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Trabajo presentado en el Encuentro Internacional Phytoma (Año Internacional de la Sanidad Vegetal. Ciencia y profesión para producir más con menos), celebrado en Córdoba el 1 y 2 de didiembre de 2021.

V Symposium on Ecological Network, Palma, 10-12 de noviembre de 2021., Understanding microbial associations through co-occurrence network analysis is a very effective tool to detect patterns of copresence and mutual exclusion among members of the plant-associated microbiome. By using 16S and ITS rRNA metagenomics equencing, we studied the associations of bacterial and fungal communities present in xylem sap of plantlets and adult olive trees of 'Picual', 'Arbequina', 'Arbosana', 'Frantoio' and 'Hojiblanca' olive varieties. Results based on the analysis of amplicon sequence variant and network assembly analysis showed distinct microbial association behaviors according to olive plant age and genotype. Thus, Pseudomonas, Bosea and Meiothermus showed the highest positive associations in the xylem of adult trees whereas Azospirillium, Rhizobium and Brevundimonas showed the greatest positive interactions in olive plantlets. Furthermore, MCODE method identified clusters of highly interconnected regions for each olive genotype, where Alternaria and Phaeococcomyces genera were unique to ‘Arbequina’; Rhizobium, Bradyrhizobium and Brevibacillus were exclusive to ‘Arbosana’; Gardnerella and Rathayibacter to ‘Frantoio’; Acinetobacter and Micrococcus to ‘Hojiblanca’ and Arthrobacter, Lawsonella, Massilia, Sphingopyxis, Thermaerobacter and Variovorax to ‘Picual’. Interestingly, Pseudomonas and Anoxybacillus were positioned at the network cores obtained when data were analyzed by plant age and plant genotype, respectively. Network association analysis has revealed new knowledge regarding specific microbial associations occurring among olive genotypes at different plant age, offering the identification of keystone xylem-inhabiting microbial species that may result determinant for olive growth and health.

Trabajo presentado en la 9th Meeting of the IOBC-WPRS Working Group "Integrated Protection of Olive Crops", celebrado en Lisboa del 26 al 29 de octubre de 2021., Xylem-inhabiting plant pathogenic organisms, such as Verticillium dahliae and Xylella fastidiosa, compromise and seriously threat the health of olive crop worldwide by collapsing the xylem vessels and ultimately causing the death of the olive tree. Nowadays, the use of host resistance is the most practical and efficient control measure for vascular diseases in olive. However, research on endophytic microorganisms is gaining a special interest as a potential tool to confer protection against infection by xylem-inhabiting pathogens or suppress disease development. This work pursues i) the modification of xylem microbiome composition of cultivated olive plantlets (‘Picual’ and ‘Arbequina’) by transplanting an external xylem microbiome obtained from wild (‘Acebuche’) and cultivated (‘Picual’ and ‘Arbequina’) adult olive trees through endotherapy treatment, and ii) the evaluation of the changes in the xylem microbial composition of challenged plants over time. A total of 1,015 ASVs (grouped in 361 bacterial genera) were identified for all treatments. Globally, Massilia (12.91%) was the most abundant genus followed by Hymenobacter (9.62%), Sphingomonas (9.32%), Curtobacterium (9.05%) and Methylobacterium (6.09%). Furthermore, alpha diversity measure showed an increase of observed ASVs after endotherapy treatment from ‘Picual’ and ‘Acebuche’ xylem sap to ‘Arbequina’ olive plantlets whereas the endotherapy inoculation of ‘Arbequina’ and ‘Acebuche’ xylematic fluid to ‘Picual’ showed the opposite behaviour. Additionally, beta diversity using ASV frequencies showed some trends across the different sampling times in each olive genotype treated. These results indicated that consortia of xylem microorganisms can be artificially inoculated in xylem vessels of seedling plants modifying their native xylem microbiome with the aim to control diseases caused by xylem-inhabiting pathogens or modify olive plant physiology and growth., Study supported by Projects XF-ACTORS 727987 (EU-H2020) and AGL2016-75606-R (MICINN Spain and FEDER-EU).

Trabajo presentado en el XVI Congreso Nacional de Ciencias Hortícolas, celebrado en Córdoba del 17 al 22 de octubre de 2021., La temperatura es un factor determinante en el desarrollo de enfermedades vasculares en cultivos, como las producidas por Verticillium dahliae o Xylella fastidiosa. Los patrones de temperatura en el interior de las partes leñosas de la planta podrían diferir de los del aire debido, entre otros factores, a la inercia térmica de la madera, la radiación incidente y el sombreado por la copa. Los objetivos de este trabajo fueron describir la dinámica de la temperatura en los vasos del xilema a lo largo del tiempo, determinándose mediante modelos de regresión la relación entre la temperatura del aire y la medida por sensores situados en el xilema de ramas y del tronco, así como del suelo en olivo y almendro. Para ello, se instalaron termopares con orientación norte y sur en las ramas y a 1 y 4 cm de profundidad en el tronco en cuatro árboles de cada cultivo. La temperatura se registró a intervalos de 10 minutos durante 31 meses en la parcela de olivo (de diciembre 2017 a junio 2020) y 14 meses en la de almendro (de febrero 2018 a marzo 2019). Para ambos cultivos, tanto los análisis de los patrones de temperatura de cada sensor a lo largo del tiempo como los modelos de regresión mostraron que la temperatura del xilema en las ramas fue similar a la del aire; por el contrario, la temperatura del xilema del tronco y del sistema suelo-raíces fue inferior a la del aire. El efecto amortiguador del tronco y el suelo fue mayor para la temperatura máxima durante los meses de verano, mientras que la temperatura mínima diaria estuvo acoplada a la temperatura del aire en todas las estaciones. Nuestros resultados indican la importancia de tener en cuenta las diferencias de temperatura entre el entorno del xilema y el aire en estudios epidemiológicos de enfermedades vasculares., 727987 XF-ACTORS (H2020-UE), E-RTA2017-00004-C06-02 AEI-INIA España y FEDER, la Interprofesional del Aceite de Oliva Español y Proyecto ITS2017-095 Consejería de Medio Ambiente, Agricultura y Pesca de las Islas Baleares,España.

Nature Conferences (Harnessing the Plant Microbiome), Virtual Conference, October 22-24, 2021., Olive is one of the most important cultivated trees in the Mediterranean Basin and exhibits a wide range of noteworthy environmental attributes. Nowadays, however, its health status is seriously threatened by phytopathogenic organisms such as the fungus Verticillium dahliae and the bacterium Xylella fastidiosa that obstruct xylem sap flow leading to wilting of plant tissues. Biological control agents represent a promising strategy to control these plant pathogens. We investigated the potential of xylem-inhabiting microorganisms that may compete for infection sites, antagonize the pathogen, and/or induce host plant defense responses, thus contributing to mitigate disease development. A total of 108 bacterial strains isolated from xylem vessels of different cultivated and wild olive genotypes were used to test their antagonistic activity against three Verticillium dahliae strains isolated from olive (defoliating and non-defoliating strains) and tomato. Antagonistic activity was tested in olive xylem sap and in three predefined culture media with chemical composition similar to that of olive xy lem sap, using a high-throughput fungal-bacterial interaction screen method. Results based on a Relative Growth Index indicated that 29 strains restrict V. dahliae (RGI0.2), while 27 strains are neutral regarding V. dahliae (-0.05, Supported by Projects XF-ACTORS 727987 (EU-H2020), AGL2016-75606-R, and PID2020-114917RB-I00 (AEI-MICINN Spain and FEDER-EU)

Trabajo presentado en la 9th Meeting of the IOBC-WPRS Working Group "Integrated Protection of Olive Crops", celebrado en Lisboa del 26 al 29 de octubre de 2021., The detection of the diseases caused by the plant pathogenic bacterium Xylella fastidiosa (Xf) is difficulted by the occurrence of asymptomatic infections, its long incubation period and non-specific and diagnostic symptoms. Early detection of infected plants based on the assessment of changes in physiological plant traits due to bacterial infection might be an important tool for pathogen detection, disease management and breeding for resistance to Xf in woody crops. Therefore, the objectives of this study were to evaluate the susceptibility to four Spanish Xf strains and the response to infection of three olive cultivars widely grown in Spain by measuring plant physiological traits with different leaf-level sensors and Xf-specific molecular diagnostic assays. Leaf measurements and petiole samplings were taken at the inoculation point, and at 5, 10, 15 and 20 cm above the inoculation points at 1, 3, 6, 12 and 18 months post-inoculation. Additionally, sampling of wood tissue was performed at 8 month post inoculation at the same plant sections of leaf sampling. Molecular diagnosis based on two qPCR tests was used to determine the presence and colonization level of the bacteria into the olive xylem tissues. The Dualex Scientific leaf clip sensor and two spectrometers (PolyPen RP400 and DLP NIRscan nano) covering the range of the electromagnetic spectrum from 340 and 1700 nm were used for leaf measurements to detect physiological changes. The Spanish strain XYL1961 from Xf subsp. pauca ST80 showed the highest colonization in the different olive cultivars as compared to strains from Xf subsp. multiplex from ST81 and ST6 from Spain and the Italian strain ‘DeDonno’ from Xf subsp. pauca ST53. On the other hand, from a total of 70 vegetation indices calculated related with chlorophyll, carotenoid and flavonoids content and structural related indexes a set was selected to discriminate between olive plants infected by Xf from those non-inoculated even in the absence of visible disease symptoms., This work has received funding from 727987 XF-ACTORS (H2020-UE), E-RTA2017-00004-C06-02 (AEI-INIA Spain and FEDER), and the Spanish Olive Oil Interprofessional. Project ITS2017-095 Consejería de Medio Ambiente, Agricultura y Pesca de las Islas Baleares, Spain.

