The olive microbiome and its role in modulating host response to Verticillium dahliae: unraveling the determining and modifying factors

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The olive microbiome and its role in

modulating host response to Verticillium dahliae: unraveling the determining and modifying factors

DOCTORAL THESIS

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

MANUEL ANGUITA MAESO

Supervisors:

Blanca B. Landa del Castillo Juan A. Navas Cortés

Enero, 2023

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TITULO: The olive microbiome and its role in modulating host response to Verticillium dahliae: unraveling the determining and modifying factors

AUTOR: Manuel Anguita Maeso

© Edita: UCOPress. 2023 Campus de Rabanales Ctra. Nacional IV, Km. 396 A 14071 Córdoba

https://www.uco.es/ucopress/index.php/es/

[email protected]

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University of Córdoba

Doctoral Program in Agricultural, Food, Forestry and Sustainable Rural Development Engineering

PhD Thesis

The olive microbiome and its role in modulating host response to Verticillium dahliae: unraveling

the determining and modifying factors Manuel Anguita Maeso

Supervisors:

Blanca B. Landa del Castillo & Juan A. Navas Cortés

Córdoba, January 2023

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Universidad de Córdoba

Programa de Doctorado en Ingeniería Agraria, Alimentaria, Forestal y de Desarrollo Rural Sostenible

Tesis Doctoral

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

factores determinantes y modificadores Manuel Anguita Maeso

Directores:

Blanca B. Landa del Castillo & Juan A. Navas Cortés

Córdoba, Enero 2023

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TÍTULO DE LA TESIS: The olive microbiome and its role in modulating host response to Verticillium dahliae: Unraveling the determining and modifying factors

DOCTORANDO/A: Manuel Anguita Maeso

INFORME RAZONADO DEL/DE LOS DIRECTOR/ES DE LA TESIS

(se hará mención a la evolución y desarrollo de la tesis, así como a trabajos y publicaciones derivados de la misma).

Dra. Blanca Beatriz Landa del Castillo y Dr. Juan Antonio Navas Cortés, Investigadores Científicos del Departamento de Protección de Cultivos del Instituto de Agricultura Sostenible (IAS-CSIC) como directores de la presente Tesis Doctoral

INFORMAN:

Que en la Tesis Doctoral titulada “The olive microbiome and its role in modulating host response to Verticillium dahliae: Unraveling the determining and modifying factors” que ha llevado a cabo el Ldo. en Biología D. Manuel Anguita Maeso bajo nuestra dirección, se han completado con éxito todos los objetivos planteados en dicho trabajo de investigación. Además, se han llevado a cabo todas las actividades formativas obligatorias del programa de Doctorado “Ingeniería agraria, alimentaria, forestal y de desarrollo rural sostenible”, así como otras actividades voluntarias y complementarias.

Del mismo modo, el Ldo. M. Anguita Maeso ha realizado cuatro estancias de movilidad internacional, con un total de 11 meses; siendo tres de ellas de duración igual o superior a 3 meses. Dichas estancias han estado orientadas a la formación del doctorando en un ambiente internacional en áreas científicas punteras relacionadas con el objeto de estudio de la Tesis Doctoral, y han contribuido a complementar su formación predoctoral.

Que dicha Tesis Doctoral se va a presentar como compendio de publicaciones donde se recogen el desarrollo experimental y resultados obtenidos para dar respuesta a los objetivos perseguidos y cumple con todos los requisitos y obligaciones dispuestos en el Real Decreto 534/2013, de 12 de julio y el Reglamento 57/2020 de los Estudios de Doctorado de la Universidad de Córdoba. En cumplimiento del Art. 53 de dicha Normativa, se presentan cinco artículos científicos correspondientes a los Capítulos II, III, IV, V y VIII de la presente Tesis Doctoral:

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Publicaciones en revistas científicas indexadas derivadas de la Tesis Doctoral

1. M. Anguita-Maeso, C. Haro, J. A. Navas-Cortés, B. B. Landa, Primer choice and xylem-microbiome- extraction method are important determinants in assessing xylem bacterial community in olive trees. Plants. 11, 1320 (2022). doi: 10.3390/plants11101320. JCR Impact Factor (2021): 4.658; JIF Rank: 39/238. First Quartile (Q1) in Plant Sciences Category. (Capítulo II)

2. C. Haro*, M. Anguita-Maeso*, M. Metsis, J. A. Navas-Cortés, B. B. Landa, Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiota profiling of olive xylem sap. Front. Plant Sci. 12, 296 (2021). doi: 10.3389/fpls.2021.640829. JCR Impact Factor (2021): 6.627; JIF Rank: 20/238. First Quartile (Q1) in Plant Sciences Category.

(Capítulo III)

3. M. Anguita-Maeso, C. Haro, M. Montes-Borrego, L. De La Fuente, J. A. Navas-Cortés, B. B. Landa, Metabolomic, ionomic and microbial characterization of olive xylem sap reveals differences according to plant age and genotype. Agronomy. 11, 1179 (2021). doi: 10.3390/agronomy11061179.

JCR Impact Factor (2021): 3.949; JIF Rank: 18/90. First Quartile (Q1) in Agronomy Category.

(Capítulo IV).

4. M. Anguita-Maeso, C. Olivares-García, C. Haro, J. Imperial, J. A. Navas-Cortés, B. B. Landa, Culture- dependent and culture-independent characterization of the olive xylem microbiota: effect of sap extraction methods. Front. Plant Sci. 10, 1708 (2020). doi: 10.3389/fpls.2019.01708. JCR Impact Factor (2020): 5.754; JIF Rank: 17/235. First Quartile (Q1) in Plant Sciences Category. (Capítulo V).

5. M. Anguita-Maeso, J. L. Trapero-Casas, C. Olivares-García, D. Ruano-Rosa, E. Palomo-Ríos, R.

M. Jiménez-Díaz, J. A. Navas-Cortés, B. B. Landa, Verticillium dahliae inoculation and in vitro propagation modify the xylem microbiome and disease reaction to Verticillium wilt in a wild olive genotype. Front. Plant Sci. 12, 250 (2021). doi: 10.3389/fpls.2021.632689. JCR Impact Factor (2021): 6.627; JIF Rank: 20/238. First Quartile (Q1) in Plant Sciences Category. (Capítulo VIII).

Otras aportaciones destacables que han surgido de la presente Tesis Doctoral son:

1. M. Anguita-Maeso, C. Haro, J. L. Trapero, G. León-Ropero, M. P. Velasco-Amo, J. Imperial, B.

Jorrín, J. A. Navas-Cortés, B. B. Landa. Descifrando, modelizando y cultivando el microbioma xilemático del olivo para incrementar su resiliencia a enfermedades vasculares (SynXylComs).

Mercacei magazine, Nº. 113, pp. 108-113, 2022.

Estancias de Investigación para el Desarrollo de la Tesis Doctoral:

1. Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Belgium. 22 August – 19 November 2022 (3 months). Decipher microbial network associations in olive tree determined by ecological niche, genotype, season and field location. Supervisor: Prof.

Dr. Karoline Faust.

2. Max Planck Institute for Plant Breeding Research. Cologne, Germany. 17 May – 16 August 2021 (3 months). Genome diversity and antagonistic activity of microbial communities present in the xylem of olive trees. Supervisor: Prof. Dr. Paul Schulze-Lefert.

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3. Center of Citricultura Sylvio Moreira, Instituto Agronômico de Campinas (IAC). Cordeirópolis, (São Paulo), Brazil. 29 January - 28 April 2021 (3 months). Characterization of the microbial communities present in the xylem of the olive tree as a control tool for the phytopathogenic bacterium Xylella fastidiosa. Supervisor: Dr. Helvécio Della Coletta Filho.

4. National University of Ireland, Dept. of Microbiology. Galway, Ireland. 21 September – 29 November 2020 (2,5 months). Effect of plant genotype, plant niche and environmental conditions on the diversity and structure of microbial communities in cultivated olive trees. Supervisor: Dr.

Alexandre de Menezes.

Congresos Nacionales e Internacionales en los que se han presentado resultados de la Tesis Doctoral

1. Anguita-Maeso, Manuel; Haro, Carmen, Olivares-García, Concepción, León-Ropero, Guillermo;

Velasco-Amo, María Pilar; Arias-Giraldo, Luis F.: Trapero-Casas, José Luis; Navas-Cortés, Juan A.;

Landa, Blanca B. Factores determinantes y modificadores asociados a las comunidades microbianas presentes en el xilema del olivo. XX Congreso de la Sociedad Española de Fitopatología (SEF).

