Degradation and vulnerability of Mediterranean Soils to Drought: Role of the soil microbial Community

DEGRADACIÓN Y VULNERABILIDAD DE LOS SUELOS MEDITERRÁNEOS A LA SEQUÍA: PAPEL DE LA COMUNIDAD MICROBIANA DEL SUELO TESIS DOCTORAL SEVILLA, SEPTIEMBRE 2024 DIRECTORAS: MARÍA TERESA DOMÍNGUEZ NÚÑEZ MARÍA ELENA FERNÁNDEZ BOY LAURA MORALES SALMERÓN Degradación y vulnerabilidad de los suelos mediterráneos a la sequía: papel de la comunidad microbiana del suelo Degradation and vulnerability of Mediterranean soils to drought: role of the soil microbial community Laura Morales Salmerón Programa de Doctorado en Recursos Naturales y Medio Ambiente Departamento de Cristalografía, Mineralogía y Química Agrícola Universidad de Sevilla Sevilla, 2024 La presente Tesis Doctoral se ha llevado a cabo en el Área de Edafología y Química Agrícola del Departamento de Cristalografía, Mineralogía y Química Agrícola de la Universidad de Sevilla (US) bajo la dirección de las Doctoras María Teresa Domínguez Núñez y María Elena Fernández Boy. Este trabajo se ha enmarcado dentro del proyecto homónimo “Degradación y vulnerabilidad de los suelos mediterráneos a la sequía: papel de la comunidad microbiana del suelo” (DEGRAMED, CGL2017-85891-R), financiado por el programa Retos, y los proyectos “Vulnerabilidad de sistemas agrícolas a la sequía bajo distintos tipos de laboreo: efectos en la comunidad microbiana del suelo” (LABORSEQ, US1260627) y “Organic waste to face drought in Mediterranean agricultural systems: effects and fate of exogenous C inputs to soil” (WASTE4DROUGHT, PID2021-122628OB-I00), financiados ambos con fondos FEDER. Por otra parte, cabe agradecer al Ministerio de Ciencia, Innovación y Universidades por conceder la ayuda FPI con la que se ha financiado mi contrato predoctoral en la Universidad de Sevilla (PRE2018–084467). DECLARACIÓN DE AUTORÍA Y ORIGINALIDAD DE LA TESIS PRESENTADA PARA OBTENER EL TÍTULO DE DOCTOR Considerando que la presentación de un trabajo hecho por otra persona o la copia de textos, fotografías o gráficos sin citar su procedencia se considera plagio, yo, Dña. Laura Morales Salmerón, con DNI 05335694Q, estudiante del Programa de Doctorado en Recursos Naturales y Medioambiente de la Universidad de Sevilla, ASUMO LA AUTORÍA RESPONSABLE Y DECLARO QUE la Tesis Doctoral que presento para su exposición y defensa titulada “Degradación y vulnerabilidad de los suelos mediterráneos a la sequía: papel de la comunidad microbiana del suelo”, dirigida por las Doctoras Dña. María Teresa Domínguez Núñez y Dña. Elena Fernández Boy, es original y que todas las fuentes empleadas han sido debidamente citadas en la misma. Asimismo, acepto que los miembros de la Comisión Evaluadora podrán utilizar las herramientas de control del plagio que consideren oportunar para garantizar la autoría de esta Tesis Doctoral. Fdo.: Laura Morales Salmerón Firmado por Laura Morales Salmerón el día 09/10/2024 con un certificado emitido por FNMT A mis padres, Enrique y Margarita ÍNDICE 5 References ..................................................................................................................... 227 Supplementary material ................................................................................................ 240 Discusión general .............................................................................................................. 255 Beneficios de las enmiendas orgánicas para la recuperación de suelos degradados ... 255 Enmiendas orgánicas para mejorar la producción vegetal en suelos degradados ....... 257 Influencia del legado de manejo del suelo .................................................................... 258 Las propiedades químicas del suelo: principal motor de la funcionalidad de los suelos degradados .................................................................................................................... 260 Beneficios del laboreo de conservación para la funcionalidad de los suelos agrícolas 261 Efecto de la sequía en las emisiones de CO2 del suelo .................................................. 262 Elevada tolerancia de los suelos mediterráneos a la sequía ......................................... 263 Conclusiones ..................................................................................................................... 267 Bibliografía ........................................................................................................................ 270 Anexo I............................................................................................................................... 277 6 RESUMEN 7 Resumen La preocupación por el cambio climático y el efecto de la sequía en los ecosistemas es un tema recurrente, especialmente en los países de la región mediterránea, donde los episodios de olas de calor y sequías son cada vez más frecuentes y prolongados. De entre los suelos mediterráneos, son los suelos degradados los más vulnerables a las perturbaciones debido especialmente al menor contenido en materia orgánica, que condiciona el contenido en macro y micronutrientes y la capacidad de retención de agua del suelo, afectando en última instancia a la biodiversidad del suelo. Los procesos de degradación del suelo, entre los que se encuentran la contaminación, el empobrecimiento en materia orgánica, la erosión y la salinización, conllevan la pérdida de la biodiversidad edáfica. Debido al importante papel de la microbiota del suelo en su funcionalidad, la degradación del suelo provocaría una pérdida de su capacidad para hacer frente a las perturbaciones, incluidas las derivadas del cambio climático. Esta Tesis ha estado centrada en el estudio del efecto de las condiciones de sequía sobre el funcionamiento de los suelos mediterráneos degradados, evaluando, además, el esperado efecto positivo de diferentes actuaciones destinadas a la recuperación del suelo en la mejora de la resistencia de estos suelos frente a la sequía. Para el desarrollo de la Tesis se ha trabajado con suelos contaminados con metales pesados (Capítulos 1 y 2) y con suelos degradados como consecuencia de un uso agrícola intensivo (Capítulos 3 y 4). En cada uno de los experimentos se simularon condiciones de sequía en la mitad de los replicados mediante la reducción en un 30 % del aporte de agua con respecto al tratamiento control, ya bien a través del riego en condiciones de invernadero (Capítulos 1, 2 y 3) o mediante el uso de estructuras de exclusión de lluvia en ensayos de campo (Capítulo 4). En la evaluación del efecto de la sequía sobre la funcionalidad del suelo se tuvieron en consideración tanto los cambios en las propiedades químicas como el impacto sobre la actividad biológica y la comunidad microbiana del suelo. Asimismo, se estudió el efecto de la sequía sobre el desarrollo de la cubierta vegetal. La hipótesis de RESUMEN 8 partida es que aquellos suelos en los que las actuaciones realizadas hayan conseguido mejorar las propiedades químicas del suelo (en especial en contenido en materia orgánica) y promover una comunidad microbiana más diversa y estructurada, serán los que presentarán una mayor capacidad de resistencia a las condiciones de sequía. En el Capítulo 1 se describe un experimento llevado a cabo en macetas bajo condiciones controladas de invernadero, en el que se comparó el efecto de la adición de una enmienda a base compost de biosólidos (producido a partir de lodos de depuradora y restos de poda) a largo plazo (aplicado 20 años atrás) con una adición a corto plazo (en el momento del experimento); se compararon además dos suelos con diferente legado de exposición a la ganadería extensiva (un suelo procedente de una zona que ha estado expuesta al ganado durante 20 años y un suelo procedente de una parcela vallada a la que el ganado no puede acceder). Para este ensayo se trabajó con suelos contaminados por metales pesados procedentes del Corredor Verde del Guadiamar, afectados por el accidente de Aznalcóllar en 1998. Las macetas se sembraron con una mezcla de especies forrajeras (Lolium rigidum y Medicago polymorpha) y en la mitad de ellas se simularon condiciones de sequía tras un periodo de establecimiento de los mesocosmos. Entre los resultados obtenidos destacó el efecto positivo de la aplicación del compost de biosólidos en las propiedades químicas del suelo, así como en su capacidad de retención de agua, aumentando la producción vegetal incluso en condiciones de sequía. Por otra parte, la exposición prolongada al ganado tuvo un efecto similar sobre el suelo al de la enmienda orgánica, lo que se reflejó en la gran similitud de las comunidades microbianas en ambos suelos. No se observó un efecto significativo de la sequía sobre la actividad biológica del suelo, demostrando la alta adaptación de las comunidades microbianas de los suelos de ambientes semiáridos a las condiciones de estrés hídrico. Una vez demostrado en el Capítulo 1 el gran efecto positivo de la aplicación de materia orgánica en las propiedades del suelo y en su resistencia a las condiciones de sequía, en el Capítulo 2 se desarrollan los resultados obtenidos en un segundo experimento en macetas, llevado a cabo igualmente bajo condiciones de invernadero. En este experimento se buscó RESUMEN 9 discernir el papel de la diversidad microbiana del suelo del efecto de las propiedades químicas del suelo en la estabilidad de la funcionalidad del suelo frente a condiciones de sequía. Para ello, una vez enmendados con compost de biosólidos, se inocularon suelos de manera controlada para generar un gradiente de diversidad microbiana. Se observó, tal y como se esperaba, una relación positiva entre la diversidad de bacterias y hongos sobre la multifuncionalidad del suelo, especialmente en las macetas enmendadas con compost de biosólidos. Sin embargo, el análisis mediante un modelo de ecuaciones estructurales (SEM) reveló que este efecto de la diversidad microbiana sobre la multifuncionalidad del suelo es indirecto, siendo las propiedades químicas del suelo y la aplicación de la enmienda los principales responsables del nivel de multifuncionalidad del suelo. Por otra parte, en el Capítulo 3 se detalla el tercer ensayo que se realizó en macetas y en condiciones de invernadero. En este experimento se comparó el efecto de diferentes enmiendas orgánicas sobre la resistencia a la sequía de un suelo empobrecido en materia orgánica como resultado de la actividad agrícola intensiva. Se eligieron cuatro enmiendas de diferente calidad, basada en su relación C:N: leonardita, humus de lombriz, compost de biosólidos (generado a partir de lodos de depuradora mezclados con restos vegetales) y un abono verde de haba (Vicia faba). Los resultados obtenidos mostraron que las enmiendas de menor relación C:N tuvieron un mayor efecto en las propiedades químicas del suelo en el corto plazo, al igual que sobre la actividad biológica del suelo (actividades enzimáticas y tasa de respiración). Se observó también un incremento en la abundancia de bacterias y hongos en las macetas enmendadas con la enmienda de menor ratio C:N, sin que se observara un efecto significativo de las condiciones simuladas de sequía en la funcionalidad del suelo, ni una influencia de la abundancia relativa de hongos y bacterias en la resistencia de los suelos a la reducción de los aportes de agua. Finalmente, en el Capítulo 4 se aborda un experimento de campo llevado a cabo en la finca experimental La Hampa (IRNAS, CSIC). Este estudio aprovechó un experimento previo a largo plazo en el que se compara el efecto de dos técnicas de agricultura de conservación (laboreo reducido y no-laboreo) con un manejo de laboreo tradicional (arado de RESUMEN 10 vertedera), en un sistema de rotación de cultivos cereal-leguminosa. Sobre estas parcelas se colocaron estructuras de exclusión de lluvia que permitían reducir el aporte de agua al suelo en un 30 %. De este modo se estableció un diseño factorial para el estudio in situ del efecto combinado de la sequía y el tipo de laboreo sobre la estabilidad del suelo frente a condiciones simuladas de sequía. Este experimento se mantuvo durante dos ciclos del cultivo consecutivos para poder considerar cierta variabilidad ambiental. Los resultados obtenidos permitieron demostrar que el laboreo de conservación, y en especial el sistema de no-laboreo, mejoran la capacidad de retención de agua y la disponibilidad de materia orgánica y nutrientes en el suelo en el largo plazo, respecto al laboreo tradicional. Asimismo, los suelos sometidos a no-laboreo presentan mayores niveles de actividad biológica y una tendencia hacia una mayor diversidad de hongos, además de una comunidad de nematodos edáficos más madura. Finalmente, aunque no se observó un efecto significativo de la interacción entre el sistema de laboreo y el tratamiento de exclusión de lluvia, las condiciones de sequía simuladas tuvieron un efecto negativo sobre la tasa de respiración del suelo en los meses más secos, y sobre la abundancia de hongos descomponedores de hojarasca, posiblemente debido a la reducción de la producción de biomasa vegetal debido a la sequía. INTRODUCCIÓN 11 Introducción El suelo y sus servicios ecosistémicos El suelo es el cuerpo natural que cubre la superficie terrestre formado por un conjunto no consolidado de materiales minerales meteorizados, materia orgánica, aire y agua, formado como resultado de la acción, a lo largo del tiempo, del clima y los organismos vivos sobre los materiales parentales (roca madre). Como resultado el suelo difiere de su material