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.

Study supported by Projects XF-ACTORS 727987 (EU-H2020), AGL2016-75606-R (MICINN Spain and FEDER-EU) and COST Action CA16107 EuroXanth., The ability of Xylella fastidiosa (Xf) to acquire new genetic information, via horizontal gene transfer and homologous recombination, has been related with its capacity to colonize new hosts and environments. In this study we have performed a comparative genome analysis of the complete circularized sequences of two Xf subsp. multiplex ST6 strains (ESVL and IVIA5901) isolated from almond trees showing almond leaf scorch (ALS) symptoms, located at the Guadalest Valley in Alicante province, in mainland Spain. Genome differences detected between both strains and with the reference strain Temecula1 of Xf subsp. fastidiosa ST1 were then related with phenotypic traits differences associated to infection and disease development observed among those strains. Genome analysis of both strains indicated an average nucleotide identity at the chromosomal level of 99.99%, but main differences were due to the presence of plasmids pXF64-Hb_ESVL and pUCLA-ESVL only present in the ESVL strain. Our results showed that the ESVL strain had a higher cell motility and a greater surface attachment capability being able to form larger aggregates, while IVIA5901 strain showed a higher planktonic growth. Despite the high genetic similarity between the two strains, we have found important differences in their genomes. The presence of the two plasmids in the ESVL strain provided 48 unique genes to ESVL strain. Also, when comparing the two chromosomes, some unique genes and SNPs differences were found associated to genes potentially related with adhesion, motility, cell wall degradation, toxin-antitoxin system and the development of outer membrane protein, which might have an influence on some of the studied traits associated to pathogenicity.

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., Understanding the dispersal of Xylella fastidiosa is essential for effective management of the disease. In Puglia, Italy, surveillance is focused on buffer and containment zones established at the edge of the infected region with the aim of containing further spread. Success of this strategy will strongly depend on whether these zones are wide enough to form a barrier to long distance dispersal of the bacterium. In this presentation, I will describe our progress towards estimating the dispersal range of Xylella in Puglia using a generic spatial epidemiological model adapted to the biology of the pathosystem. The model simulates the spread of the disease across a heterogeneous landscape depending on the location and timing of introduction, the distribution of host plants, the rate of infection growth in infected olive groves and both short and long distance dispersal. Long distance dispersal seems to be a crucial feature of the Xylella epidemic, causing rapid spread of the disease over large areas but in an unpredictable manner. To calibrate the model, we used Approximate Bayesian Computation to compare model simulations to Xylella surveillance data and remote sensing of severe damage. This allows us to contrast a simple spread scenario with more complex scenarios such as anisotropic dispersal in the direction of prevailing winds and spatial variation in disease transmission. In doing so we characterise the spread and estimate the year of introduction. Finally, I will discuss potential for using the model to simulate management strategies and new outbreaks in other regions, using the UK as a case study, Support from XF-Actors & CURE-XF (EU H2020), EFSA, BRIGIT (BBSRC, Defra & Scottish Government) & Scottish Plant Health Centre.

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.

IX Reunión del grupo Microbiología de Plantas - Sociedad Española de Microbiologia (SEM), 16 y 17 de febrero de 2021., La bacteria fitopatógena Xylella fastidiosa (Xf), habitante del xilema, es una de las principales amenazas a nivel mundial para la sanidad vegetal, ya que puede infectar más de 500 especies vegetales, y causar enfermedades severas en muchas de ellas. Esta bacteria se caracteriza por un crecimiento lento (fastidioso) y restringido al xilema que es la causa, en parte, de la dificultad para su aislamiento. A pesar de los importantes esfuerzos para conocer su diversidad genética y la patogenicidad de las nuevas cepas de la bacteria identificadas en Europa, la información disponible sobre su caracterización fenotípica es escasa, lo cual es fundamental para poder establecer posibles diferencias entre cepas que permitan explicar la infección diferencial sobre determinadas plantas huésped. El genoma de esta bacteria es muy pequeño, y se ha demostrado recientemente mediante la combinación de un modelo metabólico a escala genómica y fenotipado, que su red metabólica está completa, pero es sorprendentemente minimalista y carece de robustez. En este trabajo se ha determinado la diversidad metabólica de siete cepas de Xf pertenecientes a tres subespecies (fastidiosa, multiplex, pauca), aisladas de almendro, olivo, vid y acebuche, procedentes de España, Italia y Estados Unidos, mediante un sistema de fenotipado de alto rendimiento. Este sistema permite caracterizar la capacidad metabólica de las cepas estudiadas y establecer diferencias en el aprovechamiento de distintas rutas metabólicas, lo que contribuirá a complementar la información obtenida del genoma completo circularizado de estas cepas ya disponible. Para ello, se utilizaron microplacas de fenotipado (PM 1 a 5) de Biolog, que contienen una serie de compuestos que pueden ser usados como fuentes de carbono, nitrógeno, fósforo, azufre y suplementos nutricionales. El crecimiento de cada cepa se estimó diariamente mediante medidas de la absorbancia a 600 nm con un lector de microplacas. El análisis de las curvas de crecimiento generadas para cada compuesto indicó diferencias entre las cepas de Xf en las distintas placas utilizadas. Las mayores diferencias entre cepas se obtuvieron para algunos de los compuestos usados como fuente de carbono y de nitrógeno, y la cepa de Xf subsp. pauca ‘De Donno’, aislada del foco epidémico de Xf en Italia que presentó un perfil metabólico más diferenciado respecto a las cinco cepas aisladas en los focos en España de Xf subsp. fastidiosa y subsp. multiplex y una cepa americana de Xf subsp. fastidiosa., Financiación: Proyectos 727987 XF-ACTORS H2020-UE y E-RTA2017-00004-C06-02 ede AEI-INIA Spain, the Spanish olive oil Interprofesional and European Social Fund.

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., Two major threats affecting olives groves are the bacterium Xylella fastidiosa (Xf) and the soil borne fungus Verticillium dahliae (Vd) Kleb. Both pathogens block the water flow along the xylem vessels, triggering similar physiological symptoms which are also easily confounded with water-stress induced responses. Previous remote sensing studies have attempted to independently detect Xf and Vd infections using hyperspectral and thermal imagery, but the potential discrimination between both infections has not been addressed yet. In this study, a multistage machine learning (ML) methodology is proposed to detect and discriminate the spectral changes caused by Xf and Vd infection. The multi-stage ML approach assessed spectral plant traits retrieved by physical model inversion such as leaf pigment content (carotenoids, Cx+c, chlorophyll a+b, Ca+b and anthocyanins, Anth.), canopy structural parameters (leaf area index, LAI and the leaf inclination distribution function, LIDFa), solar-induced fluorescence (SIF@760), the thermal-based Crop Water Stress Index (CWSI) and narrow-band hyperspectral indices. On each stage, key indicators were used to i) detect infection by each of the two pathogens accounting for the importance of the spectral traits, ii) reduce the detection uncertainties due to the confounding physiological responses triggered by water stress, and iii) discriminate between Vd and Xf infections. Results showed that SIF@760, Anth. and the Normalized Phaeophytinization Index (NPQI) were the most relevant traits to identify trees showing symptoms confounded with water stress. Using a Vd+Xf cross-infected balanced dataset the multi-stage ML approach yielded overall accuracies that exceeded 92% (kappa=0.8). The most sensitive spectral plant-trait indicators to discriminate between both pathogen infections will be discussed, describing operational aspects for the large-scale monitoring of olive orchards in geographic regions potentially affected by both pathogens.

Trabajo presentado en la 4th Annual Conference of the EuroXanth COST Action (Integrating Science on Xanthomonadaceae for integrated plant disease management in Europe), virtual Conference, 28–30 June 2021., Olive tree is one of the most important crops in the Mediterranean Basin. However, nowadays its viability is seriously threatened by plant pathogens such as Verticillium dahliae and Xylella fastidiosa which colonize the xylem vascular bundles and ultimately can cause the death of the olive tree. Recent studies indicated that plant-associated microbial communities play an important role in controlling vascular wilt diseases and could form the basis of sustainable biocontrol strategies for crop production. NGS approaches with advances in bioinformatics and statistical analyses represent valuable tools for characterizing the diversity of these plant-colonizing microorganisms which will lead to a better understanding of their interactions. In this work, we deciphered the bacterial and fungal microbial communities from different olive ecological niches (soil, rhizosphere, root, xylem sap, stem, leaf and fruits) using distinct bioinformatics pipelines based on the identification of operational taxonomic units (OTUs) or amplicon sequence variants (ASVs). Sequence analysis reported a greater number of taxa using ASV-based pipeline (5.891 for bacteria and 3.055 for fungi) in contrast to the OTU-based pipeline (1.269 and 553, for bacteria and fungi, respectively). Rhizosphere was the plant niche with the highest number of ASV for bacteria (5.662), whereas soil was the niche showing highest number when using the OTU pipeline (730). Interestingly, for the xylem, we estimated 689 and 2647 ASV for bacteria and fungi, respectively; whereas a much lower of OTUs was determined (i.e., 205 and 101, for bacteria and fungi, respectively). Our results showed that bioinformatic pipelines may affect significantly the characterization of the plant-associated microbiome and highlight the importance of standardizing the computational methods for downstream analysis of NGS data, especially when searching for potential microbial taxa associated to suppression of vascular plant pathogens., Study supported by Projects XF-ACTORS 727987 (EU-H2020), AGL2016-75606-R (MICINN Spain and FEDER-EU) and COST Action CA16107 EuroXanth.

