El microbioma del olivo y su papel en la respuesta de la planta a la Verticilosis causada por Verticillium dahliae: factores determinantes y modificadores

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 (<27,48 %), ausencia de secuencias de cloroplastos y el mayor número de unidades taxonómicas operacionales (OTU) bacterianas identificadas (es decir, 254 y 266, respectivamente). Curiosamente, solo 73 de 172 y 46 de 181 géneros fueron comunes a la savia y el tejido del xilema después de la amplificación con los cebadores PCR2 o PCR4, respectivamente, lo que indica un fuerte sesgo en la caracterización de las comunidades bacterianas según los cebadores utilizados. Estos resultados ofrecieron la hoja de ruta para diseñar una estrategia optimizada en la selección del kit de extracción de ADN y la pareja de cebadores de PCR más adecuados para la evaluación de las comunidades bacterianas en olivo junto con una línea de comandos bioinformáticos precisos que pueden ser utilizados para una descripción precisa de las comunidades bacterianas presentes en los vasos xilemáticos u otros nichos de plantas. En el Capítulo IV, se llevó a cabo la caracterización de la composición ionómica y metabolómica de la savia para la identificación de los requerimientos nutricionales potenciales de los microorganismos limitados al xilema. También se analizó el efecto de la edad y el genotipo de la planta sobre la composición bacteriana y química de la savia en olivo. Los resultados mostraron que la savia del olivo incluía un alto contenido de azucares (54,35%), alcoholes (28,85%), aminoácidos (8,01%), ácidos orgánicos (7,68%) y osmolitos (1,12%). Sin embargo, este perfil metabolómico vario en función de la edad y el genotipo de la planta. Los niveles de glucosa, fructosa, sacarosa y manitol, colina, B y PO4 3− fueron significativamente mayores en arboles adultos que en plantones para ambos genotipos de olivo, mientras que los contenidos de NO3 − y Rb mostraron un comportamiento opuesto. Por otro lado, los niveles de ácido aspártico, fenilalanina y Na fueron significativamente más altos en “Picual” que en “Arbequina”, y ocurrió lo contrario para Fe, pero solo para arboles adultos. El conocimiento de la composición química de la savia podrá conducir a una mejor comprensión de los complejos requisitos nutricionales de los microorganismos que habitan el xilema del olivo, incluidos los patógenos vasculares y sus posibles antagonistas, lo que puede permitir un mejor diseño y optimización de los medios de cultivo artificiales para el cultivo del microbioma del olivo. Otros aspectos relevantes de esta Tesis Doctoral incluyeron la comparación de enfoques cultivo dependientes e independientes para el análisis de la microbiota del xilema, incluido el aislamiento y cultivo in vitro del núcleo central de bacterias

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees

Vascular pathogens are the causal agents of some of the most devastating plant diseases in the world, which can cause, under specific conditions, the destruction of entire crops. These plant pathogens activate a range of physiological and immune reactions in the host plant following infection, which may trigger the proliferation of a specific microbiome to combat them by, among others, inhibiting their growth and/or competing for space. Nowadays, it has been demonstrated that the plant microbiome can be modified by transplanting specific members of the microbiome, with exciting results for the control of plant diseases. However, its practical application in agriculture for the control of vascular plant pathogens is hampered by the limited knowledge of the plant endosphere, and, in particular, of the xylem niche. In this review, we present a comprehensive overview of how research on the plant microbiome has evolved during the last decades to unravel the factors and complex interactions that affect the associated microbial communities and their surrounding environment, focusing on the microbial communities inhabiting the xylem vessels of olive trees (Olea europaea subsp. europaea), the most ancient and important woody crop in the Mediterranean Basin. For that purpose, we have highlighted the role of xylem composition and its associated microorganisms in plants by describing the methodological approaches explored to study xylem microbiota, starting from the methods used to extract xylem microbial communities to their assessment by culture-dependent and next-generation sequencing approaches. Additionally, we have categorized some of the key biotic and abiotic factors, such as the host plant niche and genotype, the environment and the infection with vascular pathogens, that can be potential determinants to critically affect olive physiology and health status in a holobiont context (host and its associated organisms). Finally, we have outlined future directions and challenges for xylem microbiome studies based on the recent advances in molecular biology, focusing on metagenomics and culturomics, and bioinformatics network analysis. A better understanding of the xylem olive microbiome will contribute to facilitate the exploration and selection of specific keystone microorganisms that can live in close association with olives under a range of environmental/agronomic conditions. These microorganisms could be ideal targets for the design of microbial consortia that can be applied by endotherapy treatments to prevent or control diseases caused by vascular pathogens or modify the physiology and growth of olive trees