parental original en su textura, estructura, consistencia, color y propiedades químicas, biológicas y físicas (Soil Survey Staff, 1999). Constituye una interfase que permite intercambios entre la litosfera, la biosfera, la hidrosfera y la atmósfera, regulando los flujos de materia y energía entre estos sistemas. Adicionalmente, se habla del suelo como ecosistema para hacer referencia a las interacciones dinámicas que se dan entre los componentes abióticos y bióticos del suelo, y que son las responsables de las distintas funciones que ocurren en el suelo. Los suelos, además de ser el soporte para la producción de alimentos y materias primas, regulan la mayoría de los procesos que ocurren en el resto de ecosistemas terrestres (Pereira et al., 2018), y se estima que albergan más de un 25 % de la biodiversidad del planeta (Decaëns et al., 2006). Entre los servicios ecosistémicos que ofrece el suelo destacan los servicios de regulación, como el reciclado de nutrientes, la purificación del agua, la degradación de contaminantes y la regulación climática. El servicio de regulación climática, de especial relevancia en la mitigación del cambio climático, reside principalmente en la capacidad del suelo de almacenar C en forma de materia orgánica estable y biomasa vegetal, y de regular los flujos entre el suelo y la atmósfera de gases de efecto invernadero (CO2, N2O y CH4) (Oertel et al., 2016). La capacidad de secuestro de C está condicionada por la estructura del suelo. Es muy importante que los suelos tengan una estructura estable y equilibrada, ya que contribuye directamente en el secuestro de C y en la creación de un microambiente óptimo para la microfauna del suelo (Six et al., 2000; Kong et al., 2011). La materia orgánica juega un papel INTRODUCCIÓN 12 crucial en el mantenimiento de la estructura del suelo ya que, junto con las raíces de las plantas, las hifas y determinados sustratos orgánicos exudados por los hongos que funcionan como agentes aglutinantes, ayuda a la estabilidad hídrica de los macro y microagregados (Tisdall y Oades, 1982). Además, las sustancias orgánicas aglutinantes de los microagregados son en sí mismos compuestos recalcitrantes, por lo que pueden contribuir al almacenamiento de C a largo plazo en el suelo (Degens y Sparling, 1995). Además, los suelos tienen la capacidad de filtrar y regular la disponibilidad de agua en los ecosistemas terrestres, mitigando los efectos de las condiciones de sequía (O'Geen, 2013). El potencial del suelo para almacenar agua está condicionado por la textura, estructura, consistencia y contenido en materia orgánica del suelo (Rawls y Pachepsky, 2002); mientras que la dinámica del agua en el suelo está influenciada, además, por la climatología de la región (Swarowsky et al., 2011). La adaptación y mitigación de los efectos del cambio climático requiere, entre otras medidas, de una gestión sostenible del suelo (como la adopción de técnicas de agricultura de conservación y la revegetación de espacios degradados) que promueva el secuestro y la estabilización del C, optimizando el balance de C en el suelo (Lal et al., 2021). En la provisión de estos servicios ecosistémicos del suelo la biodiversidad juega un papel fundamental, destacando las funciones de grupos claves de organismos como los simbiontes con plantas, los descomponedores de detritos y materia orgánica, los ingenieros del ecosistema que modifican la estructura del suelo, y los organismos implicados en el control biológico de parásitos, predadores y enfermedades (Barrios, 2007). El escenario actual de cambio climático Conocer y mitigar el impacto del cambio climático sobre los ecosistemas es uno de los principales retos actuales a nivel global. En el quinto informe de evaluación (AR5) del Panel Intergubernamental de Expertos sobre Cambio Climático (IPCC) (Kovats, 2014) se destacaba el incremento observado de las temperaturas en Europa a partir de 1980, especialmente durante los meses de verano en el caso de la Península Ibérica, además de INTRODUCCIÓN 13 un incremento en la frecuencia de episodios de calor extremo (incluidas las olas de calor). Sin embargo, la tendencia en cuanto a las precipitaciones a partir de 1950 presentaba una gran variabilidad regional, con incrementos de la precipitación anual en el norte de Europa y descensos en el sur. Las predicciones del informe AR5 sobre cambio climático apuntaban a un incremento en Europa de las temperaturas extremas y de episodios de sequía más intensos y prolongados (especialmente en la cuenca mediterránea), acompañados por eventos de precipitaciones intensas (principalmente en la zona norte) para finales del siglo XXI (Kovats, 2014). En 2022 el sexto informe de evaluación del IPCC (AR6) advertía que las temperaturas en Europa seguirán ascendiendo, incluso por encima del calentamiento global medio, lo que conllevará importantes impactos, sobre todo en la región mediterránea (Bednar-Friedl, 2022). Se prevé que el riesgo de escasez de agua en el sur de Europa será alto o muy alto con el aumento de la temperatura global en 1.5 ºC o 3 ºC, respectivamente, afectando a más de un tercio de la población de la región mediterránea. El aumento de la temperatura también afectará a la composición y funcionamiento de los ecosistemas, conllevando la pérdida de biodiversidad (Habibullah et al., 2022). Se espera que la mayor intensidad de las olas de calor provoque la extinción en masa de las especies más vulnerables, especialmente en la región mediterránea, y favorezca la expansión de las especies más tolerantes a las condiciones de mayor temperatura hacia mayores latitudes y altitudes (Hammond et al., 2022; López et al., 2022). La degradación de los bosques, humedales y turberas, que funcionan como importantes sumideros de C, podría limitar su capacidad de regulación climática (Lorenz y Lal, 2009), sobre todo en la región mediterránea. Se requiere de actuaciones de restauración (reforestando y mejorando la conectividad de los ecosistemas) para evitar la pérdida de biodiversidad y aumentar la resiliencia de los ecosistemas terrestres. A pesar de que la subida de las temperaturas podría aumentar la producción agrícola en el norte de Europa, la realidad en el sur es que las cosechas podrían reducirse hasta en un 50 INTRODUCCIÓN 14 % (en el escenario de aumento en 3 ºC de la temperatura media global) debido a la combinación de incremento de temperatura y escasez de agua (Bednar-Friedl, 2022). La disponibilidad de recursos hídricos para la agricultura estará posiblemente más limitada, por lo que se requieren estrategias de adaptación, como el uso de cubiertas vegetales y acolchados, y técnicas de laboreo más sostenibles, que permitan mejorar la eficiencia en el uso del agua. El aumento de la temperatura también incrementará la incidencia de plagas y enfermedades en los cultivos, además de la aparición de nuevas cepas de organismos patógenos y especies invasoras (Skendžić et al., 2021; Singh et al., 2023). Las condiciones de cambio climático contribuirán a la expansión de insectos plaga, además de favorecer su supervivencia durante las estaciones frías e incrementar sus tasas de reproducción (Skendžić et al., 2021). Se prevé, además, un aumento en el riesgo de incendios forestales de mayor severidad debido la combinación de olas de calor y condiciones de sequía. En la cuenca mediterránea la frecuencia de incendios debidos a las condiciones climáticas podría aumentar en un 14 % si la temperatura media global aumenta en 2.5 ºC (Ruffault et al., 2020; El Garroussi et al., 2024). En el sexto informe del IPCC (AR6) se incluyó por primera vez un capítulo dedicado exclusivamente a la región mediterránea, a la que se atribuye una mayor vulnerabilidad frente al cambio climático puesto que las nuevas predicciones se suman a las actuales condiciones de escasez de agua existentes (Ali, 2022). Con una temperatura media actual 1.5 ºC más alta que en el periodo pre-industrial, las predicciones apuntan a un aumento de la temperatura del aire (que puede alcanzar un incremento de 5.6 ºC a finales de siglo en el escenario de emisiones de mayor gravedad) y de la frecuencia, intensidad y duración de las olas de calor, incluso por encima de la media global. Aunque la reducción en las precipitaciones puede ser variable entre regiones y escenarios de emisiones (entre un 4 % y un 22 % de reducción), dicho informe destaca que los episodios de sequía, cada vez más frecuentes e intensos, seguirán la misma tendencia. En INTRODUCCIÓN 21 generación de drenajes ácidos, los cuales constituyen importantes focos de contaminación. Los suelos del entorno de la Faja Pirítica Ibérica, afectados históricamente por el drenaje ácido de las minas, presentan valores medios de pH inferiores a 5, y elevadas concentraciones totales de As, Cu, Pb, Zn y Cd, muy superiores a los valores de fondo regional (Fernández-Caliani, 2008; Caro Moreno y Jiménez Cantizano, 2014). Estos elementos traza se acumulan en los tejidos de las especies vegetales y pueden afectar negativamente a las relaciones planta-microorganismo, como es el caso del desarrollo de micorrizas arbusculares y ectomicorrizas (López-García et al., 2018; Madejón et al., 2024). La aplicación de enmiendas orgánicas en este tipo de suelos contaminados constituye una herramienta eficaz para aumentar el pH del suelo, disminuyendo la disponibilidad de los elementos traza e incrementando la biomasa y actividad de las comunidades microbianas (Janoš et al., 2010; Park et al., 2011; Khan et al., 2017; Montiel-Rozas et al., 2018). En 2006, englobada dentro del Sexto Programa de Acción de la Comunidad Europea en materia de Medio Ambiente, se adoptó la Estrategia Temática para la Protección del Suelo de la Unión Europea, que constituyó la primera medida dedicada específicamente a la conservación de los suelos a escala comunitaria (Commission of the European Communities, 2006). Esta estrategia estaba centrada principalmente en prevenir la degradación del suelo, mantener la capacidad del suelo para producir bienes y servicios ecosistémicos, y rehabilitar los suelos degradados (especialmente los suelos contaminados). Posteriormente, en el año 2021 y ligada al Pacto Verde Europeo, la Comisión Europea publicó la Estrategia de la Unión Europea para la Protección del Suelo para 2030 (European Comission, 2021), centrada en la puesta en valor de los suelos sanos para la producción de bienes y servicios ecosistémicos, incluidos los relacionados con la mitigación del cambio climático (secuestro de carbono y retención de agua, entre otros). Un suelo sano está definido por la FAO como un ecosistema vivo y dinámico que es capaz de mantener una comunidad de micro y macroorganismos que permita mantener la funcionalidad del suelo. Esta estrategia marca unos objetivos a medio plazo (2021-2030) que contribuirán a que en 2050 los suelos europeos hayan alcanzado un buen estado de INTRODUCCIÓN 22 salud que los haga más resilientes a las perturbaciones y al cambio climático. De entre estos objetivos cabe destacar la lucha contra la desertificación y la degradación del suelo, la recuperación de los suelos degradados, y el fomento del secuestro de carbono en el suelo que ayude a alcanzar el objetivo de neutralidad climática. Recientemente aprobada en junio de 2024 la Ley de Restauración de la Naturaleza de la Unión Europea (European Parliament, 2024) obliga a cada uno de los Estados miembros a restaurar al menos un 30% de sus ecosistemas degradados para el año 2030, con un objetivo final de recuperar el 90% de los ecosistemas para 2050. Esta Ley establece además objetivos específicos, entre los que destaca la puesta en marcha de las medidas necesarias para aumentar las reservas de carbono orgánico en los suelos agrícolas. En el marco de estas normativas recientes resulta imprescindible avanzar en el conocimiento de las técnicas de recuperación de suelos degradados, y en particular en las técnicas de preservación y recuperación de los niveles de materia orgánica del suelo. A pesar de las diferentes políticas adoptadas, de la mayor vulnerabilidad de los suelos de la cuenca mediterránea frente a las condiciones de cambio climático, y de la intensidad de los procesos de degradación a los que éstos están sometidos, existe muy poca información sobre las consecuencias de la pérdida de biodiversidad edáfica en la resistencia de la funcionalidad de los suelos mediterráneos degradados frente a episodios de sequía. Diversos estudios han analizado el impacto potencial del incremento de las condiciones de sequía en los suelos mediterráneos, observando un efecto positivo de la diversidad microbiana sobre la estabilidad de los suelos (Papatheodorou et al., 2004; Curiel Yuste et al., 2014; Delgado-Baquerizo et al., 2014; Delgado-Baquerizo et al., 2017). Sin embargo, estos estudios han sido desarrollados en ecosistemas naturales, sometidos a un nivel de perturbación relativamente bajo. Con el desarrollo de esta Tesis Doctoral se pretende ampliar el conocimiento sobre el funcionamiento de los suelos degradados en condiciones de sequía, así como evaluar la efectividad de una serie de medidas de restauración y conservación de suelos aplicadas, INTRODUCCIÓN 23 basadas en el aumento y la preservación de la materia orgánica del suelo, con potencial para mejorar la estabilidad de estos suelos frente al cambio climático. En concreto, se pretende estudiar la vulnerabilidad de los suelos degradados frente a las condiciones se sequía previstas en el actual escenario de cambio climático, prestando especial atención al