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 3rd European Conference on Xylella fastidiosa (Building knowledge, protecting plant health), celebrada online el 29 y 30 de abril de 2021., The development of regionalised risk models for the establishment of Xylella fastidiosa (Xf) and its subspecies requires a better knowledge of their respond to environmental factors in particular to temperature. Furthermore, since Xf is confined within the xylem, it will be relevant to determine to which extent the xylem environment differs from external conditions. Therefore, the objectives of this study were to determine the effect of temperature in growth and survival of Xf strains and to establish the dynamics of temperature in xylem vessels in olive trees. Growth and biofilm formation of 33 Xf strains representative of five subspecies and 13 STs, a wide geographic origin and hosts plants have been determined in vitro in a temperature range from 4 to 40ºC. After 7 days, planktonic cells were inoculated into 96-well plates and incubated at 28ºC for 7 days more. On the other hand, air and xylem vessels temperature was measured at 10-min intervals during 31 months by installing thermocouples in soil, branches and trunks of four 8-yr old olive trees at a 1 and 4 cm depth. Our results indicated that overall, extreme low or high temperature differentially affected growth and survival of Xf strains. Thus, when Xf bacterial cells were incubated at 4 to 10ºC showed a slower growth rate and reduced biofilm formation compared to those incubated at 16 to 32ºC. However, cells that grew at all those temperatures were able to growth further and form biofilm when incubated at favourable temperature of 28ºC in fresh medium. In contrast, incubation temperature of 36 and 40ºC for a 7-day period resulted lethal for the bacterial cells since no further cell growth was observed after incubation at 28ºC. Regression models were developed to estimate the relationship between air and xylem or soil temperature. The slope of the fitted linear models indicated a poikilothermy where air and branch temperature progress at the same rate (slope=1), but a homeothermy in trunks and the soil-root system where air temperature increases at a lower rate (slope < 1). The buffer effect of the trunk and soil are stronger for daily maximum temperature in summer and winter seasons, while minimum daily temperature was coupled with air temperature across seasons. Our results indicate the need to take into account the differences on temperature in the xylem environment when developing regionalized risk models for Xf and highlight the importance of the differential effect of low and high temperature on Xf growth., This work has received funding from 727987 XF-ACTORS (H2020-UE), E-RTA2017-00004-C06-02 from AEI-INIA Spain and FEDER and the Spanish Olive Oil Interprofessional

IX Reunión del grupo Microbiología de Plantas - Sociedad Española de Microbiologia (SEM), 16 y 17 de febrero de 2021., El uso de cultivares o portainjertos de olivo resistentes a Verticillium dahliae (Vd) es la medida más práctica y eficiente para el control de la Verticilosis en olivar. Sin embargo, el papel de las comunidades microbianas del xilema en la reacción de resistencia del olivo a Vd no ha sido explorado hasta la fecha. Este trabajo se centra en dilucidar si la propagación in vitro de las plantas de olivo puede alterar la diversidad y composición de la microbiota habitante del xilema y si esos cambios pueden modificar la reacción de resistencia en un clon de olivo silvestre al patotipo defoliante (D) altamente virulento de Vd. Los resultados indicaron diferencias en las comunidades microbianas entre los diferentes métodos de propagación, aunque los mayores cambios ocurrieron en respuesta a la inoculación con Vd, independientemente de si la planta fue infectada o no por el patógeno y que resultó en un aumento significativo en la diversidad bacteriana en el xilema de plantas inoculadas con Vd. Por otro lado, se observó que las plantas de olivo micropropagadas en condiciones in vitro (alta asepsia) desarrollaron una reacción susceptible al patotipo D de Vd, caracterizada por síntomas severos de marchitez y una incidencia de infección vascular máxima. Asimismo, las plantas micropropagadas in vitro mostraron una disminución en el número total de OTUs en el xilema en comparación con el observado en el de las plantas propagadas en condiciones no asépticas. Globalmente, se identificaron 10 géneros bacterianos clave independientemente de la inoculación por Vd y el procedimiento de propagación de las plantas (in vitro frente a vivero), siendo Cutibacterium, Pseudomonas, Anoxybacillus, Staphylococcus, Methylobacterium-Methylorubrum y Bradyrhizobium los más abundantes, en ese orden. Pseudomonas fue el género bacteriano predominante en plantas micropropagadas y Anoxybacillus se posicionó como una bacteria clave en plantas inoculadas con Vd independientemente de su forma de propagación. Nuestros resultados muestran por primera vez la rotura de la resistencia a Vd en un clon de olivo silvestre, que posiblemente esté asociada con una modificación del microbioma xilemático, lo que contribuye a un mejor conocimiento sobre el papel del microbioma del xilema puede tener en la estabilidad de la resistencia de olivo a Vd, lo que puede ser de utilidad para un manejo más eficiente de las enfermedades vasculares del olivo., Estudio financiado por los proyectos XF-ACTORS 727987 (EU-H2020) y AGL2016-75606-R (MICINN y FEDER-EU).