Oral communication. 24-26 October 2022. Valencia, Spain

2. Haro, Carmen; Anguita-Maeso, Manuel; León-Ropero, Guillermo; Quiles-Pando, Carlos; Imperial, Juan; Navas-Cortés, Juan A.; Landa del Castillo, Blanca B. Evaluación de diferentes métodos de estabilización del microbioma xilemático del olivo para su análisis mediante NGS. XX Congreso de la Sociedad Española de Fitopatología (SEF). Poster communication. 24-26 October 2022.

Valencia, Spain.

3. Haro, Carmen; Rivas-Romero, Juan C.; Trapero-Casas, Jose L.; León-Ropero, Guillermo; Navas- Cortés, Juan A.; Anguita-Maeso, Manuel; Landa del Castillo, Blanca B. Variación estacional del microbioma xilemático de cultivares de olivo. XX Congreso de la Sociedad Española de Fitopatología (SEF). Poster communication. 24-26 October 2022. Valencia, Spain.

4. Anguita-Maeso, Manuel. Determining and modifying factors associated with the microbial communities present in the xylem of olive tree. The Xylella Files. Oral communication. 13 September 2022.

5. Anguita-Maeso, Manuel; Olivares-Garcia, C.; Navas-Cortés, Juan A.; Coletta-Filho, Helvecio D;

Landa, Blanca B. Systemic distribution of Xylella fastidiosa within olive tree branches and changes on the associated xylem microbiome communities. 14th International Conference on Plant Pathogenic Bacteria (ICPPB). Oral communication. 3–8 July 2022. Assisi, Italy.

6. Anguita-Maeso, Manuel. The microbiome of the olive tree and its role in the response of the plant to Verticillium wilt caused by Verticillium dahliae: determining and modifying factors. XII meeting of IAS PhD students. Oral communication. 29 June 2022. Córdoba, Spain.

7. Anguita-Maeso, Manuel; Navas-Cortés, Juan A.; Coletta-Filho, Helvecio D; Landa, Blanca B.

Changes in the xylem microbiota associated to infection by Xylella fastidiosa in Brazilian olive groves. Congress of the Mediterranean Phytopathological Union (MPU). Oral communication.

4–8 April 2022. Limassol, Cyprus.

8. Anguita-Maeso, Manuel; Haro, Carmen; Rivas, Juan C.; Navas-Cortés, Juan A.; Landa, Blanca B.

La elección de los iniciadores de PCR para secuenciación masiva y el método de extracción de

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ADN son factores determinantes en la caracterización de las comunidades microbianas asociadas al xilema del olivo. Encuentro Internacional Phytoma-España. Poster communication. 1-2 December 2021. Córdoba, Spain.

9. Anguita-Maeso, Manuel; Velasco-Amo, María P.; Haro, Carmen; Arias-Giraldo, Luis F.; Imperial, Juan; Navas-Cortés, Juan A.; Landa, Blanca B. Unraveling the whole genome of olive antagonistic xylem-inhabiting bacteria to fight vascular plant pathogens in olive trees. Nanopore Community Meeting conference. Oral communication. 30 November - 2 December 2021.

10. Anguita-Maeso, Manuel; Haro, Carmen; Testi, Luca; Belaj, Angjelina; Navas-Cortés, Juan A.; Landa, Blanca B. Impact of plant age and genotype on xylem microbial network associations in cultivated olive trees. V Symposium on Ecological Network. Oral communication. 10-12 November 2021.

Palma de Mallorca, Spain.

11. Anguita-Maeso, Manuel; Duran, Paloma; Domínguez-Calero, Cristina; Imperial, Juan; Navas- Cortés, Juan A.; Schulze-Lefert, Paul; Landa, Blanca B. Antagonistic activity against Verticillium dahliae of the endophytic bacterial strains isolated from the xylem of olive trees. Nature Conferences. Harnessing the Plant Microbiome. Poster communication. 22-24 October 2021.

12. Anguita-Maeso, Manuel; Haro, Carmen; Testi, Luca; Belaj, Angjelina; Navas-Cortés, Juan A.;

Landa, Blanca B. Efecto de la variedad y la edad de la planta en la composición del microbioma xilemático del olivo. XVI Congreso Nacional de Ciencias Hortícolas. Oral communication. 17-21 October 2021. Córdoba, Spain.

13. Anguita-Maeso, Manuel; León-Ropero, Guillermo; Trapero-Casas, José L.; Navas-Cortés, Juan A., Landa, Blanca B. Plant endotherapy treatments enable the modification of xylem microbiome composition in olive trees. 9th IOBC-WPRS meeting on Integrated Protection of Olive Crops.

Oral communication. 26-29 October 2021. Lisbon, Portugal.

14. Anguita-Maeso, Manuel; Haro, Carmen; Montés-Borrego, Miguel; de la Fuente, Leonardo; Navas Cortés, Juan A.; Landa, Blanca B. La caracterización metabólica, ionómica y microbiana del fluido xilemático revela diferencias según la edad y el genotipo de olivo. XX Simposio científico-técnico EXPOLIVA. Poster communication. 22 - 25 September 2021.

15. Anguita-Maeso, Manuel; León-Ropero, Guillermo; Navas Cortés, Juan A.; Landa, Blanca B.

Influencia del genotipo de olivo, factores agroambientales y estacionales en la estructura y diversidad del microbioma xilemático del olivo cultivado. XXVIII Congreso de la Sociedad Española de Microbiología. Oral communication. 28 June - 2 July 2021.

16. Anguita-Maeso, Manuel; Arias-Giraldo, Luis F.; Navas-Cortés, Juan A., de Menezes, Alexandre;

Landa, Blanca B. Bioinformatic pipelines are determinant in the analysis of microbial communities from different ecological niches in cultivated olive trees. 4th Annual Conference of the EuroXanth COST Action Integrating Science on Xanthomonadaceae for integrated plant disease management in Europe. Oral communication. 28-30 June 2021.

17. Anguita Maeso, Manuel; Rivas, Juan C.; León, Guillermo; Estudillo, Cristina; Navas-Cortés, Juan A.; Landa, Blanca B. Soil physicochemical properties, seasonality, plant niche and plant genotype affect bacterial and fungal communities in olive orchard soils. Global Symposium on Soil Biodiversity (GSOBI21). Oral communication. 19-22 April 2021.

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18. Anguita Maeso, Manuel; Olivares-García, Concepción; Rivas, Juan C.; Navas-Cortés, Juan A.;

Landa, Blanca B. Broth media cultivation of xylem microbiome from cultivated olive trees. III European Conference on Xylella fastidiosa. Poster communication. 26-30 April 2021.

19. Anguita-Maeso, Manuel; Estudillo-Cazorla, Cristina; León-Ropero, Guillermo; Navas-Cortés, Juan A.; de Menezes, Alexandre and Landa, Blanca B. Co-occurrence network inference analysis allows identification of keystone microbial species associated to soil compartments and environments in cultivated olive. vEGU21. EGU General Assembly, held online. Oral communication 19-30 April, 2021.

20. Anguita-Maeso, Manuel; Trapero, José Luis; Olivares-García, Concepción; Ruano-Rosa, David;

Palomo-Ríos, Elena; Jiménez-Díaz, Rafael; Navas-Cortés, Juan A. and Landa, Blanca B. La atenuación de la microbiota del xilema modifica la reacción de resistencia de un clon de olivo silvestre a la infección por Verticillium dahliae. IX Reunión del grupo Microbiología de Plantas- SEM. Oral communication. 16-17 February 2021.

21. Anguita-Maeso, Manuel. The microbiome of the olive tree and its role in the response of the plant to Verticillium wilt caused by Verticillium dahliae: determining and modifying factors. X meeting of IAS PhD students. Online. Oral communication. 29 October 2020. Córdoba, Spain.

22. Anguita-Maeso, Manuel; Rivas, Juan Carlos; León, Guillermo; Estudillo, Cristina; Navas-Cortés, Juan Antonio; Landa, Blanca Beatriz. Soil microbial communities from olive cultivars are shaped by seasonality and geographical scales. European Geosciences Union (EGU2020: Sharing Geoscience Online). EGU2020-4224, 2020. Oral communication. 4-8 May 2020. Viena, Austria.

doi: 10.5194/egusphere-egu2020-4224

23. Anguita-Maeso, Manuel; Navas-Cortés, Juan Antonio; Landa, Blanca Beatriz. The olive microbiome based on its genotype and its ecological niche. VII Scientific Congress of Research Formation of the University of Córdoba. ISBN 978-84-9927-508-6. Oral communication. 18-19 February 2020.