papel de la comunidad microbiana del suelo en la resistencia del funcionamiento del suelo frente a este motor de cambio global. Objetivos e hipótesis generales Los Capítulos 1 y 2 están centrados en el estudio del efecto de la sequía sobre suelos degradados por contaminación por elementos traza; mientras que en los Capítulos 3 y 4 se evalúa el efecto de la sequía sobre suelos empobrecidos en materia orgánica como resultado de una actividad agrícola intensiva. El objetivo del Capítulo 1 es analizar el efecto de la aplicación de una enmienda orgánica (compost de biosólidos) a corto y largo plazo en un suelo degradado, teniendo en cuenta la influencia del legado de manejo del suelo, sobre su funcionalidad bajo condiciones de sequía. En el Capítulo 2 se evalúa la relación entre la diversidad microbiana del suelo y su nivel de multifuncionalidad, en suelos enmendados y no enmendados con compost de biosólidos, con el fin de discernir el papel de la comunidad microbiana en el control de la funcionalidad de suelos degradados y en el proceso de recuperación bajo condiciones de sequía. El objetivo del Capítulo 3 es estudiar el efecto de la aplicación de enmiendas orgánicas de diferente calidad (indicada por la relación C:N) a suelos agrícolas pobres en materia orgánica, evaluando los cambios inducidos en la abundancia de bacterias y hongos, y su efecto sobre la estabilidad del funcionamiento del suelo frente a la sequía. Por último, en el Capítulo 4 se compara, en un experimento de campo, el efecto de dos sistemas de manejo de agricultura de conservación (laboreo reducido y no laboreo) frente al sistema del laboreo tradicional (que emplea arado de vertedera) en la resistencia del funcionamiento del suelo en condiciones de sequía. INTRODUCCIÓN 24 La hipótesis general de partida es que los factores de degradación del suelo (contaminación por elementos traza o pérdida de materia orgánica) originan pérdidas de la biodiversidad edáfica, y que estas pérdidas de diversidad por sí mismas (independientemente de los efectos de la degradación en las propiedades físicas y químicas del suelo) conllevan una reducción de la estabilidad de la funcionalidad del suelo frente a eventos de sequía. Por otra parte, se plantea la hipótesis de que es posible mejorar la estabilidad de los suelos degradados frente a la sequía aplicando medidas de restauración, en particular mediante técnicas que generen un aumento en el contenido en materia orgánica del suelo (mediante la aplicación de enmiendas orgánicas o reduciendo el laboreo intensivo), y que los efectos de estas medidas sobre la estabilidad del suelo estarán mediados, al menos parcialmente, por cambios en la estructura de las comunidades microbianas del suelo. Suelos de estudio y metodologías aplicadas La Tesis se ha desarrollado con suelos afectados por dos factores de degradación: la contaminación por elementos traza resultado de la actividad minera (capítulos 1 y 2) y el empobrecimiento del suelo en materia orgánica por un uso agrícola intensivo (capítulos 3 y 4). Los suelos contaminados con elementos traza se recogieron del Corredor Verde del Río Guadiamar (Sevilla, 37°26'14.7"N 6°13'02.8"W). Esta área fue afectada por el conocido desastre de Aznalcóllar en 1998, en el que la rotura de una balsa minera provocó el vertido de 6 hm3 de lodos y aguas ácidas (con pH cercano a 3) al río Guadiamar, y la contaminación con metales pesados (mayoritariamente Pb, Zn, As y Cu) de una superficie mayor de 4000 ha (Grimalt et al., 1999). A pesar de las labores de limpieza llevadas a cabo tras el accidente, en las que se retiraron los lodos y la capa superficial del suelo, las concentraciones de elementos traza siguieron siendo muy superiores a los valores genéricos de referencia de fondo geoquímico y el pH del suelo tenía valores entre 2 y 4 (Cabrera et al.; 1999; Burgos et al., 2003). Tras la limpieza la zona se reforestó con especies autóctonas típicas de bosques de ribera (álamo, fresno, sauce y almez) y monte mediterráneo (encina, INTRODUCCIÓN 25 alcornoque, acebuche y algarrobo), y se destinó a un uso recreativo, permitiéndose, además, el pastoreo de ganadería extensiva (Figura 1). Figura 1. Estado actual del Corredor Verde del Río Guadiamar, entorno de la finca El Vicario (Sanlúcar la Mayor, Sevilla). En el año 2002 se delimitó una parcela experimental situada a 10 km al sur de la mina en la que se evaluó la efectividad de la aplicación de diferentes enmiendas en la reducción de la movilidad de los contaminantes en el suelo a largo plazo (Madejón et al., 2006). Estudios posteriores determinaron que los efectos de las enmiendas perduraron en el tiempo y que, además de producirse un aumento del pH y la inmovilización de los elementos traza, las enmiendas sirvieron para restaurar la estructura y fertilidad del suelo, y tuvieron un efecto positivo sobre la comunidad microbiana del suelo (Burgos et al., 2006; Montiel-Rozas et al., 2018). De igual manera se delimitaron parcelas testigo en las que no se retiró el lodo ni se llevó a cabo ninguna medida de restauración, manteniéndose como controles experimentales negativos (Carreira et al., 2008). INTRODUCCIÓN 26 Por otra parte, los suelos agrícolas que han sido objeto de estudio en esta Tesis Doctoral fueron recogidos de dos parcelas dentro de la finca experimental “La Hampa” (IRNAS, CSIC) localizada en Coria del Río (Sevilla, 37°17'0.1"N 6°03'54"W). La primera de ellas se trata de una parcela dedicada tradicionalmente al cultivo en rotación de cereal y leguminosa, típico del Valle del Guadalquivir, con un cultivo de haba (Vicia faba L.) en el momento de la recogida del suelo. La segunda parcela corresponde a un ensayo de largo plazo establecido en 2008 en el que se estudia el efecto de las técnicas de laboreo de conservación (laboreo reducido y no laboreo) frente al laboreo tradicional en un cultivo en rotación de cereal y leguminosa (Panettieri et al., 2020). En cada experimento llevado a cabo en la Tesis Doctoral se estudiaron varios tratamientos, realizando al menos seis replicas por tratamiento, y se simularon condiciones de sequía a través de la reducción del aporte de agua de riego en experimentos de mesocosmos (Capítulos 1, 2 y 3) o mediante la colocación de estructuras de exclusión de lluvia en condiciones de campo (Capítulo 4) en la mitad de los replicados. Las estructuras de exclusión de lluvia, con unas dimensiones de 2.5 m × 2.5 m (Figura 2), se instalaron en octubre de 2020 y se mantuvieron durante dos ciclos del cultivo consecutivos (cultivo de haba en la temporada 2020-2021 y cultivo de triticale en la temporada 2021-2022). En todos los casos el objetivo fue simular una reducción en el aporte de agua en un 30 % con respecto al agua aportada en el tratamiento control (sin sequía). Esta reducción del 30 % se corresponde con los valores predichos por el IPCC de reducción de la precipitación media anual en el sur de la Península Ibérica para el periodo 2081-2100 (Kovats, 2014; Ali, 2022). Los niveles de humedad del suelo en los primeros 5 cm (Capítulos 1, 2 y 3) o a lo largo del perfil del suelo hasta una profundidad de 1 m (Capítulo 4) se midieron de forma periódica a lo largo de los experimentos, y especialmente tras los episodios de lluvia en el caso del ensayo en campo (Capítulo 4). INTRODUCCIÓN 27 Figura 2. Estructuras de exclusión de lluvia instaladas en el experimento llevado a cabo en la finca experimental La Hampa (IRNAS-CSIC) sobre un cultivo de triticale. Con el fin de estudiar el efecto de las condiciones de sequía sobre el suelo, en cada ensayo realizado en mesocosmos (Capítulos 1, 2 y 3) se llevó a cabo un primer muestreo previo al establecimiento del tratamiento de sequía y un segundo muestreo un mes después del inicio del periodo simulado de sequía. Por otro lado, dado que se conoce que en sistemas agrícolas de secano el efecto de la disponibilidad de agua (o de la sequía) es especialmente crítico durante la etapa de crecimiento del cultivo, cuando los requerimientos de agua son mayores en comparación con las etapas de germinación y maduración (Allan et al., 1998), en el experimento en campo (Capítulo 4) los muestreos de suelo se llevaron a cabo en las estaciones de otoño (previo a la siembra) y primavera, realizándose cuatro muestreos a lo largo del ensayo: en otoño de 2020 y primavera de 2021 (durante el cultivo de haba), y en otoño de 2021 y primavera de 2022 (durante el cultivo de triticale). De forma general, en cada muestreo se analizaron las principales propiedades químicas del suelo (pH, conductividad eléctrica, contenido en materia orgánica, C y N totales, contenido en P disponible, y concentración de otros macro y micronutrientes y elementos traza disponibles). Por otra parte, se midieron diferentes índices de actividad biológica del suelo. INTRODUCCIÓN 28 Se analizaron los niveles de actividad de cinco enzimas presentes en el suelo: deshidrogenasa (involucrada en la oxidación biológica de la materia orgánica), βglucosidasa, N-acetil-glucosaminidasa (relacionadas ambas con el ciclo del C), leucinaaminopeptidasa y fosfatasa (ligadas al ciclo del N y del P, respectivamente). Se midió además la respiración basal del suelo como indicador del nivel de actividad microbiana, y del contenido y tasa de descomposición de la materia orgánica. Además, en el ensayo en campo (Capítulo 4) se midió tasa de respiración del suelo in situ de forma periódica (generalmente después de episodios de precipitación). De las muestras de suelo recogidas se extrajo el ADN total. Posteriormente, en los experimentos descritos en los Capítulos 2 y 3 se cuantificaron las abundancias absolutas de bacterias y hongos mediante PCR cuantitativa; y para los experimentos de los Capítulos 1, 2 y 4 se recurrió a servicios externos (StabVida y Novogene) para la secuenciación masiva de bacterias (región V3/V4 del gen 16S) y hongos (región ITS) a través de la plataforma Illumina HiSeq2000. Los datos resultantes de la secuenciación se procesaron mediante técnicas bioinformáticas para finalmente llevar a cabo el estudio de la composición de las comunidades microbianas del suelo. Adicionalmente, en el experimento correspondiente al Capítulo 4 se llevó a cabo el estudio de la comunidad de nematodos edáficos, empleándolos como bioindicador del estado de salud del suelo (Bongers y Ferris, 1999; Ferris et al., 2001). Para ello se extrajeron los nematodos y, tras el recuento e identificación taxonómica mediante técnicas morfológicas, se calcularon los índices clásicos de ecología de nematodos (índices de madurez e índices de la red trófica), además de los niveles de riqueza y diversidad de taxones. Finalmente, en los ensayos llevados a cabo en mesocosmos (Capítulos 1, 2 y 3) se sembró una mezcla de especies forrajeras (Lolium spp. y Medicago polymorpha) al inicio del experimento con el objetivo de poder estudiar el efecto de los tratamientos sobre la tasa de germinación y su influencia sobre la resistencia frente a la sequía de la producción de biomasa de cada una de las dos especies. INTRODUCCIÓN 29 Publicaciones derivadas de la Tesis Doctoral Los resultados obtenidos en cada uno de los diferentes capítulos de la Tesis han sido descritos en diversas publicaciones científicas, aceptadas o enviadas a revistas internacionales: Capítulo 1: Morales-Salmerón, L., Fernández-Boy, E., Madejón, E., Domínguez, M.T., 2024. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought. Applied Soil Ecology, 195, 105226. Capítulo 2: Morales-Salmerón, L., Fernández-Boy, E., Herrador, B., León, R., Domínguez, M.T. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? Enviado a Biology and Fertility of Soils. Capítulo 3: Morales-Salmerón, L., Fernández-Boy, E., León, R., Navarro-Fernández, C.M., Domínguez, M.T. Effect of the quality of applied organic amendments on soil resistance against drought conditions. Enviado a Journal of Environmental Management. Capítulo 4: Domínguez, M.T., Morales-Salmerón, L., Navarro-Fernández, C., Madejón, E., Madejón, P., Fernández-Boy, E. Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought. En preparación. Participación en otras publicaciones Madejón, P., Fernández-Boy, E., Morales-Salmerón, L., Navarro-Fernández, C., Madejón, E., Domínguez, M.T., 2023. Could conservation tillage increase the resistance to drought in Mediterranean faba bean crops? Agriculture, Ecosystems and Environment 349, 108449. Madejón, P., Fernández-Boy, E., Madejón, E., Morales-Salmerón, L., Domínguez, M.T. Managing climate change impacts on crops: The influence of soil tillage on a triticale crop under water stress conditions. Annals of Applied Biology 1–14. INTRODUCCIÓN 30 Presentaciones en congresos European Geosciences Union General Assembly 2020: Effects of a simulated dryingrewetting cycle on microbial activity in soils degraded by post-fire erosion. Póster. Elena Fernández Boy, M. Belén Herrador Esquinas, Violeta Ordóñez, Laura Morales Salmerón, Óscar González Pelayo, Jan Jacob Keizer, M. Teresa Domínguez Núñez. 16th European Society for Agronomy Congress (2020): Could conservation agriculture land practices enhance soil resistance against drought events? Póster. Laura Morales Salmerón, María Teresa Domínguez Núñez, María Belén Herrador Esquinas, Violeta Ordóñez, Engracia Madejón Rodríguez, Elena Fernández Boy. European Geosciences Union General Assembly 2021: Effect of conservation agriculture practices on the resistance of Mediterranean soils to the predicted seasonal drought events. Comunicación oral. Laura Morales Salmerón, María Teresa Domínguez Núñez, María Belén Herrador Esquinas, Engracia Madejón Rodríguez, Elena Fernández Boy. IX Simposio Nacional sobre Control de la Degradación y Recuperación de Suelos (2021): Respuesta de la comunidad microbiana de suelos contaminados por elementos traza de la cuenca Mediterránea a un ciclo de sequía-rehidratación. Póster. Elena Fernández Boy, M. Belén Herrador Esquinas, Violeta Ordóñez, Laura Morales Salmerón, Óscar González Pelayo, Jan Jacob Keizer, M. Teresa Domínguez Núñez. IX Simposio Nacional sobre Control de la Degradación y Recuperación de Suelos (2021): Efecto de medidas de restauración post-incendio en suelos forestales sobre la actividad microbiana bajo condiciones simuladas de sequía. Comunicación oral. Laura Morales Salmerón, Elena Fernández Boy, M. Belén Herrador Esquinas, Violeta Ordóñez, Jan Jacob Keizer, Óscar González Pelayo, M. Teresa Domínguez Núñez. 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Soil Biology and Biochemistry 123, 21-32. 42 Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 43 Capítulo 1. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 44 Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 45 Resumen La aplicación de enmiendas orgánicas es una práctica común en la restauración de suelos contaminados y degradados. Diseñamos un experimento en macetas para estudiar el efecto del uso de enmiendas orgánicas (comparando una adición a corto plazo de compost de biosólidos con suelos enmendados hace 17 años) y del legado del suelo (comparando suelos expuestos o no a ganadería extensiva durante un proceso de remediación) en la resistencia de un suelo mediterráneo degradado frente a un evento simulado de sequía. Las macetas fueron sembradas con una mezcla forrajera (Lolium rigidum y Medicago polymorpha), y la resistencia del suelo fue evaluada a través de las propiedades químicas del suelo, la actividad biológica (actividades enzimáticas y tasa de respiración), la composición de la comunidad microbiana del suelo y la producción vegetal. Los resultados mostraron un efecto positivo del uso de la enmienda orgánica y de la exposición a ganadería extensiva en las propiedades del suelo, incrementando la retención de agua en el suelo y la estabilidad de la producción vegetal bajo condiciones de sequía. La adición de la enmienda a largo plazo evitó las consecuencias negativas de la sequía en la producción de L. rigidum (la especie con la tolerancia más baja al estrés hídrico), mientras que la biomasa de M. polymorpha en los suelos expuestos a la ganadería fue 12 veces superior a la biomasa en los suelos no-enmendados y no-expuestos a la ganadería. Sin embargo, la actividad biológica del suelo (indicada por el nivel de actividades enzimáticas y la tasa de respiración), así como la diversidad microbiana, no estuvieron limitadas por las condiciones simuladas de sequía bajo ningún tipo de legado de manejo del suelo, demostrando la gran adaptación de las comunidades microbianas a las condiciones de estrés hídrico en los suelos mediterráneos semiáridos. Los suelos expuestos a aportes de materia orgánica a largo plazo, mediante la adición de enmiendas o la exposición a la ganadería, mostraron altas similitudes en términos de composición de la comunidad de bacterias y hongos. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 46 Abstract The application of organic amendments is a common practice in the restoration of contaminated and degraded soils. We designed an experiment in pot mesocosms to study the effect of organic amendment (comparing a short-term addition of biosolid compost with soils amended 17 years ago) and soil legacy (comparing soils exposed or not to extensive grazing over a remediation process) on the resistance of a degraded Mediterranean soil against a simulated drought event. Pots were sown with a forage mixture (Lolium rigidum and Medicago polymorpha), and soil resistance was evaluated by measuring soil chemical properties, biological activity (soil enzyme activities and respiration rate), soil microbial community composition and plant production. Our results showed a positive effect of the organic amendment and the exposure to extensive grazing on soil properties, increasing soil water retention and the stability of plant production under drought conditions. The long-term amendment addition avoided the negative consequences of drought on L. rigidum production (the species with the lowest tolerance to water stress), while M. polymorpha biomass in soils exposed to grazing was 12 times the biomass in the non-amended and non-exposed soil. However, soil biological activity (enzyme activities and respiration rate) as well as microbial diversity were not limited by the simulated drought conditions under any type of soil management legacy, demonstrating the great adaptation of the microbial communities to water stress conditions in semiarid Mediterranean soils. Soils exposed to organic matter inputs in the long-term, through amendment addition or exposure to grazing, showed high similarities in terms of bacterial and fungal composition. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 53 h (Hoogsteen et al., 2015). Total C and N were analysed using a micro elemental analyzer (Leco Truspect CHNS Micro). Available P was determined by Olsen method (Olsen et al., 1954). Ca, Mg, K and Na were extracted with pH 7 1 M ammonium acetate and measured using an atomic absorption spectrometer (Ojea and Carballas, 1976). Ca and Mg were determined by atomic absorption spectroscopy and K and Na by atomic emission spectroscopy (PerkinElmer AAnalyst 100). Available Fe, Mn, Cu, Zn, Pb and Cd were extracted with 0,01 M CaCl2 (Novozamsky et al., 1993) and quantified by atomic absorption spectroscopy. Total content of Ca, Mg, K, Na, Fe, Mn, Cu, Zn, Pb and Cd in the biosolid compost was measured by inductively coupled plasma-mass-spectrometry (ICP-MS, Agilent 7800), after a microwave digestion in a 1:3 HNO3:HCl solution, and total C and N and bioavailable Fe, Mn, Cu, Zn, Pb and Cd were analysed in triplicate following the same methodology applied to soil samples. b) Soil biological activity Soil dehydrogenase activity (DHA) was measured by molecular absorption spectrometry using 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT) as substrate (Benefield et al., 1977). Leucine-aminopeptidase, β-glucosidase, phosphatase and N-acetylglucosaminidase activities were extracted in a 50 mM sodium acetate buffer solution at pH 5.5 and determined by fluorometry using 7-amino-4-methyl coumarin (AMC) and 4methylumbelliferone (MUB) as fluorogenic substrates (Marx et al., 2001). Soil DHA was expressed as μg INTF g dry soil−1 h−1 and the other enzymes as nmol AMC g dry soil−1 h−1 (leucine-aminopeptidase) and nmol MUB g dry soil−1 h−1. Soil respiration rate was determined with an infra-red gas analyzer (EGM-4, PP Systems) after incubating 1 g of soil at 25 °C for 24 h in a closed glass vial, sealed with a septum (Bekku et al., 1995). Soil respiration rates were reported as g C-CO2 g dry soil−1 day−1. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 54 c) DNA extraction and characterization of soil microbial communities Genomic DNA was extracted from 0.25 g of soil using the DNeasy PowerSoil Kit (Quiagen) and DNA concentration was quantified with a Qubit fluorometer (ThermoFisher). Illumina high-throughput sequencing for 16S rDNA (341F/805R) and ITS rDNA (ITS86F/ITS4) were applied to DNA extracts from the last soil sampling (post-drought) to study soil bacterial and fungal communities, respectively, in a private sequencing service (Stab Vida, Portugal). The Divisive Amplicon Denoising Algorithm (DADA2 R package, version 1.22.0) (Callahan et al., 2016) was applied to denoise raw sequences and infer the amplicon sequence variants (ASVs). The software FIGARO (Weinstein et al., 2019) was applied to 16S sequences to define the optimal trimming parameters. Cutadapt 4.0 (Martin, 2011) was used to remove the primers. After identifying and removing chimeric sequences, taxonomy was assigned to the resulting ASVs using the SILVA version 138.1 (Quast et al., 2012, Yilmaz et al., 2013, Glöckner et al., 2017) and UNITE (Abarenkov et al., 2022) databases for bacteria and fungi, respectively, and fungal taxa were classified into functional groups following the study of Põlme et al. (Põlme et al., 2020). Those ASVs which represented less than a 0.005 % of the sequences were filtered (Bokulich et al., 2013). The resulting ASVs were clustered using VSEARCH v2.21.1 (Rognes et al., 2016) and the LULU package (Frøslev et al., 2017), and singletons were removed. Function rarefy in the vegan R package (Oksanen et al., 2022) was applied to compare the observed taxa richness with the expected richness in a theoretical sample with 1,000,000 reads. Rarefaction analysis showed that every sample curve from both data sets (bacterial and fungal) reached plateau, not being necessary to rarefy to a minimum number of reads per sample (McMurdie and Holmes, 2014). It was calculated that the observed ASV richness was over 96.53 % and 98.09 % of the expected bacterial and fungal ASV richness, respectively. Fungal and bacterial indicator species associated with each soil type were determined using the Indicator Value (IndVal) index, that measures the association between a species and a Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 55 specific site group (Dufrene and Legendre, 1997). Finally, Shannon and Whittaker indices were calculated to study bacterial and fungal alpha and beta-diversity among soil types. Statistical analyses All statistical analyses were carried out in R version 4.1.3 (RCoreTeam, 2022). Linear mixed models were used to study the effect of the biosolid compost application (LT and ST soils analysed separately), the drought conditions and the interaction of both factors on soil chemical properties, soil microbial activity and plant performance. Significant effects of the different experimental treatments on soil moisture were evaluated using mixed linear models (nlme package in R), with each experimental pot considered as a random factor to account for repeated measurements. For each model residuals were evaluated in order to check the model assumptions and to validate it. When homogeneity of variance was not met, a variance coefficient was introduced in the model to account for heteroskedasticity among different factor levels, using the varIdent function of the nlme package (Pinheiro and Bates, 2000). F statistic and p-value of each model are shown in Supplementary Tables S2, S3 and S4. In addition, correlation analyses between soil moisture and each plant species biomass were carried out. Non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarities and permutational multivariate ANOVA (PERMANOVA) analyses were carried out to study differences in the microbial community composition among treatments, using the vegan package in R. Results Soil moisture Before the beginning of the simulated drought period soil moisture in LT pots was higher in LT-NA than in LT-BC (Fig. S1a), while in ST pots the biosolid compost increased soil moisture (Fig. S1b). This pattern continued during the whole simulated drought event in Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 56 both LT and ST soils, so that in the LT soils moisture was significantly lower in the amended than in the non-amended treatment, while the opposite effect was observed in the ST soils (Fig. S1). The mixed linear model showed a significant effect of the organic amendment and the drought treatment, but not of the interaction, on soil moisture in both LT and ST soils. Soil moisture measured just after pot watering showed that, during the drought period, maximum soil moisture in control pots was around 16 % in LT-BC, 15 % in LT-NA, 18 % in ST-BC and 16 % in ST-NA. However, maximum soil moisture in pots belonging to the drought treatment was around 14 % in ST-BC and 13 % in the others. Plant cover performance In LT pots, L. rigidum germination rate was significantly higher in LT-BC (43 ± 10 %) than in LT-NA (35 ± 9 %), while the germination of M. polymorpha was not significantly different in both treatments (average of 50 ± 14 % for both treatments pooled, Table S2). L. rigidum biomass was not affected by the amendment (1.7 ± 0.3 g on average considering both treatments, Table S3) but M. polymorpha biomass was highly increased in LT-BC (0.81 ± 0.41 g), compared to LT-NA (0.05 ± 0.02 g). In ST pots, germination rates of L. rigidum (33 ± 8 %) and M. polymorpha (53 ± 12 %) were not affected by the application of compost (Table S2). However, for both species growth was enhanced by the BC application (Table S4). L. rigidum dry biomass was higher in ST-BC (2.2 ± 0.9 g) than in ST-NA (1.6 ± 0.7 g), while M. polymorpha had also a higher growth in ST-BC (0.9 ± 0.3 g) compared to ST-NA (0.60 ± 0.17 g). Biomass production in the two nonamended soils with a different legacy of management (LT-NA and ST-NA) was also different, in particular for M. polymorpha. In ST-NA pots, filled with soil collected from the no-fenced area and exposed to grazing, the biomass of this legume was 12 times higher than in LTNA, belonging to soil collected from the experimental and fenced plot. The impact of the drought treatment on plant growth depended on the species and on soil management (Fig. 1, Tables S3 and S4). L. rigidum biomass was significantly affected by Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 57 drought and by the amendment × drought interaction, so that drought reduced plant growth in the LT-NA, ST-BC and ST-NA treatments, but not in the LT-BC soils. In contrast, M. polymorpha biomass was affected by drought conditions in the LT-BC soils only, but no differences were observed in LT-NA, ST-BC or ST-NA pots (Fig. 1). Figure 1. Mean (bars) and standard deviation (lines) of the Lolium rigidum and Medicago polymorpha aboveground dry biomass produced in each treatment. * above bars indicates significant differences (p<0.05) between the drought (DR, 30 % reduction of water supply) and control (CT, 100 % water supply) treatments within each soil type. LT: long-term amended soil, ST: short-term amended soil, BC: soil amended with biosolid compost, NA: non-amended soil. A correlation analysis between soil water content at the end of the drought period and the plant cover biomass resulted in a positive Pearson correlation (r = 0.32, p = 0.052) between soil moisture and the L. rigidum biomass. Soil chemical properties The initial soil chemical characterization (pre-seed sampling, Table 1 and Table S2) showed that LT amended soils (LT-BC) had significantly higher values of soil organic matter (SOM), total C and N, and bioavailable P, K and Ca contents than non-amended soils (LT-NA). In contrast, LT-NA soils had higher electrical conductivity (EC) and available Fe, Mn and Zn contents compared to LT-BC. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 58 Table 1. Initial chemical characterization (mean values ± standard deviation) of the four soil types used in the experiment (pre-seed sampling). * Indicates significant differences between the amended and the non-amended soil at the 0.05 probability level, for LT and ST soils analysed separately. Detailed results of the statistical analysis are given in Supplementary Material, Table S2. Long-term experiment (LT)† Short-term experiment (ST)‡ Parameters BC§ NA# BC§ NA# pHH2O 7.12 ± 0.08 4.56 ± 0.04 * 6.59 ± 0.09 6.78 ± 0.05 * pHKCl 6.76 ± 0.07 4.20 ± 0.05 * 6.40 ± 0.05 6.34 ± 0.02 EC (dS m-1) 0.33 ± 0.02 1.02 ± 0.07 * 0.92 ± 0.03 0.25 ± 0.02 * SOM (g kg-1) 91 ± 1 61 ± 1 * 97 ± 12 62 ± 4 * C (g kg-1) 54 ± 3 18 ± 2 * 40 ± 10 28 ± 1 N (g kg-1) 4.03 ± 0.29 0.92 ± 0.17 * 4.45 ± 0.69 2.57 ± 0.04 * P (mg kg-1) 129.8 ± 6.4 20.1 ± 0.9 * 97.3 ± 6.1 21.2 ± 0.3 * K (mg kg-1) 241 ± 41 138 ± 11 * 422 ± 12 157 ± 5 * Na (mg kg-1) 318 ± 40 275 ± 20 420 ± 150 285 ± 13 * Ca (g kg-1) 5.5 ± 1.0 2.5 ± 0.4 * 4.7 ± 0.8 3.2 ± 0.3 * Mg (mg kg-1) 147 ± 50 168 ± 30 285 ± 60 147 ± 50 * Fe (mg kg-1) 2.13 ± 0.15 6.27 ± 0.81 * 1.60 ± 0.26 3.37 ± 0.55 * Mn (mg kg-1) 8.83 ± 0.06 69.33 ± 1.15 * 7.90 ± 0.40 5.13 ± 0.29 * Cu nd nd nd nd Zn (mg kg-1) 0.83 ± 0.35 18.17 ± 0.67 * 1.17 ± 0.06 1.50 ± 0.17 * Pb nd nd nd nd Cd nd nd nd nd †Soils collected from the long-term experimental field (fenced 17 years ago and not exposed to grazing). ‡Soils collected from the unfenced area and exposed to extensive grazing for over 17 years. §Soil amended with biosolid compost. #Non-amended soil. nd: Below detection limit. Soil pHH2O in LT-BC was significantly higher than in LT-NA. ST-BC soils were also initially characterized by higher contents of SOM, total N, bioavailable P, K, Ca, as well as by a lower bioavailability of Fe and Zn compared to ST-NA soils. Higher contents of available Na and Mg were also found in ST-BC in comparison to ST-NA. However, soil pHH2O was significantly Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 59 lower in ST-BC compared to ST-NA, and the short-term biosolid compost application increased soil EC and bioavailable Mn in ST-BC. In all the four soil types, bioavailable Cu, Pb and Cd levels were below the detection limit. In the pre-drought (one month after soil amendment, Table S5) and post-drought samplings (two months after soil amendment and one month after the establishment of the drought treatment, Table S6) soil chemical properties were still highly influenced by the biosolid compost application, showing the same pattern as in the initial sampling (Tables S2, S3 and S4). Measurements from the post-drought sampling showed that most of the analysed soil chemical properties were not significantly affected by the drought treatment or by the amendment × drought interaction (Tables S3 and S4), and thus data were pooled. The only difference was the decrease in bioavailable Na under the simulated drought conditions (DR, 327 ± 20 mg kg−1) compared to the control (CT, 403 ± 16 mg kg−1) in the ST soils. Soil biological activity In general, compost addition had a positive effect on enzyme activities, in particular in the ST soils (Fig. 2, Tables S2, S3 and S4). Dehydrogenase activity in LT-BC soils was clearly greater than in LT-NA soils across the three samplings. In contrast, phosphatase activity was higher in the non-amended soils than in the LT-BC soils at the second (pre-drought) and third (post-drought) samplings. In the ST soils, compost addition had a clear positive effect on β-N-acetyl-glucosaminidase across the three samplings, and on phosphatase and β-glucosidase in the first (pre-seed) and second samplings. Dehydrogenase activity, in contrast, was higher in ST-NA than in ST-BC soils. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 60 Figure 2. Mean (bars) and standard deviation (lines) of soil dehydrogenase (a), βglucosidase (b), aminopeptidase (c), phosphatase (d) and N-acetyl-glucosaminidase (e) activities at samplings 1 (pre-seed), 2 (pre-drought) and 3 (post-drought). * indicates significant differences (p<0.05) between the amended (BC) and the non-amended (NA) within each soil type, with different legacies of soil management (long-term, LTnonexposed to grazing-, short-term, ST -exposed to grazing-). ns indicates no significant differences. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 61 Without considering the amendment treatment, drought conditions significantly decreased soil β-glucosidase activity in DR (183 ± 24 nmol MUB g−1 h−1) compared to CT (234 ± 36 nmol MUB g−1 h−1) in the LT pots (Table S3). Soil dehydrogenase activity was also reduced in DR (5.4 ± 0.6 μg INTF g−1 h−1) compared to CT (6.1 ± 0.9 μg INTF g−1 h−1) in the ST pots (Table S4). No other significant effects of the drought treatment or the amendment × drought interaction were observed in LT or ST soils (Tables S3 and S4). Fig. 3 shows the relative enzyme activities in DR pots compared to the activity in CT on each soil type, where values close to 1 indicate a high resistance of the enzyme activities to drought. Figure 3. Effect of drought conditions on soil enzyme activities. Mean (and standard deviation) enzyme activity measured in pots belonging to the drought treatment (DR) in relation to mean enzyme activity recorded under control conditions (CT) for each soil type. Values below and above 1 indicate a reduction and increase in enzyme activity in DR compared to CT, respectively. LT: long-term amended soil, ST: short-term amended soil, BC: soil amended with biosolid compost, NA: non-amended soil. Although β-glucosidase and dehydrogenase activities were reduced by drought conditions in LT and ST soils, respectively, no significant effects of the amendment × drought interaction were observed. In LT, soil respiration rate was significantly increased by the biosolid compost application (3.9 ± 1.6 μg C-CO2 g−1 day−1) compared to the non-amended control (1.7 ± 1.4 μg C-CO2 g−1 day−1) at the pre-drought sampling (Table S2). This effect of the amendment treatment was also found at the post-drought sampling (3.6 ± 1.2 μg C-CO2 g−1 day−1 in LT-BC and 2.3 ± 1.4 Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 62 μg C-CO2 g−1 day−1 in LT-NA, Table S3). No differences in soil respiration rates were found at the pre-seed sampling in ST soils (average of 4.9 ± 2.1 μg C-CO2 g−1 day−1, Table S2), but at the final sampling amended soils had a slightly higher respiration rate (3.7 ± 1.2 μg CCO2 g−1 day−1) compared to the non-amended control (2.8 ± 0.3 μg C-CO2 g−1 day−1, Table S4). Soil respiration rates in both long and short-term amended treatments were not significantly affected by the drought conditions or by the interaction amendment × drought by the end of the simulated drought period (Tables S3 and S4). Soil DNA concentration Compost application had a clearer effect on soil DNA in the LT soils than in the ST soils (Tables S2, S3 and S4). LT-BC soils had 11.5 ± 1.5 and 10.7 ± 1.9 μg DNA g−1 soil at the pre and post-drought samplings, respectively, more than three times fold than LT-NA soils (3.2 ± 0.8 and 2.7 ± 0.6 μg DNA g−1, respectively). However, after the short-term addition of compost (ST) total DNA did not significantly change (averages of 9.2 ± 1.8 and 11.3 ± 1.4 μg DNA g−1 soil for ST-BC and ST-NA, respectively). In addition, after the drought period total DNA in ST-BC (10.2 ± 0.9 μg DNA g−1 soil) was significantly lower than in ST-NA (12.8 ± 2.1 μg DNA g−1 soil). No significant effects of the drought conditions or the interaction between drought and compost addition were found for LT or ST soils (Tables S3 and S4). Diversity and structure of soil bacteria and fungi After the raw data purification, there were 2241 ASVs of bacteria belonging to 22 phyla, 47 classes, 100 orders, 143 families, and 252 genera. The NMDS analysis showed a high dissimilarity in bacterial composition due to compost addition in LT and in ST soils (Fig. 4a and b). Although the NMDS plots show an apparent separation between samples belonging the drought (DR) and control (CT) treatments, PERMANOVA analyses determined a significant effect of the amendment treatment in both LT (F = 87.86, p < 0.001) and ST soils (F = 13.74, p < 0.001), but drought conditions and the amendment × drought interaction did not significantly affect soil bacterial communities. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 69 metal bioavailability (Pérez-Esteban et al., 2014). In addition, it is possible that the movement of horses during grazing has promoted a higher colonization of bare soils by plants, by transporting seeds from other locations and alleviating soil compaction. It is known that a legacy of grazing exposure in disturbed grasslands enhances weed recruitment and diversity by mediating in belowand aboveground resource competition. Besides, cattle trampling promotes plant growth and germination, although this effect is limited by soil moisture and nutrient availability (Renne et al., 2006; Renne and Tracy, 2007). Despite the great effect of the amendment treatment on soil chemical properties, the simulated drought event did not influence soil chemistry. However, drought conditions in ST-BC reduced soil available Na. It is known that, under water stress conditions, plant roots are able to absorb and accumulate salt ions, especially K+ and Na+, to reduce their osmotic potential and promote water absorption (Huihui et al., 2021), what could explain the observed reduction in Na content in DR pots. As a result of the positive effect of the long-term application of biosolid compost on soil chemical properties, soil biological activity was also enhanced. Total DNA and soil dehydrogenase activity, considered as a sensitive indicator of the overall microbial activity in degraded Mediterranean soils (Garcia et al., 1997), were significantly higher in LT-BC soils than in LT-NA, and this effect was maintained until the end of the experiment. However, this effect was not observed in the short-term amended soils, probably due to the short period of time (two months) after the biosolid compost addition, and due to the better soil conditions in ST-NA compared to LT-NA soils. A different study carried out using the same degraded soil and biosolid compost demonstrated that the amendment enhanced microbial biomass and, six months after the application, promoted soil dehydrogenase activity and plant cover development (Pérez de Mora et al., 2005). Soil β-glucosidase and β-N-acetyl-glucosaminidase activities, positively correlated with the amount of soil organic C and total N (Deng and Tabatabai, 1996, Ekenler and Tabatabai, Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 70 2004), were positively affected by the organic amendment and immediately enhanced after the biosolid compost addition. β-glucosidase activity, considered as a good indicator of soil biological quality, quickly responds to the improvement in soil properties, especially with the increase in labile organic C (Turner et al., 2002). Therefore, the observed increase in soil β-glucosidase activity in both non-amended treatments may be explained by the development of the plant cover and the release of root exudates (Vives-Peris et al., 2020). Previous studies reported that soil leucine-aminopeptidase activity is not influenced by the addition of inorganic N but it increases when organic N compounds are added to the soil (Norman et al., 2020). In addition, a higher availability of inorganic N in the soil supresses plant dependency from organic N sources (Romero et al., 2023). In our study, there was a positive correlation between M. polymorpha biomass and the leucine-aminopeptidase activity by the end of the experiment (rho = 0.65, p = 0.007), probably due to a substrate induction response as a result of the atmospheric N fixation. Phosphatase activity is positively correlated with soil organic P, but also with soil microbial biomass and total N content (Margalef et al., 2017). P-rich organic amendments stimulate microbial biomass and phosphatase activity, especially in the short-term, although the enzyme