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

Nonlinear evolution of harmonically forced perturbations on a wingtip vortex

Wingtip vortices are created by flying airplanes due to lift generation. The vortex interaction with the trailing aircraft has sparked researchers’ interest to develop an efficient technique to destroy these vortices. Different models have been used to describe the vortex dynamics and they all show that, under real flight conditions, the most unstable modes produce a very weak amplification. Another linear instability mechanism that can produce high energy gains in short times is due to the non-normality of the system. Recently, it has been shown that these non-normal perturbations also produce this energy growth when they are excited with harmonic forcing functions. In this study, we analyze numerically the nonlinear evolution of a spatially, pointwise and temporally forced perturbation, generated by a synthetic jet at a given radial distance from the vortex core. This type of perturbation is able to produce high energy gains in the perturbed base flow (10^3), and is also a suitable candidate for use in engineering applications. The flow field is solved for using fully nonlinear three-dimensional direct numerical simulation with a spectral multidomain penalty method model. Our novel results show that the nonlinear effects are able to produce locally small bursts of instability that reduce the intensity of the primary vortex.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Theoretical parameters of trailing vortices versus aspect ratio of wing models

We perform 2D-PIV measurements to characterize trailing vortices in NACA0012 wing models for aspect ratios ranging between 1 and 2.5, and for chord-based Reynolds numbers from 7000 to 40000. Firstly, and regarding the influence of the Reynolds number, the increase of this dimensionless parameter generates a more concentrated and intense vortex, presenting, therefore, an increase in all its characteristic magnitudes: maximum azimuthal velocity and vorticity. Secondly, the greater the aspect ratio, the greater the vortex strength is observed. Thirdly, the radial location of the peak of the azimuthal velocity has a strong decay as the aspect ratio increases for Re=7000, but it changes its trend for Re=40000.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.. Ministerio de Economía y Competitividad (Spain) Grant No. DPI2013-40479-P and DPI2016-76151-C2-1-R and Junta de Andalucía Grant No. P11-TEP-7776

Stability analysis of flow structures in hovering using robotic experiments and flow visualizations

We investigate the kinematics and stability of hovering flight making use of a robotic experimental device that simulate the movement of insects or birds. We carried out this analysis based on the characterization of this movement with flow visualizations and particle image velocimetry -PIV-. First, we characterized the kinematics of the robotic device inside water. Therefore, it has been verified that the robotic experiments follow the desired input signal precisely. Second, we give qualitative and quantitative information from the experimental tests as a function of frequency and angular amplitude. The kinematics of the hovering flight produces vortices that are stable or unstable around the rigid flat plate, as well as one transient regime that has been also found between these two latter states. Finally, dimensional velocity field plus associated vorticity have been characterized in these flow regimes through PIV measurements, and we also validated the reproducibility of experiment finding excellent agreement between different set of experiments.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Effect of the axial jet on the optimal response in Batchelor vortex

En este póster se estudia la respuesta óptima del torbellino de Batchelor para distintos números de onda. Se demuestra que incluso teniendo la velocidad axial, un torbellino es capaz de tener grandes amplificaciones de energía.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

On the effect of low blowing ratio continuous jets on wingtip vortex characteristics

Vortices are an unavoidable effect of flight, which appear behind the wing with a bounded length. The strength of these vortices, which are extremely stable, is due to the lift force [1]. That is the reason why this phenomenon is especially relevant during take-off and landing operations. In these situations, when aircraft are departing from or arriving to the airport runways, the following aircraft might feel two counter-rotating vortices which remain long time under normal environmental conditions. Unfortunately, this huge rotation of airflow patterns always destabilizes the following aircraft. Consequently, trailing vortices have a mighty influence on the air traffic control of airport runways, and they have justified the research interest in this topic since the 1960's [2]. However, aeronautical engineers are still searching for different technological strategies to breakdown these wingtip vortices.Short-Term Postdoctoral Stay of Talent Attraction Plan of Andalucía TECH ICE. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Wingtip vortex in a NACA0012 airfoil and its active control

Contribución mediante sesión pósterWe conduct experiments in a towing-tank to analyse the flow patterns of wingtip vortices in a NACA 0012 airfoil. In this experimental research, we provide PIV measurements and flow visualisations. Without active control, several parameters are given experimentally as function of the Reynolds number, so we compare these data with the theoretical models of Batchelor, and Moore and Saffman together with DNS. Secondly, we analyse the effect of a continuous injection in the spanwise direction. The continuous jet has a strong influence on the wing-tip vortex formation. We explore this effect at low chord based Reynolds number ranging from 7000 up to 20000. We change the aspect ratio of the injection, R, defined as the ratio of the velocities between the jet (Uj) and free-stream (U). For R=1, we find that the jet strongly affects the wingtip vortex formation with a sudden decrement of the axial vorticity and the azimuthal velocity. This technique is a challenge and a promising tool to reduce the intensity of the vortex core.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

On the optimaL response of q-vortex

Wingtip vortices represent a hazard for the stability of the following airplane in airport highways. These flows have been usually modeled as swirling jets/wakes, which are known to be highly unstable and susceptible to breakdown at high Reynolds numbers for certain flow conditions, but different to the ones present in real flying airplanes. A very recent study based on Direct Numerical Simulations (DNS) shows that a large variety of helical responses can be excited and amplified when a harmonic inlet forcing is imposed. In this work, the optimal response of q-vortex (both axial vorticity and axial velocity can be modeled by a Gaussian profile) is studied by considering the time-harmonically forced problem with a certain frequency ω. We first reproduce Guo and Sun’s results for the Lamb-Oseen vortex (no axial flow) to validate our numerical code. In the axisymmetric case m = 0, the system response is the largest when the input frequency is null. The axial flow has a weak influence in the response for any axial velocity intensity. We also consider helical perturbations |m| = 1. These perturbations are excited through a resonance mechanism at moderate and large wavelengths as it is shown in Figure 1. In addition, Figure 2 shows that the frequency at which the optimal gain is obtained is not a continuous function of the axial wavenumber k. At smaller wavelengths, large response is excited by steady forcing. Regarding the axial flow, the unstable response is the largest when the axial velocity intensity, 1/q, is near to zero. For perturbations with higher azimuthal wavenumbers |m| > 1, the magnitudes of the response are smaller than those for helical modes. In order to establish an alternative validation, DNS has been carried out by using a pseudospectral Fourier formulation finding a very good agreement.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

PIV measurements of the effect of pulsed blowing jet on a NACA0012 wing model

Wingtip vortices are present in taking off, and landing operations and their presence in airport runways must be reduced. To that end, several strategies have been considered in the last decades, being the active control one possible technical solution. To compute the effectiveness of active control that corresponds to pulsed low-blowing-ratio transverse jet for the reduction of the wingtip vortex strength, we carry out 2D-PIV measurements in a towing tank for chord-based Reynolds numbers 15000 and 20000. We consider two cases: (i) no active control Rjet=0 and (ii) pulsating radial jet of blowing-ratio Rjet smaller than 1.7 (or momentum coefficient lower than 0.12) and different Strouhal numbers ranging from 0.27 to 0.94. Our observations show that the best reduction of wingtip vortex strength takes place at the lowest Strouhal number tested. We use the maximum azimuthal velocity and vorticity together with the circulation to quantify this decrease in the vortex strength. Besides, we define the spatial evolution of a disturbance parameter which allow us to detect again the optimal frequency that leads to vortex destruction.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech