: Native and alien pests in agriculture and forestry).

Trabajo presentado en el 32nd Symposium European Society of Nematologists, celebrado en Braga del 28 de agosto al 1 de septiembre de 2016., This work has studied the diversity of plant-parasitic nematodes infecting olive in a wide-region in southern Spain (Andalusia). Our data indicate that there is a high diversity of plant-parasitic nematodes (including root-knot = Meloidogyne spp., root-lesion = Pratylenchus spp., cystforming = Heterodera mediterranea, stubby- root = Trichodorus spp., reniform = Rotylenchulus sp., dagger and needle = Longidorus spp., Xiphinema spp., ring = Criconemoides spp., Ogmo spp., and other migratory ectoparasitic nematodes viz. Amplimerlinius spp., Helicotylenchus spp., Neodolichorhynchus spp., Paratrophurus spp. Paratylenchus spp., Rotylenchus spp., Trophurus sp., Tylenchorhynchus spp.) associated with olive in Southern Spain that can exert different damage to olive roots depending on the olive variety and their abundance. The highest number of species was detected in Xiphinema and Longidorus, with description of eight and seven new species, respectively, apart from several new records for Spain. In addition, seven Meloidogyne species were detected in wi ld and cultivated olive, from which one is being described as a new species. Multivariate analyses allowed determining the agronomic and environmental factors that drive the structure and diversity of plant-parasitic nematodes infecting olive in Southern-Spain, that included: soil physicochemicol factors (texture, pH, sand and doy, and extractable K), climatic variables (mínimum and maximum temperatures) and the olive cultivar. Nematode abundance and diversity indexes (Richness, Shannon, Evenness and plant-parasitic index) were influenced by olive cultivar, and orchard and soil management predices. The plant-parasitic nematode abundance was significantly higher in orchards managed organically compared to those managed conventionally. These findings could be the result of the influence of the herbaceous plants that compase the soil cover, which may affect plant-parasit ic nematodes at the species level.

Trabajo presentado en la 3rd International Conference on Microbial Diversity (MD2015, "The Challenge of Complexity"), celebrada en Perugia del 27 al 29 de octubre de 2015., The biochemistry and microbial ecology of PDO Aloreña de Málaga table olive fermentations were investigated in two different industries (1 and 2) of province of Málaga (Spain). Monitoring of the physicochemical parameters such as pH, acidity, salt, color and texture of fruits was carried out during the entire process. No differences were detected in physicochemical arameters, which were characteristics for this type of elaboration. The microbial ecology was investigated using both culture-dependent and culture-independent methods in fruit and brine. The initial yeast population was different between both industries. While industry (1) showed counts around 4 - 5 log10 cfu/mL during the first month of fermentation, industry 2 presented lower oncentrations (1 – 3 log 10 cfu/mL), although, both locations reached the same yeast populations at the end of storage period (>5 log 10 cfu/mL). Bar - coded pyrosequencing analysis of the ITS rDNA region identified 109 and 118 operational taxonomic units (OTUs) in fruits and brines, espectively, and revealed the presence of yeast genera previously identified in table olives such as Pichia, Candida, Debaryomyces and Sacharomyces sp., but also, this is the first time that other predominant genera such as Zygostorulaspora, Penicillium and Aspergillus have been identified in this type of table olives. Small differences were found between most abundant yeasts in fruits and brines, during the fermentation process and between both industries.

Trabajo presentado en el Rhizosphere 4: Stretching the interface of life, celebrado en Maastricht del 21 al 25 de junio de 2015., During the last two decades the phytosanitary status of olive crop in Spain is being severely threatened by Verticillium wilt (VW). Suppressive soils have been described worldwide for many different pathogens, however for the fungal pathogen Verticillium dahliae (VD) the information is very scarce. In this study a collection of rhizosphere soils from 93 olive orchards in a wide region of southern Spain, were characterized by their level of suppressiveness to VW. Results indicated that 25% of soils showed a high level of suppressiveness to VW. The objective of this study was to unravel the biotic and abiotic factors associated with this phenomenon. Using some “Classical” approaches we identified that soil texture (clay content) was highly associated to soil suppressiveness. Using ‘classical’ approaches we identified that microbial factors were also involved in this specific suppression including: 1) Transference of soil suppressiveness by adding small amounts of suppressive soil to conducive soil. 2) The treatment of suppressive soils with heat demonstrated reduction of suppressiveness. 3) Representative bacteria from suppressive soils show in vitro and in vivo biocontrol activity against VD. In a second step we used bar-coded pyrosequencing and characterized by abundance and identity, all bacterial communities associated to the rhizosphere of plants growing in 27 soils in the presence or not of the pathogen. NMDS, Classification and Regression Trees and PLS-DA analysis have allowed identifying some climatic parameters and physicochemical soil characteristics that are differentially associated to the level of suppressiveness of those soils as well as to determine which OTUs are specially enriched in the suppressive soils. Furthermore, specific OTUs have been identified as being transferred from the suppressive into the conducive soils which might be responsible of this ‘transferable’ suppressive effect.

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 [Int Microbiol 2015; 18(1):13-24]., We acknowledge financial support from the EU grant ICA4-CT-2001-10005 and an ‘Intramural Project’ to B. B. Landa from the Spanish National Research Council (CSIC), as well as CNPq for a scholarship to J. R. S. Lopes in Brazil.

Trabajo presentado en la 3rd International Conference on Microbial Diversity (MD2015, "The Challenge of Complexity"), celebrada en Perugia del 27 al 29 de octubre de 2015., The objective of this research was to characterize the biochemistry and microbiology of spontaneous PDO Aloreña de Málaga table olive fermentations and define the bacterial biodiversity during the process. The study was carried out in two different locations in Malaga province (Spain) following the fermentation during 4 months. Biochemical analysis of olives showed a progressive increase in free acidity as consequent of a spontaneous fermentation, with pH and salt values characteristic of this type of product. Both locations showed a similar trend in microbiology enumeration by plating in selective media. Yeasts were the most abundant microorganisms during the time, while lactic acid bacteria (LAB) cell count revealed low concentration or even were not detected. The novel of this research is the study of the microbial ecology of olives fermentation using bar-coded pyrosequencing of 16S rDNA to characterize bacterial diversity. The bacterial α- and β- diversity differed between fruits and brines being higher for fruits especially at the beginning of the fermentation process and the opposite for the brines. Bacterial diversity changed during fermentation time, and a total of 323 and 150 operational taxonomic units (OTUs) were identified in fruits and brines, respectively. The predominant genera were Celerinantantimonas, Pseudomonas, Propionibacterium, and diverse Acetobacteraceae. Some differences in the bacterial diversity were found between industries, special at the beginning of the process, whereas no differences were found for the rest of physicochemical parameters such as color and texture in the final product.

Trabajo presentado en el Rhizosphere 4: Stretching the interface of life, celebrado en Maastricht del 21 al 25 de junio de 2015., Plant disease epidemics result from interactions of a susceptible host, a virulent pathogen and conducive environment. Shifts in any component can change disease expression to still unknown directions. Verticillium wilt (VW) of olive caused by the soilborne fungus Verticillium dahliae (Vd) is of major concern for olive industry in the Mediterranean basin. We carried out experiments using olive cvs. Picual and Arbequina, which grew in soil infested by the defoliating (D) or non-defoliating (ND) Vd pathotypes at 20, 24 and 28ºC and CO2 levels of 386, 550 and 750 ppm, representative of current and future SRES-IPCC A2 and B2 scenarios for southern Spain. Surface response models quantified the combined effects of temperature and CO2 on VW intensity and plant growth, demonstrating a differential effect of biotic and abiotic factors. Optimum VW development occurred at 20-24°C and current CO2, being faster and severe in 'Picual'/D. Raising CO2 delayed VW and Vd infection. The transcriptomic profile of olive cultivars in response to Vd infection and the three climate SRES scenarios was assessed using a 12-plex microarray of 37,449 olive unigenes (OLEAGEN project). Main differences in the expression profiles were due to olive genotype, followed by the climatic conditions at the three SRES scenarios and in a lesser extent by Vd infection. Thus, plants growing at current climate showed higher number of differentially expressed genes compared to plants growing at SRES-IPCC A2 and B2 scenarios with a general trend to decrease the number of significantly transcribed genes as temperature and CO2 increased. Furthermore, a higher number of genes (up to 10x) were expressed differentially in 'Arbequina' than in 'Picual' in response to environmental changes as well as to infection by the D pathotype with a low number of genes being common for both cultivars.

Durante los últimos años, el Grupo AGR136 Sanidad Vegetal del IAS-CSIC y la UCO ha centrado sus proyectos de investigación de financiación privada o pública en el desarrollo de conocimeintos y tecnologías sobre medidas de lucha aplicables para la gestión integrada de verticilosis del olivo. En este artículo presentamos sucintamente algunos de los objetivos perseguidos en dichos proyectos y los resultados más destacables alcanzados en el desarrollo de los mismos., Las investigaciones de los autores referidas en este artículo han sido financiadas por los proyectos AGL2008-00344 (Ministerio de Ciencia e Innovación), AGL2011-24935 y AGL2012-37521 (Ministerio de Economía y Competitividad), P08-AGR 3528, P10-AGR-5908, P10-AGR 6082, y P12- AGR 1486 (Consejería de Economía, Innovación y Ciencia, Junta de Andalucía), cofinanciados por fondos FEDER; y en parte por la Organización Interprofesional del Aceite de Oliva Español-CITOLIVA'.