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Olive tree (Olea europaea subsp. europaea) is one of the oldest cultivated trees in the world and plays a critical role in the sustainability of Mediterranean ecosystems. As an agricultural system with important economic, sociological and environmental roles its cultivation should be maintained and preserved. However, nowadays, the health of the olive groves is seriously threatened by a notable increase, both in incidence and severity of diseases caused by various pathogens which are affecting its growth and production. Among olive diseases, those caused by the vascular plant pathogenic bacterium Xylella fastidiosa (in particular from subspecies multiplex and pauca) and the soilborne vascular fungus Verticillium dahliae are, without a doubt, global threats to olive production worldwide. The most practical and economically efficient method for the management of olive vascular diseases is the use of resistant cultivars. However, most olive cultivars widely grown in the Mediterranean Basin including Spain, are moderate to highly susceptible to the most virulent strains of these pathogens, which include V. dahliae defoliating (D) pathotype or X. fastidiosa subsp. pauca ST53. Thus, an integrated management strategy is recommended to prevent the spread and reduce the incidence and severity of those pathogens. This approach combines preventive and palliative measures to mitigate the development of the disease among which the exploitation of the beneficial plant-associated microbiome by using biological control agents might represent a long-term sustainable and environmentally friendly strategy. In nature, healthy plants live in permanent association and interact with a myriad of microorganisms, collectively called the plant microbiome, which are known to play essential roles in plant health. Thus, endophytes, known as bacteria and fungi that live within plants where they establish nonpathogenic relationships with their hosts, can control the growth of pathogens through inter-microbial interactions and by stimulating host plant immunity. The potential role that microbial communities may play in the resistant response to olive vascular pathogens has been overlooked and remains unexplored to date. Therefore, a thorough knowledge of the microbial communities inhabiting the xylem vessels of olive trees may be crucial for understanding their potential influence on the healthy growth of this tree as well as on the resistance shown by specific olive genotypes against vascular plant pathogens. This Doctoral Thesis has been focused on the characterization of the microbial communities inhabiting the xylem vessels of the olive tree, optimizing the methodological approaches for its study and cultivation, determining the main biotic and abiotic factors that determine and modify the structure, diversity and the interactions existing among the members of these microbial communities. For this, as a first step, a Next Generation Sequencing (NGS) protocol was optimized for analysis of xylem-associated microbial communities, which included the evaluation of: i) the procedure for extracting the microbiome when using xylem sap or xylem tissue, ii) the influence of the DNA extraction kits (Chapter II), iii) the choice of PCR primers targeting 16S rRNA (Chapter III). In Chapter II it was shown the significant effect that the DNA extraction protocol has on xylem sap bacterial community assessment. Thus, significant differences in the alpha (Richness) and beta (UniFrac distances) diversity measures of xylem-inhabiting bacterial communities were found among 12 DNA extraction kits, which could be clustered in four groups. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, 21 families, and 14 genera, some specific taxa, particularly those identified at low frequency, were only detected by some DNA extraction kits. The most accurate recovery and assessment of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant and PowerSoil DNA extraction kits. Chapter III addressed another important drawback in metabarcoding studies, such is the primer choice for amplification of 16S rRNA. For that, four PCR-primer pairs targeting a different región of the 16S rRNA were compared for their efficacy to avoid the co-amplification of mitochondria and chloroplast plant rRNA. The highest yields of mitochondria and chloroplast reads were obtained when using xylem woody chips and the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. On the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (76% of reads). Among the genera identified using NGS, 14 (41.2%) were also recovered in the culture collection, whereas 20 (58.8%) of the isolated bacteria were not detected by the NGS approach. In Chapter VI, we evaluated six different broth media (SXM, XVM2, XF26, PD3, 3G10R and XDM2) mimicking xylem sap composition for their ability to sustain growth of olive xylem-inhabiting bacteria. A total of 66 olive xylem-inhabiting bacterial genera could be cultured in vitro, of which 28 (42.4%) were previously described as endophytes of the plant stem in other studies; but 38 of them, were described, for the first time, as cultivable plant-endophytic bacteria. Alpha and beta-diversity measures of bacterial communities developed during cultivation indicated that the main differences were due to the broth media used, followed by the olive genotype from which the xylem sap was extracted, with no effect (Richness and Shannon alpha-diversity) or a minor effect (UniFrac beta-diversity distance) of incubation time. PD3 was the medium that best supported bacterial growth but enriched for the lowest number of bacterial amplicon sequence variants (ASVs); whereas XVM2 medium showed the highest number of ASVs detected when using sap extracted from “Picual” and “Arbequina” genotypes (258 in both), followed by 3G10R (244) in “Picual” sap and XDM2 (244) in “Arbequina” sap. These culture media can facilitate the in vitro cultivation of synthetic microbial communities that can be later used to modify the plant xylem microbiome to enhance plant resilience to vascular pathogens. Additionally, this Doctoral Thesis elucidated whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent D pathotype of V. dahliae (Chapter VII). Results from this chapter indicated that although there were differences in microbial communities among the different plant propagation methods, most substantial changes occurred when plants were inoculated with V. dahliae, regardless of whether the infection process of the stem took place. Thus, a significant increase in the diversity of bacterial communities occurred when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D V. dahliae, characterized by severe wilting symptoms and a 100% of stem vascular colonization. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus was revealed as a keystone bacterium in V. dahliae-inoculated plants, irrespective of their propagation process. Our results showed a breakdown of resistance to V. dahliae in a wild olive genotype that could potentially be related to a modification of its xylem microbiome. These results have contributed to expand our knowledge of the role of indigenous xylem-associated microorganisms on host resistance, which can be of use to fight against the main vascular diseases of olive. Finally, this Doctoral Thesis characterized the structure and diversity of the olive microbiome under natural conditions and their potential determinant and modifying factors including plant-associated host factors such as plant niche and olive genotype, and the influence of environment including climate, soil and agronomic conditions of the orchard and the season of sampling (Chapter VIII). This chapter resulted in the identification of a total of 7,132 bacterial ASVs, distributed in 28 phyla and 3,469 genera, whereas 1,356 ASVs were identified for fungal communities that were composed of 10 phyla and 714 genera. Proteobacteria was the most abundant bacterial phylum (45.21%) followed by Actinobacteriota (25.22%); whereas Pseudomonas and Sphingomonas (7.37% and 5.11%, respectively) were the dominant genera. For fungal communities, Ascomycota (87.81%) followed by Basidiomycota (9.02%) were the most abundant phyla; whereas Aureobasidium and a member of the order Saccharomycetales (18.54% and 15.00%, respectively) were the dominant fungal genera. Alpha diversity showed main significant differences for the Richness and Shannon indexes according to the plant niche, both for bacterial and fungal communities. Similarly, ANOSIM analysis of beta diversity weighted UniFrac distances indicated a significant main effect of the plant niche, followed by the field location and season of sampling, with a minor effect of the olive genotype. Network analysis identified co-presence or mutual exclusion associations between the above- and below-ground compartments of olive trees. Interestingly, specific ASVs were identified showing different relative number of positive and negative associations with other ASVs in the network analysis within each studied factor. Our results are pioneer in describing the olive holobiont and its main shaping factors including the plant niche, environmental conditions (soil physico-chemical properties, climate and seasonality) and host genotype. These results will contribute to facilitate the exploration and selection of specific keystone microorganisms that can live in close association with olive under a range of environmental/agronomic conditions and could be ideal targets for the design of biofertilizers, biostimulants and biocontrol agents for management of olive diseases. To conclude, this Doctoral Thesis has settled the methodological approaches to unravel the biotic and abiotic factors that affect the xylem microbial communities and have characterized some members of the core xylem microbiome, establishing the basis to isolate and culture them. These isolated microorganisms could be used to produce a consortium of xylem-inhabiting microorganisms that can be artificially inoculated into xylem vessels of olive plantlets to modify their native xylem microbiome to obtain plants more resilient to infection by xylem-inhabiting pathogens or to enhance olive plant physiology and growth. El olivo (Olea europaea subsp. europaea) es uno de los árboles cultivados más antiguos del mundo y desempeña un papel fundamental en la sostenibilidad de los ecosistemas mediterráneos. Al ser un sistema agrícola con importantes funciones económicas, sociales y ambientales, su cultivo debe mantenerse y preservarse. Sin embargo, en la actualidad, la salud de los olivares se está viendo seriamente amenazada por un notable incremento, tanto en incidencia como en severidad, de enfermedades causadas por diversos patógenos que están afectando tanto a su desarrollo como producción. Entre las enfermedades del olivo, las causadas por la bacteria patógena Xylella fastidiosa (en particular de las subespecies multiplex y pauca) y el hongo vascular del suelo Verticillium dahliae son, sin duda, las principales amenazas globales para la producción del olivar a nivel mundial. El método más practico y económicamente eficiente para el manejo de las enfermedades vasculares del olivo es el uso de cultivares resistentes. Sin embargo, la mayoría de los cultivares de olivo más ampliamente utilizados en la cuenca mediterránea, incluida España, presentan una reacción de moderada a altamente susceptible a las variantes más virulentas de estos patógenos, que incluyen el patotipo Defoliante (D) en V. dahliae o la subespecie pauca ST53 en X. fastidiosa. Por lo tanto, es necesaria una estrategia de gestión integrada para poder prevenir la propagación y reducir la incidencia y severidad de esos patógenos. Este enfoque debe combinar medidas preventivas y paliativas para mitigar el desarrollo de la enfermedad, entre las que se incluye la explotación del microbioma beneficioso asociado a las plantas mediante el uso de agentes de control biológico que puede representar una estrategia sostenible y respetuosa con el medio ambiente a largo plazo. En la naturaleza, las plantas sanas viven en asociación permanente e interactúan con una gran variedad de microorganismos, denominados microbioma vegetal, que desempeña funciones esenciales en la salud de las plantas. Así, los microorganismos endofitos, entre los que se incluyen las bacterias y hongos que viven en el interior de las plantas estableciendo relaciones no patogénicas con su huésped, pueden controlar el crecimiento de patógenos a través de interacciones inter-microbianas y al estimular la inmunidad de la planta huésped. El potencial papel que pueden desempeñar las comunidades microbianas en la respuesta de resistencia en olivo a patógenos vasculares no ha sido considerada y permanece inexplorado. Por tanto, un mejor conocimiento de las comunidades microbianas que habitan en los vasos del xilema del olivo puede ser crucial para comprender su influencia potencial en el crecimiento saludable de la planta, así como en la resistencia que muestran genotipos específicos de olivo frente a patógenos vasculares. Esta Tesis Doctoral se ha centrado en la caracterización de las comunidades microbianas que habitan el xilema del olivo optimizando los enfoques metodológicos para su estudio y determinando el efecto de los principales factores bióticos y abióticos que son claves en la configuración