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., Since the first detection of Xylella fastidiosa (Xf) in the Balearic Islands and Alicante province (mainland Spain), research has provided new insights on genetic diversity and host range of the strains present in the different Spanish outbreaks. The high genetic diversity of Xf in Spain (three subspecies and four STs) and the high host range (over 30) in total implies a risk for several host of agronomic interest if these genotypes spread to areas where they are not present yet. The main goal of this study was to evaluate the pathogenicity and the systemic colonization of four Spanish strains on three olive cultivars representative of the most widely grown in Spain. Two experiments are being conducted in a confined growth chamber (26ºC day/24ºC night), where 10-month-old self-rooted micro-propagated olive cultivars (Arbequina, Hojiblanca and Picual) were inoculated with the strains XYL1961 of Xf subsp. pauca ST80 and the Italian strain De Donno of Xf subsp. pauca ST53 (Control), strain XYL1966 of Xf subsp. multiplex ST81 isolated from olive trees in the Balearic Islands, and the strains IVIA5901 and ESVL of Xf subsp. multiplex ST6 isolated from almond trees in the Guadalest valley (Alicante). Leaf samplings were carried out at the inoculation point (IP), and at 5, 10, 15 and 20 cm above the IP, at 1, 3, 6, 12 and 18 months post-inoculation (mpi), respectively. Furthermore, destructive sampling of different sections of the main stem was performed at 9 mpi at the same levels of leaf sampling. qPCR tests were used to determine the presence and systemic colonization of the bacterium. After 2 years, no clear or specific symptoms associated to Xf infection has been observed. Our results show that all the strains of Xf subsp. multiplex were detected in the leaf petioles at the IP and 5 cm above it although at low frequency ( 34) indicating that the strains had some trouble colonizing the different olive genotypes. On the contrary, Xf subsp. pauca strains could be detected on all leaf petiole samples even up to 20 cm over the IP, although the frequency of positive samples and bacterial inoculum decreased drastically from the IP. When sampling the main stem, results indicated that the Spanish strain of Xf subsp. pauca ST80 colonized a higher number of plants and multiplied better than the Italian strain of Xf subsp. pauca ST53 which indicated the risk that this strain may represent for olive. No clear differences could be observed among the patterns of infection of the different Xf strains on the three olive cultivars tested., This work has received funding from 727987 XF-ACTORS (H2020-UE), E-RTA2017-00004-C06-02 (AEI-INIA Spain and FEDER), and the Spanish Olive Oil Interprofessional.

XXVIII Congreso Nacional de Microbiología, 28 de juanio al 2 de julio de 2021., Los microorganismos endófitos pueden actuar como una defensa natural innata para hacer frente a la infección por organismos fitopatógenos que habitan el xilema como son Verticillium dahliae y Xylella fastidiosa. Hoy en día, se desconoce la gran mayoría de los microorganismos que residen en el xilema del olivo. Por lo tanto, este trabajo se centra en la caracterización de las comunidades bacterianas y fúngicas presentes en la savia y vasos xilemáticos del olivo mediante el uso de técnicas metagenómicas, y determinar el efecto del genotipo de olivo (Picual, Arbequina y Frantoio), variables agro-ambientales (fincas en Úbeda, Baena y Antequera, Andalucía) y estacional (otoño y primavera). Se determinaron un total de 993 ASVs bacterianos y 692 ASVs fúngicos que variaron principalmente en función del componente ambiental (localización geográfica), estacionalidad, y genotipo, en este orden. Se identificó una mayor riqueza en diversidad de bacterias en primavera y de hongos en otoño. Las muestras de xilema del tallo del cv. Picual creciendo en la finca de Úbeda mostraron la mayor riqueza fúngica. Globalmente, Cutibacterium (17.11%), Pseudomonas (7.00%) y Anoxybacillus (4.56%) fueron los géneros bacterianos más representativos mientras que un Ascomycota no cultivable fue el hongo más abundante (42.85%), seguido de Aureobasidium (28.96%) y Phaeococcomyces (6.99%). Nuestros resultados contribuyen a mejorar la caracterización del microbioma xilemático de olivo determinando los principales factores diferenciadores, lo que puede ser de gran utilidad en la búsqueda de microorganismos habitantes del xilema para un manejo más eficiente de las enfermedades vasculares del olivo., Financing: Estudio financiado por los proyectos XF-ACTORS 727987 (EU-H2020), AGL2016-75606-R (MICINN y FEDER-EU) y 2020AEP125 (CSIC).

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) strains from Mallorca belong to subspecies fastidiosa Sequence Type (ST) 1. Isolates from Xf subsp. fastidiosa cause Pierce’s Disease on grapes and leaf scorch disease on almonds in California. Xf colonization and disease development in host plants have been shown to be related to the size of cell aggregates, bacterial motility, and biofilm formation, which are mediated by Type I and Type IV pili, among other traits. In this study several Xf subsp. fastidiosa strains from Spain were phenotypically characterized and compared with strains from the USA. The four Spanish Xf strains used included IVIA5235, IVIA5770, XYL461, R2XF4358 that were isolated from cherry tree, grapevine and Rhamnus alaternus. The Californian strains included Temecula 1 (reference strain), WM1-1 and CFBP7970 that were isolated from grapevines; M23, CFBP8073, CFBP7969, CFBP8068, CFBP8082 isolated from almonds, Coffea canephora, Vitis rotundifolia, Ulmus sp., and Ambrosia artemifolia, respectively. To study bacterial behavior and phenotypic characteristics several experiments were performed to determine adhesion force to substrate, biofilm formation, movement, cell–cell aggregation, twitching motility and patterns of bacterial growth. Taking together our results showed that Spanish strains do not have twitching motility as M23, CFBP7969, CFBP8068, CFBP8082 strains, whereas the remaining strains have. Standardized area under the growth progress curve showed that IVIA5770 and IVIA5235 have similar growth rate and planktonic growth than M23, but lower biofilm formation as compared to the remaining strains. Future research will be performed to determine the relationship between those phenotypic traits and virulence level on host plants of agronomic interests., This work has received funding from COST Action CA16107 EuroXanth supported by European Cooperation in Science and Technology 727987 XF-ACTORS (H2020-UE) and E-RTA2017-00004-C06-02 from AEI-INIA Spain and the Spanish olive oil Interprofesional.

Trabajo presentado en el XVI Congreso Nacional de Ciencias Hortícolas, celebrado en Córdoba del 17 al 22 de octubre de 2021., Los microorganismos endófitos representan una estrategia innovadora para el manejo de las enfermedades vasculares en el olivo actuando como una defensa natural innata para hacer frente a la infección por patógenos que colonizan el xilema como son Verticillium dahliae y Xylella fastidiosa. Sin embargo, pocos estudios han caracterizado el efecto de la variedad y la edad de la planta en la estructura y diversidad microbiana del xilema del olivo. Por lo tanto, este trabajo se ha centrado en la caracterización mediante un enfoque metagenómico de las comunidades bacterianas y fúngicas presentes en el fluido xilemático de plantones jóvenes y olivos adultos de las variedades de olivo ‘Picual’, ‘Arbequina’, ‘Arbosana’, ‘Frantoio’ y ‘Hojiblanca’. Se han identificado un total de 440 ASVs bacterianos y 96 ASVs fúngicos que variaron en función de la edad de la planta alcanzando una diversidad (alfa) microbiana mayor en plantas adultas que en los plantones jóvenes y presentando una estructura microbiana (beta-diversidad) diferente en función de la edad, pero no de la variedad. Por otra parte, las variedades ‘Arbosana’ y ‘Hojiblanca’ mostraron una mayor riqueza bacteriana y fúngica, respectivamente. Globalmente, Meiothermus (24.61%), Anoxybacillus (18.21%) y Paenibacillus (13.15%) fueron los géneros bacterianos más representativos mientras que Aureobasidium fue el hongo más abundante (50.51%), seguido de Plectosphaerella (6.94%) y Malassezia (6.38%). Estos resultados contribuyen a mejorar la caracterización y el conocimiento de la variabilidad del microbioma xilemático de olivo, generando una información muy útil para optimizar el manejo de las enfermedades vasculares del olivo mediante el uso de sus propios microrganismos endofíticos como agentes control biológico., Estudio financiado por los proyectos XF-ACTORS 727987 (EU-H2020) y AGL2016-75606-R (MICINN y FEDER-EU).