activity gradually decreases with time (Criquet et al., 2007). These results are consistent with our results and explain the increase in phosphatase activity in ST-BC, but not in the soils from the long-term experiment. Indeed, in these soils phosphatase activity was much higher in LT-NA. These soils, with a low content of total N, where characterized by a very low pH. Other studies have demonstrated that microbial communities in N-limited acidic soils tend to be more dominated by fungi, what leads to a higher phosphatase activity (Margalef et al., 2017). Biosolid compost application also increased soil respiration rate, as found in previous works (Montiel-Rozas et al., 2016). The low soil respiration in LT-NA is consistent with the lower soil quality and the lower microbial biomass under this treatment compared to LT-BC, but the development of the plant cover increased soil respiration by the end of the experiment, Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 71 as found in other experiments with degraded soils (Oyonarte et al., 2012). The addition of the organic amendment at short-term increased soil respiration in ST-BC two months after the compost application. This result is supported by others obtained in a previous study which demonstrated that an increase in soil organic matter enhances microbial respiration in a degraded soil (Kowaljow and Mazzarino, 2007). Under the experimental conditions, soil biological activity (total DNA, the analysed enzyme activities and soil respiration rate) was not limited by the simulated drought conditions, and no effects of the drought treatment (except for a light reduction in dehydrogenase and β-glucosidase activities, Fig. 3) or the amendment × drought interaction were recorded. Our hypothesis that the application of organic amendments promotes a higher stability of soil functioning against drought was therefore only confirmed for the productivity of the plant species with a lower resistance to water stress (L. perenne), but not for M. polymorpha or soil biological activity. It must be considered that, according to data from the National Agency for Meteorology (AEMET), the area where soils were collected from has an average monthly temperature of 27 °C and average monthly precipitation of 5.6 mm during the summer season (June, July, and August, period 1981–2010), leading to severe water stress conditions. Soil microbial communities in such semi-arid Mediterranean regions are adapted to drought, and a previous study showed that soil biological activity in this kind of soils was only reduced when soil moisture was below 2 % (Hueso et al., 2011). A previous experiment carried out in the same experimental area showed that mean soil moisture (calculated after drying the soil samples at 105 °C for 24 h) was around 7 % in March, while it was reduced to a 2.5 % in June, at the beginning of the dry season (Madejón et al., 2019). However, under our experimental conditions, mean soil moisture of samples belonging to the drought treatment was over 5.5 % at the third soil sampling. This fact could have limited the effect of the simulated drought conditions, as well as the potential positive effect of the organic amendment on soil biological activity resistance against water stress. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 72 Effects on bacterial and fungal communities The study of soil microbial community showed a great effect of the long-term compost addition, but also of the long-term exposure to extensive grazing in ST soils. Several studies have demonstrated that the application of different types of organic amendments can lead to a shift in soil bacterial community composition. In a previous experiment the long-term application of an organic manure stimulated the abundance of Chitinophagaceae, associated to the cellulose decomposition and Beijerinckiaceae families (Gautam et al., 2020). Pirellulaceae population was also increased by soil amendment with sewage sludge (Li et al., 2021b), and an increase in Vicinamibacteraceae abundance was observed after the application of an organic mulch (Xu et al., 2022). Vicinamibacteraceae and Sphingomonadaceae were also enhanced by the application of corn straw biochar and seaweed compost, respectively (Yin et al., 2022). The addition of biochar (Cheng et al., 2017) and a manure-based compost (Deng et al., 2022) increased Sphingomonadaceae abundance. These results support the dominance of these bacterial families in the longterm amended soil (LT-BC) and the soils that had been exposed to organic matter inputs trough livestock activity (ST-BC and ST-NA). The dominance of Ktedonobacteraceae and Sphingomonadaceae in the non-amended LTNA treatment may be explained by the low soil pH. Both families are found in acidic soils (pH around 4), and their relative abundance was increased after the addition of biochar (Xu et al., 2014; Jutakanoke et al., 2023) or the use of cover crops (Shen and Lin, 2021). The WD2101 soil group, present in the four soil types, is known to have a wide distribution in terrestrial habitats and a special association with some grass species has been demonstrated (Florian et al., 2021; Lewin et al., 2021). Fungal communities in LT-BC and both ST treatments were dominated by the same families and fungal traits. The observed fungal taxa belonging to Nectriaceae and Pleosporaceae families were mainly plant pathogens, taxa from family Phaeosphariaceae were plant pathogens and litter saprotrophs, and taxa belonging to family Cladosporiaceae were litter Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 73 saprotrophs (Põlme et al., 2020). The higher abundance of dung saprotrophs in LT-BC and in soils exposed to grazing (ST-BC and ST-NA) compared to LT-NA was remarkable, which is consistent with the presence of organic matter inputs in these treatments. In LT-NA the lower relative abundance of plant pathogens and litter saprotrophs compared to the other soil types may be the result of the scarce development of the plant cover under this treatment. Fungal taxa belonging to family Filobasidiaceae were mainly unspecified saprotrophs and the family Didymosphaeriaceae was dominated by wood saprotrophs (Põlme et al., 2020). Pollen constitutes a source of nutrient-rich fine particulate organic matter, with a special relevance in oligotrophic systems, and pollen grains are colonized by saprophytic fungi (Wurzbacher et al., 2014). This fact could explain the high abundance of pollen saprotrophs in LT-NA soils, poor in N and organic matter. Arbuscular mycorrhizal fungi (AMF) relative abundances were also ten times lower in LTNA soils than in the rest of the treatments. Nevertheless, the observed abundances of AMF were lower than the expected in a grassland (Honnay et al., 2017). The ITS86F/ITS4 primer pair is useful for the analysis of fungal communities from soil samples, but it is biased for Ascomycota, Basidiomycota and Zygomycota clades, which may result in the underestimation of AMF abundance (Vancov and Keen, 2009). The great number of indicator species (bacteria and fungi) exclusive and ubiquitous in LTNA samples indicates the high difference in soil microbial community composition under this treatment, non-amended and excluded from the cattle effect, compared to the other soil types. Bacterial and fungal alpha and beta diversity were also lower in LT-NA than in LT-BC, probably due to the more adverse soil chemical properties. The PERMANOVA analysis showed a significant effect of the amendment treatment on bacterial and fungal community compositions in both LT and ST soils. However, microbial communities in ST-BC and ST-NA were very similar and no effect of the biosolid compost Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 74 application was observed on alpha and beta diversity, what indicates that effects of organic amendments on soil microbial communities may only be noticed in the long-term. It is also very important to point out that the simulated drought conditions or the interaction amendment × drought, did not influence soil microbial community composition and diversity. Soil microbial communities from soils exposed to a recurrent drought conditions, as the Mediterranean soils, have developed strategies against water stress and are more resistant to drought events (Evans and Wallenstein, 2012). Long-term dryrewetting episodes result in shifts in the composition of soil bacterial and fungal communities, increasing the abundance of tolerant taxa (Meisner et al., 2018). Conclusion The application of biosolid compost increased soil organic matter and enhanced water retention in the short-term. The organic amendment also had a positive effect on the plant cover, promoting plant production. It was remarkable the positive effect of the long-term application of biosolid compost on the stability of plant productivity under drought conditions, especially of species with a high vulnerability to water stress (L. rigidum). The positive effects of biosolid compost on soil chemical properties (increases in soil pH, soil organic matter, total C and N content, and macronutrient bioavailability) were apparent 17 years after soil amendment and were also observed after the short-term application. However, the long-term legacy of extensive grazing also improved soil quality, promoting the natural remediation of the soil without the aid of the addition of amendments. Regarding the soil microbial community, our results showed a clear effect of the long-term organic amendment, as well as the long-term exposure to livestock grazing, on soil microbial community, showing great similarities in bacterial and fungal composition. Furthermore, microbial community in the non-amended and non-grazed soil was driven by the high soil acidity and the low N and organic matter content. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 75 Nevertheless, under our experimental conditions, soil biological activity, as well as microbial community composition and diversity, were not conditioned by the simulated drought event, probably due to microbial adaptations to water stress as consequence of a long-term exposure to natural dry-rewetting episodes. Once demonstrated the positive effect of organic C increase (via biosolid compost addition or a long-term exposure to extensive grazing) on soil chemical properties and microbial activity, further work that simulates harder drought conditions is necessary to evaluate the effect of these improvements on the resistance against drought events in semi-arid soils. Also, future studies using a higher number of soils with a gradient of organic matter content (via organic amendment application or taking advance of different soil legacies) could be useful to infer the effect of soil chemical properties on the composition and diversity of the microbial community and the resistance to drought events. Acknowledgements This study was funded by the Spanish Ministry of Science and Innovation (projects CGL2017-85891-R DEGRAMED and PID2021-122628OB-I00 WASTE4DROUGHT) and European funds. Laura Morales thanks the Ministry of Science and Innovation for the FPI fellowship (PRE2018-084467). References Abarenkov, K., Zirk, A., Piirmann, T., Pöhönen, R., Ivanov, F., Nilsson, R.H., Kõljalg, U., 2022. UNITE general FASTA release for Fungi. Version 16.10.2022. UNITE Community. Arenas, J.M., Carrero, G., Galache, J., Mediavilla, C., Silgado, A., Vázquez, E.M., 2001. Work carried out following the Aznalcóllar accident. Boletín Geológico y Minero. 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Chemical characterization of the applied biosolid compost. Total and available (when measured) contents of the main chemical elements are shown. Elements Total Available C (g kg-1) 170 N (g kg-1) 18 P (g kg-1) 10.5 0.2 K (g kg-1) 4.4 Na (mg kg-1) 488 Ca (g kg-1) 29.3 Mg (g kg-1) 3.4 Fe (mg kg-1) 19200 2.8 Mn (mg kg-1) 500 6.9 Cu (mg kg-1) 121 nd Zn (mg kg-1) 414 1.2 Pb (mg kg-1) 347 nd Cd (mg kg-1) 0.6 nd nd: below detection limit. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 86 Table S2. F statistic and p-value of the models used to study the effect of the long and short-term amendment treatment on plant germination rate, soil chemical properties, and biological activity at the first (pre-seed) and second (pre-drought) soil samplings. Long-term (LT) † Short-term (ST) ‡ Sampling Parameters F p-value F p-value Pre-seed pHH2O 6379 < 0.001 8.1 0.047 pHKCl 2782 < 0.001 5.2 0.085 Electric conductivity 306.1 < 0.001 1340 < 0.001 Organic matter 1620 < 0.001 25.6 0.007 Total C 283 < 0.001 6.4 0.065 Total N 220.9 < 0.001 22.3 0.009 Olsen P 863.7 < 0.001 472.5 < 0.001 K 18.8 0.014 1379 < 0.001 Na 2.7 0.174 10.6 0.047 Ca 22.2 < 0.001 8 0.047 Mg 0.4 0.554 8.9 0.040 Fe 17.8 0.014 25.1 0.007 Mn 8215 < 0.001 94.4 < 0.001 Zn 1591 < 0.001 10 0.034 Dehydrogenase 2540 < 0.001 10.4 0.048 β-glucosidase 67.5 0.015 149.9 0.007 Aminopeptidase 0.8 0.424 0.005 0.946 Phosphatase 1.1 0.357 24.2 0.016 N-acetyl-glucosaminidase 6.2 0.130 57.5 0.017 Pre-drought L. rigidum germination 4.4 0.048 1.6 0.217 M. polymorpha germination 0.3 0.606 0.1 0.824 Organic matter 18.7 0.012 90.9 < 0.001 Total C 4.8 0.094 100 < 0.001 Total N 8.3 0.045 37.7 0.004 Olsen P 102 0.002 1754 < 0.001 K 70.9 0.001 7.4 0.053 Na 0.2 0.706 0.7 0.457 Ca 6.8 0.080 15.6 0.017 Mg 2.5 0.158 11.1 0.029 Dehydrogenase 376.4 < 0.001 18.7 0.002 β-glucosidase 1.1 0.312 6 0.041 Aminopeptidase 0.9 0.375 104.7 < 0.001 Phosphatase 39.4 < 0.001 5.2 0.046 N-acetyl-glucosaminidase 0.01 0.895 8.4 0.020 Respiration rate 6.4 0.030 4.5 0.062 Total DNA 78 < 0.001 2.8 0.171 †Soils collected from the long-term experimental field (fenced 17 years ago and not exposed to grazing). ‡Soils collected from the unfenced area and exposed to extensive grazing for over 17 years. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 87 Table S3. F statistic and p-value of the models used to study the effect of the amendment and drought treatments, and the interaction between both factors, on plant biomass, soil chemical properties, and microbial activity and diversity at the final (post-drought) soil sampling in soils from the long-term experiment. Long-term experiment† Amendment‡ Drought§ Interaction Parameters F p-value F p-value F p-value L. rigidum biomass 0.5 0.476 7.8 0.019 7.7 0.020 M. polymorpha biomass 46.9 < 0.001 7.3 0.023 6.6 0.028 Organic matter 304.1 < 0.001 4.0 0.081 3.3 0.105 Total C 28.2 < 0.001 0.2 0.695 0.04 0.839 Total N 90.9 < 0.001 3.1 0.119 0.2 0.656 Olsen P 2268.7 < 0.001 0.9 0.386 0.5 0.499 K 0.4 0.551 1.2 0.307 0.8 0.389 Na 3.1 0.119 1.1 0.326 0.4 0.569 Ca 17.5 0.003 1.0 0.348 0.0 0.993 Mg 8.7 0.018 4.3 0.072 1.4 0.266 Fe 6.6 0.034 0.04 0.851 0.2 0.692 Mn 144.3 < 0.001 3.1 0.114 2.9 0.129 Zn 607.1 < 0.001 1.6 0.236 1.4 0.277 Dehydrogenase 451.5 < 0.001 0.008 0.933 0.02 0.888 Β-glucosidase 2.9 0.132 6.6 0.037 0.1 0.754 Aminopeptidase 97.5 < 0.001 1.8 0.233 3.2 0.136 Phosphatase 507.9 < 0.001 5.3 0.062 3.1 0.131 N-acetylglucosaminidase 0.3 0.584 0.07 0.800 0.8 0.409 Respiration rate 6.0 0.040 4.4 0.070 0.3 0.616 Total DNA 73.3 < 0.001 0.005 0.945 0.1 0.808 Bacterial α-diversity 653.1 < 0.001 2.3 0.171 0.03 0.858 Fungal α-diversity 7.5 0.026 1.1 0.331 0.5 0.517 †Soils collected from the long-term experimental field (fenced 17 years ago and not exposed to grazing). ‡Biosolid compost amendment compared to a non-amended control treatment. §Control (100 % water supply) compared to drought (30 % reduction of water input) treatment. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 88 Table S4. F statistic and p-value of each model used to study the effect of the amendment and drought treatments, and the interaction between both factors, on plant biomass, soil chemical properties, and microbial activity and diversity at the final (post-drought) soil sampling in soils from the short-term experiment. Short-term experiment † Amendment‡ Drought§ Interaction Parameters F p-value F p-value F p-value L. rigidum biomass 8.1 0.010 27.8 < 0.001 0.4 0.546 M. polymorpha biomass 5.7 0.027 1.3 0.266 1.0 0.325 Organic matter 44.5 < 0.001 1.4 0.278 0.2 0.708 Total C 6.113 0.039 0.4 0.566 3.6 0.095 Total N 10.232 0.013 0.5 0.487 3.2 0.109 Olsen P 552.7 < 0.001 1.0 0.384 0.9 0.390 K 64 < 0.001 0.2 0.639 5.8 0.043 Na 0.7 0.417 0.1 0.732 3.3 0.106 Ca 66.892 < 0.001 0.002 0.962 0.8 0.398 Mg 43.768 < 0.001 1.8 0.217 2.6 0.143 Fe 0.819 0.400 1.0 0.348 2.2 0.186 Mn 9.049 0.020 0.2 0.646 2.0 0.197 Zn 0.351 0.572 2.8 0.140 0.0 1.000 Dehydrogenase 5.385 0.049 5.4 0.049 0.1 0.818 Β-glucosidase 0.207 0.665 3.7 0.102 1.0 0.346 Aminopeptidase 1.211 0.313 0.08 0.791 2.2 0.191 Phosphatase 3.577 0.101 0.4 0.567 3.7 0.096 N-acetylglucosaminidase 7.461 0.029 1.7 0.237 5.1 0.059 Respiration rate 4.600 0.069 0.4 0.540 5.2 0.056 Total DNA 7.128 0.028 0.2 0.688 1.0 0.344 Bacterial α-diversity 2.1 0.184 0.2 0.633 1.1 0.318 Fungal α-diversity 0.6 0.476 0.1 0.721 0.1 0.784 †Soils collected from the unfenced area and exposed to extensive grazing for over 17 years. ‡Biosolid compost amendment compared to a non-amended control treatment. §Control (100% water supply) compared to drought (30% reduction of water supply) treatment. Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought | Capítulo 1 89 Table S5. Soil chemical properties before the simulated drought event (pre-drought sampling). Mean values and standard deviation of total C and bioavailable Na, Ca and Mg. * indicates significant differences between the amended and the non-amended soil at the 0.05 probability level, for LT and ST soils analysed separately. Long-term experiment † Short-term experiment ‡ Parameters BC § NA # BC § NA # SOM (g kg - 1 ) 77 ± 4 54 ± 8 * 94 ± 3 60 ± 6 * C (g kg - 1 ) 41 ± 5 25 ± 12 67 ± 8 21.8 ± 1.3 * N (g kg - 1 ) 4.0 ± 0.3 2.3 ± 1.0 * 6.6 ± 1.3 2.0 ± 0.1 * P (mg kg - 1 ) 132.4 ± 15.1 18.7 ± 0.2 * 94.8 ± 1.6 21.8 ± 2.6 * K (mg kg - 1 ) 174 ± 10 87 ± 15 * 232 ± 41 100 ± 38 * Na (mg kg - 1 ) 316 ± 50 295 ± 80 278 ± 40 231 ± 70 Ca (g kg - 1 ) 5.6 ± 0.5 4.7 ± 0.1 5.8 ± 0.9 3.4 ± 0.6 * Mg (mg kg - 1 ) 199.3 ± 0.6 306 ± 80 366 ± 120 133 ± 30 * †Soils collected from the long-term experimental field (fenced 17 years ago and not exposed to grazing). ‡Soils collected from the unfenced area and exposed to extensive grazing for over 17 years. §Soil amended with biosolid compost. #Non-amended soil. Capítulo 1 | Soil legacy and organic amendment role in promoting the resistance of contaminated soils to drought 90 Table S6. Soil chemical properties after the simulated drought event (post-drought sampling). Mean values and standard deviation of soil organic matter (SOM), total C and N, and available P, K, Na, Ca, Mg, Fe, Mn, Cu, Zn, Pb and Cd. * indicates significant differences between the amended and the non-amended soil at the 0.05 probability level, for LT and ST soils analysed separately. Long-term experiment † Short-term experiment ‡ Parameters BC § NA # BC § NA # SOM (g kg - 1 ) 85 ± 6 49 ± 3 * 100 ± 10 68 ± 5 * C (g kg - 1 ) 37 ± 7 17 ± 5 * 34 ± 12 22 ± 4 * N (g kg - 1 ) 3.37 ± 0.40 1.75 ± 0.19 * 3.83 ± 1.16 2.38 ± 0.35 * P (mg kg - 1 ) 130.3 ± 5.5 19.6 ± 0.9 * 88.5 ± 5.6 20.3 ± 1.2 * K (mg kg - 1 ) 118 ± 45 101 ± 51 356 ± 57 165 ± 40 * Na (mg kg - 1 ) 210 ± 50 285 ± 80 365 ± 50 320 ± 13 * Ca (g kg - 1 ) 6.1 ± 0.9 4.3 ± 0.4 * 5.0 ± 0.5 3.1 ± 0.2 * Mg (mg kg - 1 ) 156 ± 30 222 ± 60 * 259 ± 60 109 ± 11 * Fe (mg kg - 1 ) 1.73 ± 1.12 7.25 ± 4.64 * 1.08 ± 0.62 0.77 ± 0.53 Mn (mg kg - 1 ) 3.68 ± 0.72 69.50 ± 15.87 * 7.86 ± 1.11 5.13 ± 1.78 * Cu nd nd nd nd Zn (mg kg - 1 ) 1.75 ± 0.80 23.23 ± 2.09 * 1.22 ± 0.33 1.35 ± 0.41 Pb nd nd nd nd Cd nd nd nd nd †Soils collected from the long-term experimental field (fenced 17 years ago and not exposed to grazing). ‡Soils collected from the unfenced area and exposed to extensive grazing for over 17 years. §Soil amended with biosolid compost. #Non-amended control soil. nd: below detection limit. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 91 Capítulo 2. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 92 Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 93 Resumen Una gran proporción de los suelos mediterráneos se encuentran amenazados debido a las pérdidas de materia orgánica y biodiversidad, lo que podría comprometer el suministro de servicios ecosistémicos y la estabilidad de los ecosistemas frente al cambio climático. En este trabajo exploramos varias hipótesis relacionadas con el papel de los aportes de C y la diversidad microbiana en la multifuncionalidad de los suelos mediterráneos degradados, y su resistencia a la sequía. Hemos llevado a cabo un experimento en mesocosmos (macetas) con un diseño factorial para evaluar los efectos de la adición de C orgánico y de la diversidad microbiana (empleando cuatro inóculos con diferente abundancia y diversidad de microorganismos del suelo), en la resistencia de la funcionalidad del suelo frente a condiciones simuladas de sequía. Las macetas fueron sembradas con una mezcla forrajera (Lolium rigidum y Medicago polymorpha), y la producción vegetal, las propiedades físicoquímicas del suelo y la actividad y diversidad microbianas fueron medidas antes y después de un evento simulado de sequía. La enmienda orgánica incrementó la humedad del suelo, mejorando la estabilidad de la producción de biomasa de M. polymorpha. La inoculación con diferentes comunidades microbianas tuvo un efecto muy limitado sobre la resistencia de la actividad biológica frente a la sequía. En efecto, la actividad microbiana fue muy resistente a la reducción en el aporte de agua, lo que podría estar relacionado con la predominancia de bacterias Gram positivas en estos suelos. La riqueza y diversidad microbianas estuvieron significativamente influenciadas por la enmienda y el tratamiento de inoculación, al igual que por la interacción enmienda × inóculo, mientras que no se detectaron efectos del tratamiento de sequía. Las relaciones entre la diversidad microbiana y la multifuncionalidad del suelo solo fueron significativas en los suelos enmendados. Un modelo de ecuaciones estructurales confirmó que el aumento de la multifuncionalidad tras la aplicación de la enmienda fue debida al efecto directo del C orgánico sobre la retención de agua y la fertilidad del suelo. En estos suelos degradados las limitaciones físico-químicas, en vez de la diversidad bacteriana o fúngica, son los principales responsables de la multifuncionalidad del suelo. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 94 Abstract A large fraction of the Mediterranean soils is threatened by losses of organic matter and soil biodiversity, which could compromise the provision of soil ecosystem services and the stability of ecosystems in the face of climate change. In this work we explore several hypotheses related to the role of C inputs and microbial diversity on soil multifunctionality and its resistance to drought in degraded Mediterranean soils. We designed a factorial experiment to test the effects of the addition of an organic amendment and of microbial diversity (using four inoculants with different abundance and diversity of soil microbiota), on the resistance of soil functionality against simulated drought conditions in pot mesocosms. Pots were sown with a forage mixture (Lolium rigidum and Medicago polymorpha), and plant productivity, soil chemical properties and microbial activity and diversity were measured before and after a simulated drought event. The organic amendment increased soil moisture, enhancing the stability of the biomass production of M. polymorpha. Inoculation with different microbial communities had a very limited effect on the resistance of biological activity to drought. Indeed, microbial functioning was highly resistant to reduced water inputs, which could be related to the dominance of Gram positive bacteria in these soils. Microbial richness and diversity were significantly influenced by the amendment and the inoculum treatments, as well as by the amendment × inoculum interaction, while no effects of the drought treatment were detected. Relationships between microbial diversity and soil multifunctionality were significant only in the amended soils. Structural equation modeling confirmed that the enhancement of multifunctionality after soil amendment was due to the direct effect of organic C on soil water retention and chemical fertility. In these degraded soils physico-chemical limitations, rather than bacterial or fungal biodiversity, are the major drivers of soil multifunctionality. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 101 activities and respiration rate), finally, 3 g of soil were frozen and stored at -80 ºC to analyze soil microbial DNA. a) Soil chemical properties A 1:2.5 soil:water suspension was used to measure soil pH, and soil electrical conductivity (EC) was measured in a 1:5 soil:water suspension. Total C, N and S contents were analyzed with a micro elemental analyzer (Leco Truspect CHNS Micro). Bioavailable fractions of Ca, Mg, K and Na were extracted in ammonium acetate at pH 7 (Ojea and Carballas, 1976). Ca and Mg contents were measured by atomic absorption spectroscopy, and K and Na by atomic emission spectroscopy (PerkinElmer AAnalyst 100). Olsen P was extracted in 0.5 N sodium bicarbonate and quantified by molecular absorption spectrophotometry (Murphy and Riley, 1962). b) Soil microbial activity Soil dehydrogenase activity (DHA) was measured by molecular absorption spectrophotometry using 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium chloride (INT) as substrate (Benefield et al., 1977). Leucine-aminopeptidase, β-glucosidase, phosphatase and N-acetyl-glucosaminidase activities were extracted in a sodium acetate solution and determined by fluorometry using 7-amino-4-methyl coumarin (AMC) and 4methylumbelliferone (MUB) as fluorogenic substrates, respectively (Marx et al., 2001). Soil respiration rate was determined with an infra-red gas analyzer (EGM-4, PP Systems) after incubating soil samples at 25 ºC for 24 hours (Bekku et al., 1995). c) Soil genomic DNA extraction and analysis DNA was extracted from 0.25 g of soil using the DNeasy PowerSoil Pro Kit (QUIAGEN). DNA concentration was measured with a Qubit fluorometer (ThermoFisher). qPCRs (Takara Bio’s SYBR® Premix Ex Taq) of the 16S rDNA (341F/805R) and ITS rDNA (ITS86F/ITS4) regions were carried out to determine the relative abundance of bacteria and fungi within the soil Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 102 microbial community. The qPCR experiments were performed on a Mx3000P Multiplex Quantitative PCR System (Stratagene). The amplification programme started with an initial denaturation stage of 10 min at 95 ºC, followed by 40 amplification cycles (30 s at 95 ºC, 30 s at 60 ºC (16S) or 62 ºC (ITS), and 30 s at 72 ºC), and a final dissociation phase (1 min at 95 ºC, 30 s at 55 ºC and 30 s at 95 ºC). Each qPCR measurement was carried out in triplicate. To calibrate the qPCR method, samples of 16S and ITS amplicons in serial concentration dilution gradient were