de su estructura, diversidad y las interacciones existentes entre los componentes del microbioma. Para ello, como primer paso, se optimizo un protocolo de secuenciación masiva (NGS) para el análisis de comunidades microbianas asociadas al xilema, que incluía la evaluación de: i) el procedimiento de extracción del microbioma cuando se utiliza savia o tejido xilemático, ii) la influencia de los kits de extracción de ADN (Capítulo II) y iii) la elección de los cebadores de PCR dirigidos al ARNr 16S (Capítulo III). En el Capítulo II se mostró el efecto significativo del protocolo de extracción de ADN en la evaluación de la comunidad bacteriana de la savia del xilema. Así, se encontraron diferencias significativas en los valores de diversidad alfa (riqueza) y beta (distancias UniFrac) de las comunidades bacterianas que habitan en el xilema entre 12 kits de extracción de ADN, que pudieron agruparse en cuatro grupos. Aunque el número principal de taxones detectados por todos los kits de extracción de ADN incluía cuatro filos, siete clases, 12 órdenes, 21 familias y 14 géneros, algunos taxones, en particular los identificados con baja frecuencia, solo se detectaron con algunos de los kits de extracción de ADN. La recuperación y evaluación más precisa de una comunidad bacteriana inoculada artificialmente en muestras de savia se generó al utilizar los kits de extracción de ADN PowerPlant y PowerSoil. El Capítulo III abordo otro aspecto importante en los estudios de secuenciación masiva, como es la elección del cebador para la amplificación del ARNr 16S. Para ello, se compararon cuatro pares de cebadores de PCR dirigidos a diferentes regiones del ARNr por su eficacia para evitar la coamplificación de ARNr de mitocondrias y cloroplastos de la planta. Las amplificaciones más altas de secuencias de mitocondrias y cloroplastos se obtuvieron cuando se utilizó tejido xilemático con los pares de cebadores PCR1-799F/1062R (76,05 %) y PCR3-967F/1391R (99,96 %). Por el contrario, los pares de cebadores PCR2-799F/1115R y PCR4-799F/1193R mostraron la menor amplificación de ARNr 16S mitocondrial (76% de las lecturas). Entre los géneros identificados mediante NGS, 14 (41,2%) también se recuperaron en cultivo, mientras que 20 de los géneros aislados (58,8%) no pudieron ser detectados mediante NGS. En el Capítulo VI, se evaluaron seis medios de cultivo liquido diferentes (SXM, XVM2, XF26, PD3, 3G10R y XDM2) que imitan la composición de la savia del xilema por su capacidad para favorecer el crecimiento de bacterias que habitan en el xilema del olivo. Se pudieron cultivar in vitro un total de 66 géneros bacterianos habitantes del xilema del olivo, de los cuales 28 (42,4%) habían sido previamente descritos como endofitos del tallo de la planta en otros estudios, pero 38 de ellos fueron descritos por primera vez como bacterias endofitas cultivables de plantas. Las medidas de diversidad alfa y beta de las comunidades bacterianas desarrolladas durante el cultivo indicaron que las principales diferencias entre ellas se debían al medio de cultivo, seguido del genotipo de olivo del que se extrajo la savia, sin verse afectado (diversidad alfa de Riqueza y Diversidad-Shannon) o con un efecto menor (distancia de diversidad beta UniFrac) por el tiempo de incubación. PD3 fue el medio que mejor sustento el crecimiento bacteriano, pero enriqueció con el menor número de ASV bacterianos; mientras que el medio XVM2 mostro el mayor número de ASVs al utilizar savia extraída de los genotipos “Picual” y “Arbequina” (258 en ambos), seguido de 3G10R (244) en savia de “Picual” y XDM2 (244) en savia de “Arbequina”. Estos medios de cultivo pueden facilitar el cultivo in vitro de comunidades microbianas sintéticas que posteriormente pueden utilizarse para la modificación del microbioma del xilema de olivo para mejorar la reacción de este a patógenos vasculares. Además, en esta Tesis Doctoral se ha dilucidado si la propagación in vitro del olivo puede alterar la diversidad y composición del microbioma que habita en el xilema y si esos cambios pueden modificar la respuesta de resistencia que muestra un clon de olivo silvestre al patotipo D altamente virulento de V. dahliae (Capítulo VII). Los resultados indicaron que, aunque hubo diferencias en las comunidades microbianas xilemáticas en función del método de propagación de olivo, los cambios más sustanciales ocurrieron cuando las plantas se inocularon con V. dahliae D, independientemente de si se produjo