Córdoba, Spain.

24. Anguita-Maeso, Manuel; Haro, Carmen; Rivas, Juan Carlos; León, Guillermo; Estudillo, Cristina;

Costa, Joana; Ares, Aitana; Navas-Cortés, Juan Antonio; Landa, Blanca Beatriz. Characterization of olive xylem microbiome community composition by metabarcoding greatly depends on the matrix used to extract DNA and 16S universal bacterial PCR primers. II European Conference on Xylella fastidiosa. Poster communication. 29-30 October 2019. Ajaccio, France.

25. Anguita-Maeso, Manuel; Rivas, Juan Carlos; León, Guillermo; Estudillo, Cristina; Navas-Cortés, Juan Antonio; Landa, Blanca Beatriz. Plant genotype, soil and climate as drivers of the olive microbiome composition. Conference on Soil Biota driven Ecosystem Services in European Agriculture. Poster Pitch communication. 22-23 October 2019. Braunschweig, Germany.

26. Anguita-Maeso, Manuel; Olivares-García, Concepción; Velasco-Amo, María del Pilar; Rivas, Juan Carlos; Román-Écija, Miguel; Navas-Cortés, Juan A.; Landa, Blanca B. The microbiome of the olive xylem as a tool for biological control of its vascular diseases. II Congress of Young Researchers in Agrifood Sciences in Almería. ISBN 978-84-09-17547-5. Oral communication. 17 octubre 2019.

Almería, Spain.

27. Anguita-Maeso, Manuel; Haro, Carmen; Rivas, Juan Carlos; Navas Cortés, Juan Antonio; Landa, Blanca Beatriz. Differences in xylem sap composition of distinct olive tree varieties: a metabolomic

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approach. Specialized Metabolites Symposium. Oral communication. 4-5 July 2019. Gif-sur- Yvette, France.

28. Anguita-Maeso, Manuel. The microbiome of the olive tree and its role in the response of the plant to Verticillium wilt caused by Verticillium dahliae: determining and modifying factors. IX meeting of IAS PhD students. Oral communication. 14 June 2019. Córdoba, Spain.

29. Anguita-Maeso, Manuel. Cultivation of the xylem microbiome of the olive tree in artificial media.

VIII Scientific Congress of Research Formation of the University of Córdoba. ISBN 978-84-9927- 341-9. Oral communication. 7-6 February 2019. Córdoba, Spain.

En conclusión, Manuel Anguita Maeso cuenta con cinco artículos indexados derivados de su Tesis Doctoral, todos ellos en el primer cuartil de su categoría científica dentro del Journal Citation Report (Clarivate) y como primer autor, y un artículo en revista de divulgación. Además, ha participado en 29 congresos nacionales e internacionales (27 comunicaciones como primer autor) con 23 exposiciones orales y cuatro presentaciones en formato panel. Ha sido beneficiario de cuatro becas competitivas de diferentes instituciones que le han permitido realizar cuatro estancias de investigación en diversos países. También, ha sido beneficiario de un proyecto de investigación por una entidad pública y ha participado en labores de docencia siendo miembro de tribunal de un trabajo fin de máster. Del mismo modo, ha obtenido una amplia formación científica y técnica con la asistencia y aprovechamiento a 33 cursos y seminarios. Asimismo, ha participado en la organización de nueve actividades de diferente índole científico y ha realizado nueve acciones divulgativas de la ciencia y de su trabajo realizado durante la Tesis Doctoral.

Por todo ello, consideramos finalizada dicha Tesis Doctoral, y que puede ser presentada y tramitada para su exposición y defensa en la Comisión de Doctorado de la Universidad de Córdoba con mención internacional en la modalidad por compendio de publicaciones.

Córdoba, 4 de enero de 2023

Firma de los directores

Fdo.: Blanca B. Landa del Castillo Fdo.: Juan A. Navas Cortés

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TÍTULO DE LA TESIS: The olive microbiome and its role in modulating host response to Verticillium dahliae: Unraveling the determining and modifying factors

DOCTORANDO/A: Manuel Anguita Maeso

INFORME RAZONADO DEL TUTOR (Ratificando el informe favorable del director.

Sólo cuando el director no pertenezca a la Universidad de Córdoba).

Dra. María Esperanza Sánchez Hernández, como tutora del Ldo. Manuel Anguita Maeso perteneciente al Programa de doctorado de “Ingeniería agraria, alimentaria, forestal y de desarrollo rural sostenible”

informa que:

Durante su periodo como doctorando del programa, desde marzo de 2018, hasta la fecha de emitir este informe ha cumplido todas las actividades formativas obligatorias del programa, así como otras actividades voluntarias y complementarias, con diligencia y dedicación. En consecuencia, como tutora de la Tesis Doctoral que comprende dicha investigación considero que el doctorando ha logrado una evolución y desarrollo satisfactorio, lo que le ha permitido alcanzar el nivel de especialización requerido para optar al grado de doctor.

Por todo ello, considero que puede ser presentada para su exposición y defensa pública y ratifico la consideración favorable emitida por los Directores de la Tesis Doctoral a tal efecto.

Córdoba, cuatro de enero de dos mil veintitrés

Firma de la tutora

Fdo.: María Esperanza Sánchez Hernández Catedrática de Producción Vegetal

Dpto. Agronomía-Unidad de Excelencia María de Maeztu (DAUCO), ETSIAM Firmado por SANCHEZ HERNANDEZ MARIA ESPERANZA - 51897600D el dï¿½a 09/01/2023 con un certificado emitido por AC FNMT Usuarios

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Financiación

Esta Tesis Doctoral ha sido realizada en el Grupo de Investigación Fitopatología de Sistemas Agrícolas Sostenibles perteneciente al Grupo PAIDI AGR-136 ‘Sanidad Vegetal’ del Departamento de Protección de Cultivos del Instituto de Agricultura Sostenible (IAS-CSIC). Manuel Anguita Maeso ha sido beneficiario de un contrato predoctoral BES-2017-082361 en el marco del programa para la formación de doctores del Subprograma Estatal de Formación del Programa Estatal de Promoción del Talento y su Empleabilidad, Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016. Dicho contrato predoctoral está adscrito al Proyecto de Investigación AGL2016-75606-R “El microbioma del olivo y su papel en la respuesta de la planta a la Verticilosis causada por Verticillium dahliae: factores determinantes y modificadores” financiado por el Programa Estatal de I+D+i Orientado a los Retos de la Sociedad del Gobierno de España, la Agencia Estatal de Investigación y los fondos FEDER de la Unión Europea.

Además, los trabajos incluidos en esta Tesis Doctoral han sido parcialmente financiados por el Proyecto de Investigación 727987 ‘XF-ACTORS: Xylella fastidiosa Active Containment Through a Multidisciplinary Oriented Research Strategy’ del Programa Marco de Investigación Horizonte 2020 de la Unión Europea y por el Proyecto de Investigación PID2020-114917RB-I00 ‘Descifrando, modelizando y cultivando el microbioma xilemático del olivo para incrementar su resiliencia a enfermedades vasculares’ del Programa Estatal I+D+i de Generación del Conocimiento y Fortalecimiento Científico y Tecnológico del Sistema de I+D+i y de I+D+i Orientada a los Retos de la Sociedad en el marco del Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020.

Del mismo modo, la realización de estos trabajos ha sido posible gracias a la financiación de diversas instituciones orientadas a fomentar la movilidad internacional del contratado predoctoral a través de convocatorias competitivas: ‘COST Action EuroXanth: Integrating science on Xanthomonadaceae for integrated plant disease management in Europe’, Fundación Carolina, German Academic Exchange Service (DAAD) y el Campus de Excelencia Internacional en Agroalimentación (ceiA3).

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TÍTULO DE LA TESIS: The olive microbiome and its role in modulating host response to Verticillium dahliae: Unraveling the determining and modifying factors

DOCTORANDO/A: Manuel Anguita Maeso

Mención Internacional en el título de Doctor

Mediante la presentación de esta Tesis Doctoral se propone optar a la mención de Doctorado Internacional, debido a que el doctorando presenta los siguientes requisitos exigidos para tal mención según queda reflejado en el Artículo 54 del Acuerdo de Consejo de Gobierno, en sesión ordinaria de 27 de julio de 2022, por el que se aprueba la modificación del Reglamento 26/2021, aprobado en sesión ordinaria de Consejo de Gobierno de 24 de mayo de 2021 (BOUCO núm. 2021/00584, de 24 de mayo), por el que se regula la Organización y Funcionamiento interno de la Comisión Académica del Programa de Doctorado en Ingeniería Agraria, Alimentaria, Forestal y del Desarrollo Rural Sostenible por la Universidad de Córdoba y la Universidad de Sevilla.

Para tal fin, se acredita lo siguiente:

1. Informes favorables de dos doctores pertenecientes a Instituciones de Enseñanza Superior de otros países:

- Prof. Dr. Jeff Dangl, Department of Biology, University of North Carolina, Chapel Hill (Carolina del Norte, Estados Unidos).

- Prof. Dr. Timothy Paulitz, Department of Plant Pathology, Washington State University (Washington, Estados Unidos).

2. Uno de los miembros del tribunal que ha de evaluar la Tesis Doctoral pertenece a un centro de Enseñanza Superior de otro país:

- Prof. Dr. Jos Raaijmakers, Department of Microbial Ecology, Netherlands Institute of Ecology, Wageningen (Holanda).

3. La exposición y la defensa de esta Tesis Doctoral se realizarán en una de las lenguas habituales para la comunicación científica diferente a la materna: inglés.

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4. Estancia de tres meses en un centro de investigación de otro país:

- Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Bélgica. 22 Agosto – 19 Noviembre 2022 (3 meses). Tutora: Prof. Dr. Karoline Faust.

- Max Planck Institute for Plant Breeding Research. Colonia, Alemania. 17 Mayo – 16 Agosto 2021 (3 meses). Tutor: Prof. Dr. Paul Schulze-Lefert.

- Center of Citricultura Sylvio Moreira, Instituto Agronômico de Campinas (IAC).

Cordeirópolis, (São Paulo), Brasil. 29 Enero - 28 Abril 2021 (3 meses). Tutor: Dr.

Helvécio Della Coletta Filho.

Córdoba, 4 de enero de 2023 Firma del doctorando

Fdo.: Manuel Anguita Maeso

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por ANGUITA MAESO

MANUEL - 26252940G

Fecha: 2023.01.04

08:24:11 +01'00'

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TÍTULO DE LA TESIS: The olive microbiome and its role in modulating host response to Verticillium dahliae: Unraveling the determining and modifying factors

DOCTORANDO/A: Manuel Anguita Maeso

Indicios de calidad científica de la Tesis Doctoral

Según el Artículo 53 del Acuerdo de Consejo de Gobierno, en sesión ordinaria de 27 de julio de 2022, por el que se aprueba la modificación del Reglamento 26/2021, aprobado en sesión ordinaria de Consejo de Gobierno de 24 de mayo de 2021 (BOUCO núm. 2021/00584, de 24 de mayo), por el que se regula la Organización y Funcionamiento interno de la Comisión Académica del Programa de Doctorado en Ingeniería Agraria, Alimentaria, Forestal y del Desarrollo Rural Sostenible por la Universidad de Córdoba y la Universidad de Sevilla, esta Tesis Doctoral cumple el requisito establecido en tal artículo para su presentación como compendio de publicaciones, consistente en un mínimo de 3 artículos publicados en revistas incluidas en los tres primeros cuartiles del JCR (Journal Citation Reports) en la categoría y año correspondiente a cada artículo, tal y como está indicado en cada uno de ellos:

1. M. Anguita-Maeso, C. Haro, J. A. Navas-Cortés, B. B. Landa, Primer Choice and Xylem- Microbiome-Extraction Method Are Important Determinants in Assessing Xylem Bacterial Community in Olive Trees. Plants. 11, 1320 (2022). doi: 10.3390/plants11101320. JCR Impact Factor (2021): 4.658; JIF Rank: 39/238. First Quartile (Q1) in Plant Sciences Category.

2. M. Anguita-Maeso, C. Haro, M. Montes-Borrego, L. De La Fuente, J. A. Navas-Cortés, B. B.

Landa, Metabolomic, ionomic and microbial characterization of olive xylem sap reveals differences according to plant age and genotype. Agronomy. 11, 1179 (2021). doi: 10.3390/agronomy11061179.

JCR Impact Factor (2021): 3.949; JIF Rank: 18/90. First Quartile (Q1) in Agronomy Category.

3. M. Anguita-Maeso, J. L. Trapero-Casas, C. Olivares-García, D. Ruano-Rosa, E. Palomo-Ríos, R.

M. Jiménez-Díaz, J. A. Navas-Cortés, B. B. Landa, Verticillium dahliae inoculation and in vitro propagation modify the xylem microbiome and disease reaction to Verticillium wilt in a wild olive genotype. Front. Plant Sci. 12, 250 (2021). doi: 10.3389/fpls.2021.632689. JCR Impact Factor (2021): 6.627; JIF Rank: 20/238. First Quartile (Q1) in Plant Sciences Category.

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4. C. Haro^*, M. Anguita-Maeso^*, M. Metsis, J. A. Navas-Cortés, B. B. Landa, Evaluation of established methods for DNA extraction and primer pairs targeting 16S rRNA gene for bacterial microbiota profiling of olive xylem sap. Front. Plant Sci. 12, 296 (2021). doi: 10.3389/fpls.2021.640829. JCR Impact Factor (2021): 6.627; JIF Rank: 20/238. First Quartile (Q1) in Plant Sciences Category.

5. M. Anguita-Maeso, C. Olivares-García, C. Haro, J. Imperial, J. A. Navas-Cortés, B. B. Landa, Culture-dependent and culture-independent characterization of the olive xylem microbiota: effect of sap extraction methods. Front. Plant Sci. 10, 1708 (2020). doi: 10.3389/fpls.2019.01708. JCR Impact Factor (2020): 5.754; JIF Rank: 17/235. First Quartile (Q1) in Plant Sciences Category.

Córdoba, 4 de enero de 2023 Firma del doctorando

Fdo.: Manuel Anguita Maeso

ANGUITA

MAESO MANUEL - 26252940G

Firmado digitalmente

por ANGUITA MAESO

MANUEL - 26252940G

Fecha: 2023.01.04

08:24:59 +01'00'

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Agradecimientos

¡Sí, sí, síiii! Ha llegado el momento que todo doctorando está deseando que llegue al embarcarse en esta aventura. Han sido cuatro años y medio muy intensos, pero también muy gratificantes, que volvería a repetir sin pensármelo dos veces. Mucha gente se ha cruzado en mi vida durante este camino, por eso quiero agradecer a todas esas personas que han colaborado a ser quien soy hoy y que sin ellos no habría sido posible el desarrollo personal y profesional que supone hacer una Tesis Doctoral.

Por todo ello, estaré siempre agradecido:

A Blanca, por confiar en mí, por darme una oportunidad y por dejarme hacer y deshacer en el laboratorio para poder crecer científicamente hasta volver a todos locos y conseguir estresarla, cosa que poca gente puede decir.

A Juan Antonio, por su apoyo, por sus análisis, por sus scripts, por sus innumerables invitaciones y por su disponibilidad para ayudar en lo que sea necesario incluso cuando el Real Madrid está jugando la Champions.

A Alex, Helvécio, Paul y Karoline por acogerme en sus grupos de investigación, por descubrir diversas técnicas y por enseñarme la manera de hacer ciencia en otros países.

A José Luis, por mantener las condiciones idóneas de los experimentos, por cuidar de las plantas y por su minuciosidad y detalle para explicar el riego necesario para cada planta.

A Guille, por su ayuda en los muestreos, por sus trucos en el montaje de los experimentos y por inculcarnos una dieta sana y saludable, aunque algunos seamos un caso perdido.

A Conchi, por estar siempre dispuesta a hacerte un favor, por sus ansiados gifs en los grupos de whatsapp y por tener todo el material de laboratorio en mente, hasta saber lo que hay en la esquina mas remota del almacén.

A Carmen, por sus discusiones con los resultados, por ver diferentes alternativas de los experimentos y por formar parte del club del microbioma.

A Pilar, por ser la divulgadora oficial de nuestros trabajos, por ser mi instagramer y twittera favorita, por ser mi estilista personal, por nuestras confidencias y por nuestros viernes de McDonald’s.

A Giovana, por nuestro encuentro en Brasil, por sus brigadeiros y por estar delante de mí en la sala de estudio con todo lo que ello supone.

A Luis Felipe, por hacerme ver que a Linux y a R se les puede coger cariño, por nuestras numerosas experiencias vividas y por su falta de orientación en las rutas de senderismo.

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A Miguel, por su gesto de aprobación, por su paladar tan exquisito en las catas, y por estar presente en toda presentación con su foto legendaria.

A Juan Carlos, por su apoyo para integrarme al grupo en mi llegada, por sus notas de aviso en el ordenador y por sus preguntas incomodas y curiosas.

A Cristina Estudillo, por ser mi alumna mas aventajada, por nuestros récords en los experimentos y por ser mi esclava a ritmo de reguetón.

A Cristina (Patricia), por mantener vivas a nuestras niñas, por ponerme mote al segundo día y por mostrarme que los dramas pueden ser divertidos.

A María José, por revelarme los misterios de la química, por sus nervios previos en las presentaciones y por nuestro paseo en Sevilla viendo palomas.

A Carlos, por ser tan metódico, por dejar anotado los protocolos en las paredes y por dar otra vida a los papeles de filtro.

En definitiva, a todos los doctorandos del IAS por las fiestas, comidas, eventos, reuniones, cotilleos, catas, ferias, viajes, playa, rafting, noches locas de la regadera, barbacoas en el jardin, mi 30 cumpleaños, rutas de senderismo, el ambiente de las salas de estudio, piraguismo por el Guadalquivir, nuestro bar clásico de la esquina, apropiacion de piscinas ajenas, noches de juegos de mesa, los elegidos que probaron mi maquina del gym y por diversas actividades difícil de relatar en todo este tiempo (no los nombro a todos porque seguro que me falta alguien pero vosotros sabéis quienes sois); al personal de administración y servicios, en especial a José Juan por hacer que las facturas y los anexos fluyan de manera eficiente; a las personas que se han cruzado en mi camino por Córdoba (a mis compañeros de piso, a la gente del gimnasio, del padel y en especial a María por sus maríadas que van a ser imposibles de olvidar); a las personas que se han cruzado también durante mis estancias en Irlanda, Brasil, Alemania y Bélgica por ayudarme a situarme y a disfrutar de la mejor manera posible, y como no, a mis amigos de Granada que me vieron en mis inicios científicos y que vamos creciendo juntos en la distancia, en especial a Viky, a Joan y a Gonzalo.

Y, por último, a mi familia, por apoyarme en este largo camino y por preocuparse que no me faltase de nada para poder hacer lo que quiero hacer, y para poder ser lo que quiero ser.

A todos, muchas gracias.

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“It is not the strongest of the species that survive, nor the most intelligent but the one most adaptive to change”

“No es la más fuerte de las especies la que sobrevive, ni la más inteligente sino la que mejor se adapta al cambio“

Charles Darwin (1809-1882)

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MICROBIAL COMMUNITIES PRESENT IN THE XYLEM OF OLIVE TREES . . . 55 1. Introduction . . . 58 2. Olive tree and its vascular pathogens . . . 59 3. The role of xylem composition and its associated microorganisms in plants . . . 60 4. Methodological approaches to study the xylem microbiota . . . 61 4.1 Methods used to extract xylem microbial communities . . . 61 4.2 Assessment of microbial communities by culture-dependent approaches . . . 62 4.3 Assessment of microbial communities by next generation sequencing . . . 63 5. Biotic and abiotic factors that influence xylem microbiota . . . 65 5.1 Plant associated factors . . . 65 5.2 Environmental factors . . . 68 5.3 Infection by vascular pathogens . . . 68 6. Future perspectives for xylem microbiome analysis . . . 69 6.1 Bioinformatic analysis: Network analysis . . . 69 6.2 Metagenomics . . . 70 6.3 Culturomics era . . . 70 6.4 Endotherapy treatments . . . 71 7. References . . . 73

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The olive microbiome and its role in modulating host response to Verticillium dahliae:

unraveling the determining and modifying factors

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CHAPTER II.

EVALUATION OF ESTABLISHED METHODS FOR DNA EXTRACTION AND PRIMER PAIRS TARGETING 16S rRNA GENE FOR BACTERIAL MICROBIOTA

PROFILING OF OLIVE XYLEM SAP . . . 85 1. Introduction . . . 88 2. Materials and Methods . . . 90 2.1 Xylem sap collection . . . 90 2.2 Extraction of microbial DNA from xylem sap . . . 90 2.3 Validation of a DNA extraction protocol and PCR Primers . . . 90 2.4 Sampling xylem from olive cultivars and extracting DNA . . . 91 2.5 16S rRNA gene amplification. . . 94 2.6 Library preparation and sequencing . . . 95 2.7 Data processing and bioinformatic analysis . . . 96 3. Results . . . 97 3.1 Effect of DNA extraction protocols on xylem sap bacterial community assessment. . . . 97 3.2 Validation of a DNA extraction method, primer pair performance, and taxonomy

reference databases . . . 104 3.3 Effect of primer pairs on xylem sap bacterial community assessment of two olive cul-

tivars . . . 104 4. Discussion . . . 109 5. References . . . 115

CHAPTER III.

PRIMER CHOICE AND XYLEM-MICROBIOME-EXTRACTION METHOD ARE IMPORTANT DETERMINANTS IN ASSESSING XYLEM BACTERIAL

COMMUNITY IN OLIVE TREES . . . 121 1. Introduction . . . 124 2. Materials and Methods . . . 126 2.1 Plant sampling . . . 126 2.2 DNA extraction and library sequencing . . . 126 2.3 Bioinformatics and statistical approximation . . . 128 3 Results . . . 129 3.2 Illumina output and taxa diversity of plastids and bacterial community . . . 129 3.2 Xylem-bacterial-community distribution . . . 131 3.3 Bacterial-abundance analysis . . . 133 3.4 Effects of PCR-primer pairs, xylem-extraction method and plant age on bacte-

rial-community structure . . . 135 4 Discussion . . . 135 5 References . . . 140

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Table of Contents

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CHAPTER IV.

METABOLOMIC, IONOMIC AND MICROBIAL CHARACTERIZATION OF OLIVE XYLEM SAP REVEALS DIFFERENCES ACCORDING TO PLANT AGE AND

GENOTYPE . . . 147 1. Introduction . . . 151 2. Materials and Methods . . . 152 2.1 Olive plant material and sampling . . . 152 2.2 Xylem sap extraction . . . 153 3.3 Metabolomic and ionomic analysis . . . 153 2.4 Microbiome analysis . . . 154 2.5 Statistical and bioinformatics analyses . . . 154 3. Results . . . 155 3.1 Metabolite and ion profiles in olive xylem sap . . . 155 3.2 Alpha and beta microbial diversity . . . 164 3.3 Composition and abundance of bacterial communities in olive xylem sap . . . 165 3.4 Effect of olive plant age and genotype in xylem sap chemical and microbial composition 166 4. Discussion . . . 171 5. References . . . 174

CHAPTER V.

CULTURE-DEPENDENT AND CULTURE-INDEPENDENT

CHARACTERIZATION OF THE OLIVE XYLEM MICROBIOTA: EFFECT OF SAP EXTRACTION METHODS . . . 181 1. Introduction . . . 184 2. Materials and Methods . . . 185 2.1 Sampling of olive trees . . . 185 2.2 Microbiome extraction from xylem . . . 186 2.3 Culture-dependent characterization of xylem bacteria . . . 186 2.4 Culture-independent characterization of xylem bacteria . . . 187 2.5 Statistical and bioinformatics analysis . . . 188 3. Results . . . 189 3.1 Bacterial abundance and alpha diversity measures . . . 189 3.2 Composition of xylem sap bacterial communities . . . 191 3.3 Bacterial abundance distribution . . . 193 3.4 Bacterial community structure . . . 196 4. Discussion . . . 197 5. References . . . 200

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CHAPTER VI.

TIME COURSE ANALYSIS OF OLIVE XYLEM SAP MICROBIOTA CULTIVATION REVEALS DIFFERENCES ACCORDING TO THE GROWTH MEDIA AND PLANT GENOTYPE . . . 207 1. Introduction . . . 211 2. Materials and Methods . . . 213 2.1 Plant material . . . 213 2.2 Xylem sap extraction . . . 213 2.3 Culture of xylem sap microbiome in broth culture media . . . 213 2.4 DNA extraction and 16S-PCR amplification . . . 214 2.5 Bioinformatics and statistical analysis . . . 215 3. Results . . . 216 3.1 Culture media and xylem sap chemical profiles . . . 216 3.2 Dynamics of microbial growth over time . . . 217 3.3 α- and β-bacterial diversity analysis . . . 218 3.4 Composition of xylem sap bacterial communities . . . 220 3.5 Differential abundance of culturable bacteria associated to the broth culture media . . . 225 4. Discussion . . . 229 5. References . . . 234 CHAPTER VII.

VERTICILLIUM DAHLIAE INOCULATION AND IN VITRO PROPAGATION MODIFY THE XYLEM MICROBIOME AND DISEASE REACTION TO

VERTICILLIUM WILT IN A WILD OLIVE GENOTYPE . . . 239 1. Introduction . . . 243 2. Materials and Methods . . . 245 2.1 Olive plant material . . . 245 2.2 Pathogenicity experiment . . . 246 2.3 DNA xylem microbiome extraction and sequencing . . . 247 2.4 Statistical and bioinformatics analyses . . . 248 3. Results . . . 249 3.1 Pathogenicity experiment . . . 249 3.2 Alpha and Beta diversity analysis . . . 250 3.3 Composition and abundance of olive xylem bacterial communities . . . 252 4. Discussion . . . 258 5. References . . . 261

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Table of Contents

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CHAPTER VIII.

THE OLIVE HOLOBIONT: UNRAVELING THE EFFECT OF PLANT NICHE, ENVIRONMENTAL CONDITIONS AND OLIVE GENOTYPE ON THE

MICROBIOME . . . 269 1. Introduction . . . 273 2. Materials and Methods . . . 274 2.1 Study area and olive plant genotypes . . . 274 2.2 Plant sampling for microbiome analysis, leaf ionome and soil physicochemical

analyses . . . 275 2.3. Climate-related variables . . . 276 2.4. DNA microbiome extraction and sequencing . . . 276 2.5 . Bioinformatics and statistical analysis . . . 277 3. Results . . . 279 3.1. Environmental and soil physicochemical related variables . . . 279 3.2. Leaf ionome analysis . . . 280 3.3. α- and β-diversity of bacterial and fungal communities . . . 280 3.3. Composition of bacterial and fungal communities . . . 287 3.4. Differential abundance of bacterial and fungal taxa . . . 290 3.5. Effect of environmental variables on microbial communities . . . 290 3.6. Co-occurrence network inference analysis . . . 293 4. Discussion . . . 296 5. References . . . 299 SUMMARY DISCUSSION . . . 307 References . . . 313 GENERAL CONCLUSIONS . . . 317 ANNEXES . . . 321 Annex. Chapter II . . . 323 Annex. Chapter III . . . 343 Annex. Chapter IV . . . 353 Annex. Chapter V . . . 359 Annex. Chapter VI . . . 383 Annex. Chapter VII . . . 431 Annex. Chapter VIII . . . 439 Annex. Scientific production . . . 475

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31 List of Figures

CHAPTER I.

EXPLORING THE METHODOLOGICAL, BIOTIC AND ABIOTIC FACTORS INFLUENCING THE CHARACTERIZATION OF THE MICROBIAL

COMMUNITIES PRESENT IN THE XYLEM OF OLIVE TREES

Figure 1. Number of publications including “plant microbiome” in the title and/or as keywords in Web of Science (WoS) and Scopus databases. . . . 58 Figure 2. Main abiotic, biotic and methodological factors that influence the characterization of

xylem microbiome and its interactions in olive tree. . . . 67 CHAPTER II.

EVALUATION OF ESTABLISHED METHODS FOR DNA EXTRACTION AND PRIMER PAIRS TARGETING 16S rRNA GENE FOR BACTERIAL MICROBIOTA PROFILING OF OLIVE XYLEM SAP

Figure 1. Boxplots of Richness and Shannon alpha diversity indices of olive xylem bacterial com- munities at operational taxonomic unit (OTU) taxonomic level determined by different DNA extraction kits and after taxonomic assignments with the Greengenes_13-8 and Silva_132 databases. Boxes represent the interquartile range, while the black dots inside the box define the median, and whiskers represent the lowest and highest values. P value was calculated using Kruskal–Wallis test. . . 100 Figure 2. Hierarchical clustering dendrogram analysis using Ward method and Bray–Curtis dis-

tance (A) And principal coordinate analysis (PCoA) of weighted UniFrac and Bray–Curtis distances (B) Of olive xylem bacterial communities obtained by using different DNA extraction kits and after taxonomic assignments with the Greengenes_13-8 and Silva_132 databases. Colored dots represent the four clusters obtained in the hierarchical clustering analysis.

PERMANOVA (999 permutations; P < 0.05) was performed to test significant differences according to DNA extraction kits. . . . 102 Figure 3. Taxonomic bubble plot of olive xylem bacteria relative abundance at phylum, class, and

family level present in four groups of DNA extraction kits and after taxonomic assignments with the Greengenes_13-8 and Silva_132 databases. Circle sizes represent the relative abundance, and colors indicate DNA extraction kit groups shown in Figure 2. Only abundances greater than 1% are shown. . . . 103 Figure 4. Relative abundance of ZymoBIOMICS Microbial Community Standard (ZMCS) com-

position according to the PCR primer pairs tested and the database used for taxonomic assignment [Greengenes_13-8 (gg) and Silva_132 (silva)]. Comparison with the theoretical abundances of ZMCS was based on Spearman correlation coefficient. . . . 105 Figure 5. Boxplots of Richness and Shannon alpha diversity indices of xylem bacterial commu-

nities from “Picual” and “Arbequina” olive cultivars at operational taxonomic unit (OTU) taxonomic level determined by using different PCR primer pairs. Boxes represent the interquartile range, while the black dots inside the box define the median, and whiskers represent the lowest and highest values. P value was calculated using general linear modeling (GLM). . 107

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Figure 6. Hierarchical clustering dendrogram analysis using Ward method and Bray–Curtis distance (A) And principal coordinate analysis (PCoA) of weighted UniFrac and Bray–

Curtis distances (B) Of xylem bacterial communities from “Picual” and “Arbequina” olive cultivars obtained by using different PCR primer pairs. PERMANOVA (999 permutations;

P < 0.05) was performed to test significant differences according to PCR primers used and olive genotypes. . . 108 Figure 7. Taxonomic bubble plot of bacterial relative abundance at phylum, class, and family level

present in different PCR primer pair combinations. Circle sizes represent the relative abundance, and colors indicate PCR primer pairs used. All phyla and classes were shown, while only the most abundant bacteria (98% of reads) at family level were represented. Horizontal lines indicate the taxonomy lineage from each bacterial family. . . 110 Figure 8. DESeq2 analysis of differentially enriched bacterial genera when using different PCR

protocols. PCR2, N1PCR1, and N1PCR1 were compared against PCR3. The color scale bar indicates log2 fold change. Only significant genera (P < 0.01) are shown. . . . 111 CHAPTER III.

PRIMER CHOICE AND XYLEM-MICROBIOME-EXTRACTION METHOD ARE IMPORTANT DETERMINANTS IN ASSESSING XYLEM BACTERIAL COMMUNITY IN OLIVE TREES

Figure 1. Relative abundances of mitochondria, chloroplast and bacteria in xylem sap of plantlets (SD) or adult (AD) olive plants extracted with Scholander chamber (SCh) or woody chips (WC) as determined by using four PCR-primer combinations. Number of observed OTUs are indicated between brackets. . . 129 Figure 2. Richness, Simpson, Shannon and Faith_PD diversity indices at OTUs’ taxonomic level

determined by using four different PCR-primer pairs. Dots represent the values for all samples tested in each PCR-primer pairs. . . . 130 Figure 3. Heat trees and main taxonomic ranks identified from phyla to OTU level for each

PCR-primer combination tested. Identified phyla are shown within each heat tree. . . . 132 Figure 4. Proportional elliptical Venn diagrams showing the unique and shared bacterial genera

obtained in xylem sap of plantlets (SD) or adult (AD) olive plants extracted by using the Scho- lander chamber (SCh) or macerated woody chips (WC), and for each PCR-primer pair used.

(A) Bacterial genera identified for each factor of the study. (B) Bacterial genera identified according to the plant age and xylem-extraction method shown for each PCR tested. . . . 132 Figure 5. Taxonomic bubble plot of bacterial relative abundance at genus level present in olive

xylem sap and identified for each PCR-primer combination of plantlets (SD) or adult (AD) olive plants extracted with the Scholander chamber (SCh) or from woody-chip maceration (WC). Only genera with relative abundances greater than 80% of reads are shown. . . . 134 Figure 6. Principal-coordinate plots of weighted UniFrac distances of bacterial communities at

OTUs’ taxonomic level in xylem sap of plantlets (SD) or adult (AD) olive plants extracted with the Scholander chamber (SCh) or woody chips (WC) within each PCR-primer combination tested. Points are colored by PCR-primer pairs, where xylem-sap-extraction method is represented by color intensity and olive-plant age by shape. . . . 136

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List of Figures

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CHAPTER IV.

METABOLOMIC, IONOMIC AND MICROBIAL CHARACTERIZATION OF OLIVE XYLEM SAP REVEALS DIFFERENCES ACCORDING TO PLANT AGE AND GENOTYPE Figure 1. Percentage composition of the different organic groups (A), amino acids, organic acids,

alcohols, sugars, osmolytes (B) mineral elements and inorganic ions (C) detected in all olive xylem sap samples analyzed in the study. . . . 163 Figure 2. Principal coordinates plot of weighted UniFrac distances of bacterial communities, at

ASV taxonomic level, in xylem sap of plantlets (SD) and adult (AD) olive trees of ‘Picual’

(PIC) and ‘Arbequina’ (ARB) genotypes. . . 164 Figure 3. Prevalence Venn diagrams showing the unique and shared bacterial genera in olive

xylem sap of plantlets (SD) and adult (AD) olive trees of ‘Picual’ (PIC) and ‘Arbequina’ (ARB) genotypes. . . . 165 Figure 4. Partial least squares discriminant (PLS-DA) 2D score plot and loading importance in

projection (VIP scores) in the first PLS-DA component of metabolomic and ionomic profile of olive xylem sap of plantlets (SD) and adult (AD) olive trees of ‘Picual’ (PIC) and ‘Arbequina’

(ARB) genotypes. (A) Combined analysis of all olive cultivars and plant age combinations.

(B) Separate analysis by olive plant age. . . 168 Figure 5. Partial least squares discriminant (PLS-DA) 2D score plot and loading importance in

projection (VIP scores) in the first PLS-DA component of microbiome profile of olive xylem sap of plantlets (SD) and adult (AD) olive trees of ‘Picual’ (PIC) and ‘Arbequina’ (ARB) genotypes. (A) Combined analysis of all olive cultivars and plant age combinations. (B) Separate

analysis by olive plant age. . . . 169 Figure 6. NMDS based on Bray-Curtis dissimilarity and environmental fitting test analysis (env-

fit) displaying significant chemical variables (p < 0.05) explaining bacterial ASV distribution in olive xylem sap of plantlets (SD) and adult (AD) olive trees of ‘Picual’ (PIC) and ‘Arbequina’

(ARB) genotypes. . . 170

CHAPTER V.

CULTURE-DEPENDENT AND CULTURE-INDEPENDENT CHARACTERIZATION OF THE OLIVE XYLEM MICROBIOTA: EFFECT OF SAP EXTRACTION METHODS Figure 1. Culturable bacterial population densities [colony forming units (cfu)/ml] present in

xylem sap of different olive genotypes extracted with the Scholander chamber or from woody chips macerates from three independent trees per genotype, irrespective of the culture medium used. The number of bacterial isolates selected for 16S taxonomic identification for each treatment is indicated between brackets. For each extraction method, bars with the same letter do not differ significantly among olive genotypes. The “*” indicate for each genotype the existence of significant differences at P≤ 0.05 between both xylem sap extraction methods. . . 190 Figure 2. Heat tree of the abundance of bacterial taxa at different ranks present in olive xylem sap

and determined using culture-dependent and culture-independent approaches and extracted with the Scholander chamber (SCh) or from woody chips (WC) macerates. The size and color of nodes and edges are correlated with the abundance of taxa. The central nodes are the total of all the other nodes in the tree for each phylum . . . 192

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Figure 3. Prevalence Venn diagram showing the unique and shared bacterial genera obtained using culture-dependent approaches (upper panel) or culture-independent approach (lower panel) in olive xylem sap samples when compared by olive genotype (“Acebuche,” “Arbequi- na,” and “Picual”). . . . 194 Figure 4. Prevalence Venn diagram showing the unique and shared bacterial genera obtained

using culture-dependent approaches (upper panel) or culture-independent approach (lower panel) in olive xylem sap samples when compared by xylem sap extraction method [Scholan- der chamber (SCh) or wood chips (WC) maceration]. . . . 195 Figure 5. Principal component analysis of the relative abundance of bacterial genera obtained

using culture-dependent approaches (left panel) or culture-independent approach (right panel) in olive xylem sap samples when compared by olive genotype (Ace: “Acebuche,” Arb: “Ar- bequina,” and Pi: “Picual”) or by xylem sap extraction method [Scholander chamber (SCh) or wood chips (WC) maceration]. . . 196 CHAPTER VI.

TIME COURSE ANALYSIS OF OLIVE XYLEM SAP MICROBIOTA CULTIVATION REVEALS DIFFERENCES ACCORDING TO THE GROWTH MEDIA AND PLANT GENOTYPE

Figure 1. Hierarchical clustering on principal components (HCPC) of nutritional content of xylem sap (SO) and different predefined culture media for xylem microbial growth based on the Ward clustering algorithm. Colours indicate the different clusters detected.. . . 216 Figure 2. (A) Variation over time of total bacterial growth measured by absorbance increase (600

nm) after inoculation of xylem sap extracted from “Picual” and “Arbequina” olive genotypes in different predefined broth culture media. (B) Area under the absorbance curves standar- dized by duration of incubation time in days (SAUAPC). Bars followed by the same letter do not differ significantly according to the post hoc Tukey’s honestly significant difference (HSD) test at (P < 0.05). . . . 217 Figure 3. (A) Variation over time of total bacterial number after inoculation of xylem sap extrac-

ted from “Picual” and “Arbequina” olive genotypes in different predefined broth culture media. (B) Mean number of genera in each incubation time of xylem sap extracted from “Picual”

and “Arbequina” olive genotypes across the predefined broth culture media used. . . . 218 Figure 4. Boxplots of Richness (observed) and Shannon diversity indices at the amplicon sequen-

ce variant (ASV) taxonomic level for bacterial communities developed in different culture media after inoculation of xylem sap extracted from “Picual” and “Arbequina” olive genotypes. The boxes represent the interquartile range, while the horizontal line within the box defines the median and whiskers represent the lowest and highest values of three values for each treatment combination. . . 219 Figure 5. Principal coordinate plot of weighted UniFrac distances of bacterial communities at the

ASV taxonomic level when using all data from the study (A) or separated by genotype (B) of the olive xylem sap inoculated in selected predefined culture media that sustain growth of xylem-inhabiting bacteria. Points are colored according to broth culture media and shaped by genotype. . . 221

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List of Figures

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Figure 6. Main taxonomic bacterial ranks identified from phylum to ASV level for crude xylem sap and each broth culture media. Numbers indicate the total taxa found on each level. . . . 222 Figure 7. Bar plots showing the relative abundance of bacterial taxa at the genus level in the xylem

of “Picual” and “Arbequina” olive trees before (B) and after (A) xylem sap cultivation in predefined culture media. . . . 226 Figure 8. UpSet plot of number of bacterial ASVs (shared and unique) identified in crude xylem

sap extracted from “Picual” and “Arbequina” olive genotypes and after its incubation in different broth culture media. . . 227 Figure 9. Volcano plot of DESeq2 analysis showing the bacterial-ASVs that were differentially

enriched between “Picual” or “Arbequina” olive genotypes after growth in different broth culture media. The logarithms of the Log₂FC (Fold Changes) of individual bacteria (x-axis) are plotted against the negative logarithm of their P-value to base 10 (y-axis). Red color denotes bacterial-ASV that are significant in both parameters (P-value and Log₂FC). Cut-off scores are marked with dotted lines. Bacterial genera are shown with different symbol shapes. . . . 228 Figure 10. Prevalence Venn diagram showing the unique and shared bacterial genera described

as inhabitants of the olive xylem vessels by Anguita-Maeso et al. (2020) and in this Chapter (Anguita-Maeso et al., 2022) with the current repertoire of cultured plant endophytic bacteria in shoot tissues proposed by Riva et al., 2022. . . . 231 CHAPTER VII.

VERTICILLIUM DAHLIAE INOCULATION AND IN VITRO PROPAGATION MODIFY THE XYLEM MICROBIOME AND DISEASE REACTION TO VERTICILLIUM WILT IN A WILD OLIVE GENOTYPE

Figure 1. Verticillium wilt disease progression in “Ac-18” in vitro (standard and adapted) and nur- sery propagated olive plants inoculated with the defoliating pathotype of Verticillium dahliae.