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees

Vascular pathogens are the causal agents of some of the most devastating plant diseases in the world, which can cause, under specific conditions, the destruction of entire crops. These plant pathogens activate a range of physiological and immune reactions in the host plant following infection, which may trigger the proliferation of a specific microbiome to combat them by, among others, inhibiting their growth and/or competing for space. Nowadays, it has been demonstrated that the plant microbiome can be modified by transplanting specific members of the microbiome, with exciting results for the control of plant diseases. However, its practical application in agriculture for the control of vascular plant pathogens is hampered by the limited knowledge of the plant endosphere, and, in particular, of the xylem niche. In this review, we present a comprehensive overview of how research on the plant microbiome has evolved during the last decades to unravel the factors and complex interactions that affect the associated microbial communities and their surrounding environment, focusing on the microbial communities inhabiting the xylem vessels of olive trees (Olea europaea subsp. europaea), the most ancient and important woody crop in the Mediterranean Basin. For that purpose, we have highlighted the role of xylem composition and its associated microorganisms in plants by describing the methodological approaches explored to study xylem microbiota, starting from the methods used to extract xylem microbial communities to their assessment by culture-dependent and next-generation sequencing approaches. Additionally, we have categorized some of the key biotic and abiotic factors, such as the host plant niche and genotype, the environment and the infection with vascular pathogens, that can be potential determinants to critically affect olive physiology and health status in a holobiont context (host and its associated organisms). Finally, we have outlined future directions and challenges for xylem microbiome studies based on the recent advances in molecular biology, focusing on metagenomics and culturomics, and bioinformatics network analysis. A better understanding of the xylem olive microbiome will contribute to facilitate the exploration and selection of specific keystone microorganisms that can live in close association with olives under a range of environmental/agronomic conditions. These microorganisms could be ideal targets for the design of microbial consortia that can be applied by endotherapy treatments to prevent or control diseases caused by vascular pathogens or modify the physiology and growth of olive trees

Primer Choice and Xylem-Microbiome-Extraction Method Are Important Determinants in Assessing Xylem Bacterial Community in Olive Trees

Understanding the unique and unexplored microbial environment of xylem sap is starting to be of relevant importance for plant health, as it could include microbes that may protect plants against xylem-limited pathogens, such as Verticillium dahliae and Xylella fastidiosa. In this study, we evaluated the effects that the method for extracting the xylem bacterial communities, the plant age and the PCR primers may have on characterizing the xylem-bacterial-community composition by using an NGS approach. Xylem sap was extracted from xylem vessels by using a Scholander pressure chamber, or by macerating wood shavings that were obtained from xylem tissues by using branches from 10-year-old olive trees, or the entire canopy of 1-year-old olive plantlets. Additionally, we compared four different PCR-primer pairs that target 16S rRNA for their efficacy to avoid the coamplification of mitochondria and chloroplast 16S rRNA, as this represents an important drawback in metabarcoding studies. The highest amplifications in the mitochondria and chloroplast reads were obtained when using xylem woody chips with the PCR1-799F/1062R (76.05%) and PCR3-967F/1391R (99.96%) primer pairs. To the contrary, the PCR2-799F/1115R and PCR4-799F/1193R primer pairs showed the lowest mitochondria 16S rRNA amplification (60% of reads) included Anoxybacillus, Cutibacterium, Pseudomonas, Spirosoma, Methylobacterium-Methylorubrum and Sphingomonas; however, their relative importance varied, depending on the matrix that was used for the DNA extraction and the primer pairs that were used, with the lowest effect due to plant age. These results will help to optimize the analysis of xylem-inhabiting bacteria, depending on whether whole xylematic tissue or xylem sap is used for the DNA extraction. More importantly, it will help to better understand the driving and modifying factors that shape the olive-xylem-bacterial-community composition