colonización del tallo por el patógeno. Así, se produjo un aumento significativo en la diversidad de comunidades bacterianas en plantas crecidas en suelo infestado por el patógeno. Además, se observo que las plantas de olivo multiplicadas en condiciones in vitro desarrollaron una reacción susceptible a V. dahliae D, caracterizada por síntomas severos de marchitez y un 100% de infección vascular del tallo. Además, esas plantas propagadas in vitro mostraron un microbioma de xilema alterado con una disminución en el número total de OTUs en comparación con las plantas multiplicadas en condiciones no asépticas. Pseudomonas spp. fue el género bacteriano predominante en plantas micropropagadas y Anoxybacillus se revelo como una bacteria clave en las plantas inoculadas con V. dahliae D independientemente de su método de propagación. Nuestros resultados mostraron una ruptura de la resistencia a V. dahliae D en un genotipo de olivo silvestre que podría estar potencialmente relacionada con una modificación de su microbioma xilemático. Estos resultados han contribuido a ampliar nuestro conocimiento sobre el papel de los microorganismos asociados al xilema en la resistencia del huésped, lo cual puede ser útil para luchar contra las principales enfermedades vasculares del olivo. Finalmente, en esta Tesis Doctoral se caracterizó la estructura y diversidad del microbioma del olivo en condiciones naturales y sus posibles factores determinantes y modificadores, incluidos diversos factores asociados a la planta huésped, como el nicho de esta y su genotipo, así como la influencia del medio ambiente incluyendo las condiciones climáticas, del suelo y agronómicas del olivar y la estación del año en que se lleva a cabo el muestreo (Capítulo VIII). En este capítulo se identificaron un total de 7.132 variantes de secuencia de amplicón (ASV) bacterianos, distribuidos en 28 filos y 3.469 géneros, mientras que se identificaron 1.356 ASV fúngicos que se encuadraban en 10 filos y 714 géneros. Proteobacteria fue el filo bacteriano más abundante (45,21%) seguido de Actinobacteriota (25,22%); mientras que Pseudomonas y Sphingomonas (7,37% y 5,11%, respectivamente) fueron los géneros dominantes. Para las comunidades fúngicas, Ascomycota (87,81%) seguido de Basidiomycota (9,02%) fueron los filos prevalentes; mientras que Aureobasidium y un miembro del orden Saccharomycetales (18,54% y 15,00%, respectivamente) fueron los géneros fúngicos dominantes. La diversidad alfa mostro diferencias significativas para los índices de Riqueza y Shannon según el nicho de la planta, tanto para las comunidades bacterianas como fúngicas. De manera similar, el análisis ANOSIM de disimilaridad UniFrac de la diversidad beta indico que el nicho de la planta fue el factor de mayor influencia, seguido de la localidad y el momento de muestreo, con un menor efecto del genotipo del olivo. El análisis de redes identifico asociaciones de copresencia o exclusión mutua entre los compartimentos del olivo en la parte aérea y suelo. Curiosamente, este análisis permitió identificar ASV específicos que mostraban un numero relativo diferente de asociaciones positivas y negativas con otros ASV para cada factor estudiado. Nuestros resultados son pioneros en la descripción del holobionte del olivo y sus principales factores de configuración, incluidos el nicho de la planta, las condiciones ambientales (clima y estacionalidad) y el genotipo del huésped. Estos resultados contribuirán a facilitar la exploración y selección de microorganismos clave específicos que pueden vivir en estrecha asociación con el olivo en una diversidad de condiciones ambientales/ agronómicas, y podrían ser componentes idóneos para el diseño de biofertilizantes, bioestimulantes y agentes de biocontrol para el manejo de enfermedades en olivar. Para concluir, esta Tesis Doctoral ha establecido los enfoques metodológicos para esclarecer los factores bióticos y abióticos que afectan a las comunidades microbianas del xilema de olivo. Asimismo, se han caracterizado algunos componentes clave del microbioma del xilema, sentando las bases para su aislamiento y cultivo. Estos microorganismos podrían utilizarse para la creación de un consorcio de microorganismos habitantes del xilema que pueden inocularse artificialmente en los vasos xilemáticos de plantones de olivo para modificar su microbioma xilemático nativo y así hacer frente a la infección por patógenos vasculares o mejorar su desarrollo fisiológico y su crecimiento.