“Picual” and “Ac-15” olive genotypes were used as positive controls to determine the inoculation success and development of the disease. Each point represents the mean disease severity (0–4 scale: 0 = healthy, 4 = dead plant) of data and error bars show the standard error from six plants per treatment. . . 249 Figure 2. Boxplots of Richness, Shannon, and Evenness alpha diversity indices at OTU taxonomic

level in olive xylem from Verticillium dahliae (Vd)-inoculated and non-inoculated “Ac-18”

plants following in vitro (standard and adapted) and nursery propagation methods. Boxes represent the interquartile range, while the horizontal line inside the box defines the median and whiskers represent the lowest and highest values of six values for each treatment combi- nation. For all three indexes and propagation methods, values on Vd-inoculated plants were significantly higher compared to that on non-inoculated treatments according to the Schei- rer–Ray–Hare test at P<0.05. . . 251 Figure 3. Principal coordinates plot of weighted UniFrac distances of bacterial communities

at OTU taxonomic level in olive xylem from Verticillium dahliae (Vd)-inoculated and non-inoculated “Ac-18” plants following in vitro (standard and adapted) and nursery propagation methods. Points are colored by plant inoculation treatment and shaped by propagation methods. . . . 252

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Figure 4. Prevalence Venn diagram showing the unique and shared bacterial genera in olive xylem from the core microbiome (A) or by each propagation approach (B) obtained from Verticillium dahliae (Vd)-inoculated and non-inoculated (NI) “Ac-18” plants following in vi- tro (standard and adapted) and nursery propagation methods. . . . 254 Figure 5. Bar plots showing the relative bacterial abundance taxa at phylum (A) and genera (B)

level present in olive xylem from Verticillium dahliae (Vd)-inoculated and non-inoculated

“Ac-18” plants following in vitro (standard and adapted) and nursery propagation methods. . 255 Figure 6. Cladogram representation from LEfSe analysis showing the taxonomic ranks from the

innermost phylum ring to the outermost genera ring. Each point is a member within each taxonomic rank. Significant taxa (P < 0.05) appearing as dominant for each treatment when comparing each propagation method by inoculation treatment (A,B), or each inoculation treatment by propagation method (C–E) are shown in different colors (red, green, or blue) associated to the legend of each individual cladogram. . . . 257 CHAPTER VIII.

THE OLIVE HOLOBIONT: UNRAVELING THE EFFECT OF PLANT NICHE,

ENVIRONMENTAL CONDITIONS AND OLIVE GENOTYPE ON THE MICROBIOME Figure 1. Boxplots of Richness (observed) and Shannon diversity indices for bacterial communi-

ties at the amplicon sequence variant (ASV) taxonomic level according to the plant ecological niche, olive genotype, field location and sampling season. The boxes represent the interquartile range, while the horizontal line within the box defines the median and whiskers represent the lowest and highest values of four values for each treatment combination. Letters above boxes indicate the olive genotype as follows: “Arbequina” (A), “Frantoio” (F) and “Picual”

(P). . . . 282 Figure 2. Boxplots of Richness (observed) and Shannon diversity indices for fungal communities

at the amplicon sequence variant (ASV) taxonomic level according to the plant ecological niche, olive genotype, field location and sampling season. The boxes represent the interquartile range, while the horizontal line within the box defines the median and whiskers represent the lowest and highest values of four values for each treatment combination. Letters above boxes indicate the olive genotype as follows: “Arbequina” (A), “Frantoio” (F) and “Picual” (P). . . . 283 Figure 3. Principal coordinate plots of weighted UniFrac distances of bacterial communities at

the ASV taxonomic level according to the plant ecological niche, olive genotype, field location and sampling season. Points are colored by ecological niche and field location and shaped by plant genotype and sampling season. . . 285 Figure 4. Principal coordinate plots of weighted UniFrac distances of fungal communities at the

ASV taxonomic level according to the plant ecological niche, olive genotype, field location and sampling season. Points are colored by ecological niche and field location and shaped by plant genotype and sampling season. . . 286 Figure 5. Bar plots showing the relative abundance of bacterial taxa at the genus level according

to the plant ecological niche, olive genotype, field location and season of sampling. . . . 288 Figure 6. Bar plots showing the relative abundance of fungal taxa at the genus level according to

the plant ecological niche, olive genotype, field location and season of sampling. . . . 289

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List of Figures

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Figure 7. Constrained ordination analysis based on capscale as a distance-based redundancy me- thod that determine significant environmental variables (P < 0.05) explaining bacterial ASV distribution in olive according to the plant ecological niche, olive genotype, field location and sampling season. . . . 291 Figure 8. Constrained ordination analysis based on capscale as a distance-based redundancy me-

thod that determine significant environmental variables (P < 0.05) explaining fungal ASV distribution in olive tree according to the plant ecological niche, olive genotype, field location and sampling season. . . . 292 Figure 9. General co-occurrence network inference plot showing interconnected patterns of bac-

terial and fungal communities (A) and selected five ASV with the highest positive and negative degree (B) in above- and belowground compartment of olive tree. Nodes are colored by phylum taxa. Copresence (green) and mutual exclusion (red) are shown as the edges between the nodes. . . . 294 Figure 10. General co-occurrence network inference cluster plot based on the MCODE method

showing interconnected patterns of bacterial and fungal communities. Nodes are colored by phylum taxa. Edge weight indicated by color intensity from red (negative exclusion) to green (copresence) reflects the strength of the relationship between ASVs. . . . 295

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39 List of Tables

CHAPTER II.

EVALUATION OF ESTABLISHED METHODS FOR DNA EXTRACTION AND PRIMER PAIRS TARGETING 16S rRNA GENE FOR BACTERIAL MICROBIOTA PROFILING OF OLIVE XYLEM SAP

Table 1. Characteristics of the DNA extraction protocols used in the study. . . 92 Table 2. PCR protocols used in the study, with primer sequences, hypervariable region of 16S

rRNA gene amplified and expected product size. . . . 94 Table 3. Number of reads and OTUs derived from NGS analysis of xylem sap microbiome from

DNA samples extracted with 12 extraction protocols, amplified with the 967F+1193R primer pair (PCR2) and taxonomic assignment using the Greengenes 13-8 and Silva 132 reference databases . . . 98 Table 4. Number of reads and OTUs derived from NGS analysis of xylem sap microbiome from

DNA samples extracted with the PowerPlant protocol, amplified with four PCR protocols and taxonomic assignment using the Silva 132 reference database. . . 106 CHAPTER IV.

METABOLOMIC, IONOMIC AND MICROBIAL CHARACTERIZATION OF OLIVE XYLEM SAP REVEALS DIFFERENCES ACCORDING TO PLANT AGE AND GENOTYPE Table 1. Mean content (µM) and range of the main groups of metabolites identified in xylem

sap from plantlets and adult olive trees of ‘Picual’ and ‘Arbequina’ genotypes and results of ANOVA analysis (p-values of F test) to determine the effects of plant age, genotype and its interaction. For each treatment mean values and standard derivation are shown. Detection of each compound in the total samples tested is displayed in parentheses. . . 156 Table 2. Mean content (µM) and range of the main groups of elements and inorganic ions present

in xylem sap from plantlets and adult olive trees of ‘Picual’ and ‘Arbequina’ genotypes and results of ANOVA analysis (p-values of F test) to determine the effects of plant age and geno- type. For each treatment mean values and standard derivation are shown. Detection of each compound in the total samples tested is displayed in parentheses. . . . 160

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41 List of Abbreviations

Ac Acebuche (wild olive)

AD Adults

ALSD Almond leaf scorch disease AMF Arbuscular mycorrhizal fungi

AN Nutrient agar

ANOVA Analysis of variance

ARB Arbequina

ASV Amplicon sequence variant AUDPC Area under disease progress curve BCYE Charcoal-yeast extract medium BLAST Basic local alignment search tool

C Carbon

Ca Calcium

CaCO3 Calcium carbonate

CFU Colony-forming unit

CITIUS University of Seville Research, Technology and Innovation Centre CSIC Spanish National Research Council

CTAB Cetyltrimethylammonium bromide

Cu Copper

cv. Cultivar

D Defoliating

DA Demarcated area

dai Days after inoculation

db-RDA Distance-based redundancy analysis DESeq2 Differential gene expression analysis

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DSF Diffusible signalling factor

EC Electrical conductivity

EFSA European food safety authority

EPPO European and Mediterranean Plant Protection Organization