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Figure S1: Agarose gels showing traC gene amplification for selected X. fastidiosa strains from the collection at the Instituto de Agricultura Sostenible (IAS-CSIC) using ND116-pRIV5-F1/ND117-pRIV5-R1 primer pair (expected amplicon size of 521bp). PAC = Positive amplification control contained boiled cells of IVIA5235 strain. NAC= Negative amplification control contained DNA from a Xf strain known not to harbor the traC gene. L= 10 Kb GeneRuler™ DNA Ladder Mix (Thermo Scientific™); Figure S2: Alignments of all traC sequences obtained in this study and those retrieved from GenBank using MAFFT. single nucleotide polymorphisms are indicated in relation to the consensus sequence. The subspecies of the Xf strain is indicated between brackets (F= fastidiosa, M= multiplex, P= pauca, M= morus; S= sandyi, S/M= sandyi/morus)

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., Xylella fastidiosa (Xf) has the capacity of uptaking extracellular DNA from the environment and incorporate it into its genome by homologous recombination, process known as Natural Competence. The process of genetic acquisition and recombination of extracellular DNA could be related to the acquisition of new traits such as antibiotic resistance and virulence factors, giving rise to the origin of new pathotypes. We took a step forward in comparative genomics analysis of Spanish populations of Xf by determining the complete genome of several isolates from Alicante, Mallorca and Ibiza and established potential recombination events that may have occurred with isolates from other places in the world. For this purpose, we used combined Illumina and Oxford Nanopore sequencing platforms and combined assembly approaches to obtain complete circularized genomes. Our results showed that olive and almond isolates belonging to subspecies pauca ST80 from Ibiza (Balearic Islands), the only place where this ST has been detected, showed an unusual level of recombination, being so far the isolates showing the highest number of recombination events among all the Xf isolates sequenced to date. On the order hand, we have confirmed the high degree of homology among genomes from Xf strains from Mallorca and California belonging to subspecies fastidiosa ST1 and subspecies multiplex ST81 isolated from almond and grapes, and we have also identified specific long-size genomic rearrangements in isolates of Xf subspecies multiplex ST6 from Alicante, compared to their homologous strains from the Americas. Research is now underway to determine the source and the functionality of these recombinant genes and their role in pathogenicity.

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. Acknowledgement 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.

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.

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).

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.