Culture-Dependent and Culture-Independent Characterization of the Olive Xylem Microbiota: Effect of Sap Extraction Methods

Microbial endophytes are well known to protect host plants against pathogens, thus representing a promising strategy for the control of xylem-colonizing pathogens. To date, the vast majority of microbial communities inhabiting the olive xylem are unknown; therefore, this work pursues the characterization of the xylem-limited microbiome and determines whether the culture isolation medium, olive genotype, and the plant material used to analyze it can have an effect on the bacterial populations retrieved. Macerated xylem tissue and xylem sap extracted with the Scholander chamber from olive branches obtained from two cultivated and a wild olive genotypes were analyzed using culture-dependent and -independent approaches. In the culture-dependent approach using four solid culture media, a total of 261 bacterial isolates were identified after performing Sanger sequencing of 16S rRNA. Culturable bacteria clustered into 34 genera, with some effect of culture media for bacterial isolation. The cultivated bacteria belonged to four phyla and the most abundant genera included Frigoribacterium (18.8%), Methylobacterium (16.4%), and Sphingomonas (14.6%). On the other hand, in the culture-independent approach conducted using Illumina MiSeq 16S rRNA amplicon sequencing [next-generation sequencing (NGS)] of the xylem extracts, we identified a total of 48 operational taxonomic units (OTUs) belonging to five phyla, being Sphingomonas (30.1%), Hymenobacter (24.1%) and Methylobacterium (22.4%) the most representative genera (>76% of reads). In addition, the results indicated significant differences in the bacterial communities detected in the xylem sap depending on the genotype of the olive tree studied and, to a minor extent, on the type of sap extraction method used. Among the total genera identified using NGS, 14 (41.2%) were recovered in the culture collection, whereas 20 (58.8%) in the culture collection were not captured by the NGS approach. Some of the xylem-inhabiting bacteria isolated are known biocontrol agents of plant pathogens, whereas for others little information is known and are first reported for olive. Consequently, the potential role of these bacteria in conferring olive tree protection against xylem pathogens should be explored in future research.This study was funded by project 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 Project XF-ACTORS (grant 727987) from the European Union's Horizon 2020 Framework Research Programme. MA-M is a recipient of a research fellowship BES-2017-082361 from the Spanish Ministry of Economy and Competitiveness

Transmission and distribution of Xylella fastidiosa subsp. pauca in olive trees as a parameter for managing olive quick decline syndrome

Systemic bacteria such as Xylella fastidiosa can be vertically transmitted by using diseased propagative material during nursery tree production. We tested the hypothesis that X. fastidiosa is not transmitted by symptomless olive tree sprouts. In addition, we investigated the distribution of bacteria in olive plants (different parts of canopy and root system) with initial leaf scorch symptoms and with symptoms spread throughout the canopy. In both studies, the presence of bacteria was tested by quantitative real-time PCR (qPCR). For the first hypothesis, sprouts from a symptomless olive tree were rooted and checked for X. fastidiosa infection 32 months later. A total of 57.7% (41 of 71) of rooted seedlings were positive for X. fastidiosa (Ct from 24.25 to 31.82) but only one plant had scorched leaves. Regarding the distribution of the bacteria in the diseased plants, results based on qPCR showed that X. fastidiosa was systemically distributed through the olive plant canopy (30% to 77% of sampled tissues, with Ct values from 19.56 to 31.59). Samples from trees showing symptoms of olive quick decline syndrome (OQDS), taken 3 m above the lateral branch with symptoms, were positive for the presence of X. fastidiosa, as was the root system. The results demonstrate that vegetative material from olive plants used for rooting must be carefully selected for the absence of X. fastidiosa. Also, based on the qPCR-positive results, pruning may have a limited effect on eliminating the bacteria from diseased plants, even from the ones showing few OQDS symptoms.This work was funded by Horizon 2020 (XF Actors grant 727987) and FAPESP (São Paulo Research Foundation grant 2016/02176-7). The authors are very grateful to Cristina Maria Vicentine for supporting the sampling of the olive plants. H.D.C.F. thanks the National Council for Scientific and Technological Development (CNPq) for the research fellowship (project 308164/2021-0). Manuel Anguita-Maeso acknowledges the research stay fellowship from SEGIB - Carolina Foundation.Peer reviewe