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).

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 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., The plant-pathogenic bacterium Xylella fastidiosa, widely distributed in the Americas and with several recent outbreaks in EU, emerged as a serious threat to olive crop and the typical Mediterranean landscape. This xylem-limited plant pathogen spreads by xylem sap-feeding insects, causes symptoms of marginal leaf scorch, leaf chlorosis, defoliation, and a general decay in olive trees. Research on plant-associated microorganisms has gained importance in the last decade as a key component in plant health. However, the role of the xylem microbiome and its contribution to plant health is still scarce. Therefore, this work is focused on mapping X. fastidiosa infection and deciphering the changes in the composition of microbial communities on symptomatic and asymptomatic olive tree branches. Three olives trees of cultivar Grappolo growing at Sao Paulo state (Brazil) were selected and within each tree three main branches were sampled showing different severity of symptoms development; one branch presenting typical X. fastidiosa symptoms of pathogen infection in contrast to the other two branches that were asymptomatic. X. fastidiosa infection was verified by qPCR. Leaves and stem samples were collected in a gradual scale above and below the symptomatic branches. Chips extracts of xylem tissue from its stems and leaf veins and petioles were used for DNA extraction to undergo metagenomic analysis (NGS) for microbial composition estimation and to diagnose the presence of the pathogen by real-time PCR, respectively. These results will help to understand the occurrence of step-wise changes in olive xylem microbiome as a consequence of X. fastidiosa infection and determine which microorganisms respond faster or can be associated to a symptom progression on infected trees., Study supported by Projects 727987 XF-ACTORS (EU-H2020) and AGL2016-75606-R (MEIC Spain and FEDER-EU) and SEGIB – Carolina Foundation.

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 xylem-inhabiting plant pathogenic bacterium Xylella fastidiosa (Xf) represents one of the major phytopathological threats to olive crop worldwide, due to its devastating effects on agricultural yields losses and high tree mortality that causes profound socioeconomic and environmental impacts. Endophytes play an essential role on plant growth and its physiological status, but they can also act as an innate natural defense to cope against infection by xylem-inhabiting pathogenic organisms. Today, vast majority of microorganisms residing in olive xylem are unknown; therefore this work pursues the characterization of the olive microbiome through culture-dependent and independent (NGS) techniques as a tool for identifying potential biological control agents for this pathogen. Hence, four cultivated olive genotypes (Arbequina, Arbosana, Koroneiki and Grappolo) located in Sao Paulo state (Brazil) showing visual Xf symptoms or asymptomatic-non-infected were selected. Xf infection was verified by qPCR. For the culture-dependent approach, chips extracts of xylem tissue from branches and roots were plated in two solid media (R2A and R2A supplemented with plant extract). For culture independent approach, total DNA extracted from xylem tissue was analyzed by metagenomic analysis of 16S and ITS region to characterize the xylem-inhabiting bacterial and fungal communities. Preliminary culture results indicated differences in the frequency of microbial communities depending on the olive genotype and the type of plant material analyzed, as well as, the presence or absence of Xf symptoms on the sampled trees that correlated with Xf infection. These results will help to expand our knowledge on the olive xylem microbiome community composition and understand its driving factors when Xf infection occurs and more importantly to identify xylem-inhabiting microorganisms with potential to combat this harmful bacterium., Study supported by Projects 727987 XF-ACTORS (EU-H2020) and AGL2016-75606-R (MEIC Spain and FEDER-EU) and SEGIB – Carolina Foundation.

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

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.

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).

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 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).

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.

International audience

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).