Número 334 dedicado a: 2020: Año Internacional de la Sanidad Vegetal.

Every year, different harmful organisms (pests and pathogens) significantly reduce the potential yield of agricultural crops and threaten the sustainability of the planet’s forests. At present, this situation is compounded by the harmful effects of climate change. It is essential to generate new knowledge and technologies for a more efficient control and management of strategic diseases and pests that constitute a threat in key productive sectors for the world economy, and in particular the Spanish one.

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., Nowadays there is no culture media described for axenic growth of olive xylem microbiome which can be essential to understand the ecological interactions of xylem-inhabiting pathogens. This work focused on the study of culture media of defined composition to sustain growth of xylem-inhabiting microorganisms. Xylem sap extracted with a Scholander pressure chamber from two olive cultivars were inoculated in SMX, XVM2, XF26, PD3, 3G10R and XDM2 liquid culture media for two weeks at 28°C. The increase in absorbance was measured daily and aliquots were sampled every two days to analyze changes in bacterial composition by Illumina MiSeq metagenomic analysis. Globally, the most abundant genus of culturable bacteria was Massilia (21.25%), followed by Sphingomonas (20.54%), Curtobacterium (9.80%), Pseudomonas (7.51%) and Frigoribacterium (7.12%), although there were substantial differences in microbial communities over time and among culture media. Thus, SMX and XF26 displayed the highest bacterial OTU richness variation on time (33-9 and 30-6, respectively), followed by XVM2 (29-11), XDM2 (25-9), 3G10R (19-9) and PD3 (9-6). Currently, we have evaluated the possibility to modify the xylem microbiome composition of olive seedlings by transplanting a microbiome artificially cultured in liquid media by an endotherapy treatment and monitored the stability of this modification over time. Results indicated that consortia of xylem microorganisms can be cultivated in vitro using predefined culture media, and that those can artificially introduced in xylem vessels of seedling plants modifying their native xylem microbiome. This technique, when optimized, can settle the basis to inoculate ad-hoc selected microorganisms with the aim to control diseases caused by xylem inhabiting pathogens such as Xylella fastidiosa or modify olive plant physiology and growth., Research financially supported by Projects XF-ACTORS 727987 (EU-H2020) and AGL2016-75606-R (MICINN Spain and FEDER-EU).

Taabajo presentado en el Global Symposium on Soil Biodiversity, celebrado del 19 al 22 de abril de 2021., This study 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 Projects SOILMAN (01LC1620) and XF-ACTORS (727987) from the European Union's Horizon 2020 Framework Research Programme.

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

Potential impact of Xylella fastidiosa subsp. pauca in European olives: a bio-economic analysis

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., Two Xylella fastidiosa(Xf) subsp. multiplexstrains IVIA5901 and ESVL were isolated from symptomatic almond trees in Alicante (Spain). Even if these Xf strains show an average nucleotide identity at the chromosomal level of 99.99%, they differ by the presence of two plasmids pXF64-Hb_ESVL and pUCLA-ESVL, only found in strain ESVL. Xf colonisation and disease development in host plants have been shown to be related to the size of cell aggregates, bacterial motility and biofilm formation, which are mediated by type I and type IV pili, among other traits.The goal of this study was to characterise the phenotypic characteristics of these strains that may be related to their differences in plasmid content.The Spanish Xf subsp. multiplexstrains were compared with Xf subsp. multiplexstrains Alma-Em3 and BB08-1 isolated from blueberry and the reference strain Xf subsp. fastidiosaTemecula1 from grapes, all isolated in the US. To study bacterial behaviour and phenotypic characteristics, several experiments were performed to determine adhesion force to substrate, biofilm formation, movement, cell–cell aggregation, twitching motility and patterns of bacterial growth. Additionally, a virulence assays were conducted in the greenhouse using tobacco plants cvs. SR1 and Xanthi. We also determined the presence of genes coding for type I and type IV pili in the Spanish strains. Our results show that the two Spanish isolates of Xf subsp. multiplexhave lower motility, less capacity of aggregation, make less biofilm, and cause lower disease severity when compared with the US isolates used in this study. Also, there were significant differences in disease severity between Xf Spanish strains that varied according to the tobacco cultivar, with Xanthi being the most susceptible one. Besides, our resultsindicate that strain ESVL exhibits stronger attachment to substrate than IVIA5901, but no differences in biofilm formation, cell–cell aggregation or twitching motility were found between them. The role of plasmids in the biology of these Spanish Xf strains will be further investigated., Study supported by Project 727987 XF-ACTORS (EU-H2020), COST Action CA16107 EuroXanth and E-RTA2017-00004-C06-02 from AEI-INIA Spain and FEDER.