Evaluation of Established Methods for DNA Extraction and Primer Pairs Targeting 16S rRNA Gene for Bacterial Microbiota Profiling of Olive Xylem Sap

Next-generation sequencing has revolutionized our ability to investigate the microbiota composition of diverse and complex environments. However, a number of factors can affect the accuracy of microbial community assessment, such as the DNA extraction method, the hypervariable region of 16S rRNA gene targeted, or the PCR primers used for amplification. The aim of this study was to assess the influence of commercially available DNA extraction kits and different primer pairs to provide a non-biased vision of the composition of bacterial communities present in olive xylem sap. For that purpose, branches from “Picual” and “Arbequina” olive cultivars were used for xylem sap extraction using a Scholander chamber device. The DNA extraction protocol significantly affected xylem sap bacterial community assessment. That resulted in significant differences in alpha (Richness) and beta diversity (UniFrac distances) metrics among DNA extraction protocols, with the 12 DNA extraction kits evaluated being clustered in four groups behaving differently. Although the core number of taxa detected by all DNA extraction kits included four phyla, seven classes, 12 orders, 16 or 21 families, and 12 or 14 genera when using the Greengenes or Silva database for taxonomic assignment, respectively, some taxa, particularly those identified at low frequency, were detected by some DNA extraction kits only. The most accurate depiction of a bacterial mock community artificially inoculated on sap samples was generated when using the PowerPlant DNA extraction kit, the combination of 799F/1193R primers amplifying the hypervariable V5–V7 region, and the Silva 132 database for taxonomic assignment. The DESeq2 analysis displayed significant differences among genera abundance between the different PCR primer pairs tested. Thus, Enterobacter, Granulicatella, Prevotella, and Brevibacterium presented a significant higher abundance in all PCR protocols when compared with primer pair 799F/1193R, while the opposite was true for Pseudomonas and Pectobacterium. The methodological approach followed in this study can be useful to optimize plant-associated microbiome analysis, especially when exploring new plant niches. Some of the DNA extraction kits and PCR primers selected in this study will contribute to better characterize bacterial communities inhabiting the xylem sap of olives or other woody crop species.This study was funded by the project AGL2016-75606-R (Programa Estatal de I+D Orientado a los Retos de la Sociedad from Spanish Government, the Spanish State Research Agency, and FEDER-EU) and Project XF-ACTORS (grant 727987) from the European Union’s Horizon 2020 Framework Research Programme. MA-M is a recipient of a research fellowship BES-2017-082361 from the Spanish Ministry of Economy and Competitiveness

Culture and metagenomic approaches for the identification of olive xylem microbial communities as a biological control tool to cope against Xylella fastidiosa infection

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

La elección de los iniciadores de PCR para secuenciación masiva y el método de extracción de ADN son factores determinantes en la caracterización de las comunidades microbianas asociadas al xilema del olivo

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

La elección de los iniciadores de PCR para secuenciación masiva y el método de extracción de ADN son factores determinantes en la caracterización de las comunidades microbianas asociadas al xilema del olivo

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