used. PCRs were used to amplify both target regions (34 amplification cycles, annealing temperature of 60 ºC or 62 ºC for the 16S and ITS regions, respectively) from one random soil DNA extract. PCR products were loaded in an agarose gel, and 16S and ITS amplicons were purified using the GeneJET Kit (Thermo Scientific™). DNA concentrations were determined using a Qubit fluorometer (ThermoFisher). Subsequently, serial dilutions were done (10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7) and the corresponding Ct values used to draw the calibration curve, were determined by qPCRs (same protocol described above). Finally, the same primer pairs were used in an Illumina high-throughput sequencing process to study the composition of bacterial and fungal communities. Paired-end reads were merged using the software FLASH version 1.2.7 (Magoč and Salzberg, 2011), and the resulting raw data were filtered following the Qiime (version 1.7.0) quality controlled process (Caporaso et al., 2010). After removing chimera sequences, effective tags with ≥ 97 % similarity were clustered into the same Operational Taxonomic Unit (OTU). Bacterial and fungal OTUs were assigned to the corresponding taxonomic information using SILVA (Yilmaz et al., 2013) and UNITE (Abarenkov et al., 2022) reference databases, respectively. Fungal taxa were finally classified into functional groups following the database of Põlme et al. (Põlme et al., 2020). Statistical analyses All statistical analyses were performed in R version 4.1.3 (RCoreTeam, 2022). Linear mixed models were used to study the effect of the biosolid compost application, the inoculation, Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 103 the drought conditions and the interaction of these factors (amendment × inoculum × drought) on soil chemical properties, microbial activity, bacterial and fungal abundance and diversity, and plant development. Tukey tests were used as post hocs (p < 0.05). The effect of the experimental treatments on soil moisture was evaluated using mixed linear models (nlme package in R), with each pot considered as a random factor to account for repeated measurements. The residuals of each model were studied before the model validation. In those cases where the model residuals showed heteroscedasticity among the different factor levels, a variance coefficient was introduced using the varIdent function of the nlme package (Pinheiro, 2000). Non-metric multidimensional scaling (NMDS) analyses based on Bray-Curtis dissimilarities were carried out to spatially visualize samples according to the composition of their microbial community. Differences among groups and the effect of the different treatments on the bacterial and fungal community structure were tested by permutational multivariate ANOVA (PERMANOVA) analyses and pairwise post-hoc tests were used to analyse differences among groups. In order to integrate the information of soil biological activity and plant productivity in a single index, and to synthetize the influence of amendment addition, drought and biodiversity levels on soil functioning, a soil multifunctionality index (Byrnes et al., 2014) was calculated. This index included total plant production and soil biological activity (dehydrogenase, leucine-aminopeptidase, β-glucosidase, phosphatase and N-acetylglucosaminidase activities, and soil respiration rate). Among the different ways to measure multifunctionality, the averaging approach is calculated by averaging the standardized values of the multiple ecosystem functions (Byrnes et al., 2014). Data were log-transformed and subsequently z-transformed to standardize the seven soil functions included in this index. Pearson`s correlations between the resultant soil multifunctionality index and abundance, diversity and taxa richness of soil bacterial and fungal populations were evaluated. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 104 Finally, structural equation modelling (SEM) was used to explore the structure of direct and/or indirect effects of the application of the organic amendment and microbial diversity on soil multifunctionality. Based on previous observations, main soil chemical properties (total C and N, and available P) were selected as expected relevant predictors of soil multifunctionality and were tested in separate models together with the bacterial and fungal diversity values to achieve the model with the best fit. Soil moisture was also included as predictor of soil multifunctionality since our study is based on the effect of drought on soil functioning. Each model goodness-of-fit was evaluated according to three indexes. The Comparative Fit Index (CFI) and the Tucker-Lewis Index (TLI), that indicate the best fit of the postulated model compared to a baseline model, were tested for an acceptable fit (CFI ≥ 0.90 and TLI ≥ 0.95). In addition, the Root Mean Squared Error of Approximation (RMSEA) index was used to evaluate the badness-of-fit of the proposed model, being values < 0.05 considered as acceptable. Finally, the model with the best fit and the highest p-value in the chi-square test was selected. The chi-square tests the null hypothesis that the predicted and the observed covariance patterns are equal, therefore a high p-value is desired. Analysis were carried out with the lavaan package (Rosseel, 2012) and the obtained model was plotted using the package lavaanPlot (Lishinski, 2021). Results Soil moisture From the beginning of the experiment soil moisture was enhanced by the organic amendment. Before the drought period mean soil moisture before watering was 11.4 ± 2.8 %v in BC pots and 9.7 ± 1.7 %v in NA, and during the one-month drought period soil moisture was 13.5 ± 4.6 % v/v in BC and 8.3 ± 1.3 % v/v in NA. During the drought period significant effects of the amendment treatment (F = 77.68, p < 0.001), drought treatment (F = 15.22, p = 0.002) and the amendment × drought interaction (F = 8.41, p = 0.005) were observed, and, by the end of the experiment, soil moisture in the BC-CT and in the BC-DR treatment was 47% and 28% higher than in the non-amended NA-CT and NA-DR pots, Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 105 respectively. Besides, the inoculum treatment had also a significant effect on soil moisture (F = 8.51, p = 0.001), being the highest in A pots in comparison to the rest of inoculums (12.7 ± 5.0 % v/v, drought and control pooled). Nevertheless, no significant effect of the amendment × inoculum × drought interaction on soil moisture was recorded (F = 0.95, p = 0.421). Plant germination and growth The application of biosolid compost significantly decreased both species germination rates. L. rigidum germination was reduced from 14 ± 4 % to 9 ± 4 % (F = 32.73, p < 0.001), while M. polymorpha germination decreased from 31 ± 12 % to 18 ± 9 % (F = 40.17, p < 0.001) in BC and NA, respectively. In addition, the inoculum treatment (F = 3.66, p = 0.016) and the amendment × inoculum interaction (F = 4.38, p = 0.007) had a significant effect on M. polymorpha germination rate in NA pots (Supplementary Figure S1a). L. rigidum germination was not significantly influenced by the inoculum treatment (F = 1.93, p = 0.133) or the amendment × inoculum interaction (F = 0.72, p = 0.543). L. rigidum final biomass was not significantly different in BC and NA treatments (pooled mean of 1.85 ± 0.65 g, F = 0.31, p = 0.582), but the inoculum (F = 9.94, p < 0.001) and the amendment × inoculum interaction (F = 8.09, p < 0.001) significantly influenced L. rigidum growth in BC pots, so that higher biomass was recorded with inoculum C and D than with A and B (Supplementary Figure S1b). Drought conditions negatively affected L. rigidum final biomass (F = 20.21, p < 0.001), reducing biomass by 23.5 %, but the interaction amendment × inoculum × drought had no significant effect (F = 0.12, p = 0.950). M. polymorpha growth was enhanced by the biosolid compost addition (mean of 0.30 ± 0.20 g per plot) compared to the non-amended treatment (0.16 ± 0.09 g, F = 16.21, p < 0.001), and was significantly higher in B than in D (F = 3.13, 0.032). In this species biomass production was not affected by drought (F = 0.45, p = 0.507) or by the interaction between the different factors (F = 0.12, p = 0.948, Supplementary Figure S1b). Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 106 In relative terms, when comparing the plant biomass in DR pots to the biomass measured in CT pots, it was observed that the negative effect of drought on L. rigidum production (Fig. 1a) was not influenced by the amendment (F = 0.03, p = 0.867), the inoculum (F = 0.55, p = 0.654) or the interaction amendment × inoculum (F = 0.29, p = 0.836). Figure 1. Effect of drought conditions on Lolium rigidum (a) and Medicago polymorpha (b) biomass production. Values indicate mean (± standard deviation) biomass produced in pots belonging to the drought treatment (DR) divided by the mean plant biomass recorded under control conditions (CT) for each treatment. Values below and above 1 indicate a reduction and an increase in plant production in DR compared to CT, respectively. However, the relative impact of drought on M. polymorpha was ameliorated in amended soils in comparison to non-amended soils (F = 5.91, p = 0.021). The inoculum treatment also had a significant effect (F = 3.34, p = 0.032), and M. polymorpha biomass was less Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 107 affected in soils inoculated with A compared to C (Fig. 1b), while the interaction amendment × inoculum had not a significant effect (F = 1.27, p = 0.302). Soil chemical properties At the beginning of the experiment the addition of the organic amendment significantly increased soil pH and electrical conductivity, total C and N contents, and the availability of Na and macronutrients (K, Mg, Ca and P). Inoculum A also had significantly higher pH, total C and N contents, and available K and P compared to inoculum B. In addition, the sludge was characterized by a very low pH, high content of S and available Ca, and very low content of available P. Mean values of the analyzed chemical properties in the initial soil and in the different inoculums are shown in Supplementary Tables S1 and S2. Analyses performed at the second and third samplings provided very similar results regarding to the effects of biosolid compost addition on soil chemical properties: increases of pH, total C and N, available nutrients, and electrical conductivity (Table 1, Supplementary Tables S3, S4 and S5). Besides, the inoculum treatment had a significant effect on most of the analyzed soil chemical properties at both soil samplings, especially in NA soils, and the influence of the interaction amendment × inoculum on soil properties had a special relevance at the third soil sampling (Supplementary Table S5). In particular, NA soils inoculated with the A inoculum tended to have higher pH, organic C content, and P and Mg availability. One month after the establishment of the drought treatment, soil pH and Na content were increased by the drought treatment and were influenced by the amendment × inoculum × drought interaction. Soil pH was significantly higher in BC-B-DR (5.89 ± 0.05) than in BC-BCT (5.38 ± 0.12), and Na content was higher in BC-C-DR (0.27 ± 0.03 mg kg-1) than in BC-CCT (0.22 ± 0.01 mg kg-1) pots. No effect of the simulated drought conditions, nor of the amendment × inoculum × drought interaction was found on the other analyzed soil chemical properties. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 108 Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 109 Soil microbial activity As well as on soil chemical properties, the amendment treatment had a significant effect on soil biological activity at the first soil sampling, before soil inoculation. The addition of the biosolid compost enhanced soil dehydrogenase (DHA) and N-acetyl-glucosaminidase activities, while soil leucine-aminopeptidase and phosphatase activities, as well as soil respiration rate, were significantly higher in NA than in BC. Soil β-glucosidase activity was not significantly affected by the organic amendment. Comparing A and B inoculums, all biological parameters were significantly higher in A than in B, except for soil phosphatase activity which was higher in the inoculum B. The sludge showed the lowest values of Nacetyl-glucosaminidase, phosphatase and β-glucosidase activities. Mean values of the analyzed biological parameters from the first soil sampling are shown in Supplementary Tables S1 and S2. The positive effect of the organic amendment was observed at the second soil sampling on soil DHA, leucine-aminopeptidase and N-acetyl-glucosaminidase activities, as well as on soil respiration rate. However, no significant effect of the amendment treatment was observed on the other measured enzyme activities. The inoculation treatment had a significant influence on most of the indexes of biological activity (Supplementary Table S4), except for soil phosphatase activity, being soil DHA the highest in pots with inoculum A. However, Nacetyl-glucosaminidase activity was enhanced by inoculums C and D. Besides, the effect of the amendment × inoculum interaction was only significant for the leucine-aminopeptidase and N-acetyl-glucosaminidase activities (Supplementary Table S4). Finally, at the third soil sampling, all enzyme activities, as well as soil respiration rate, were significantly different in BC and NA soils. Following the same pattern as in the previous soil sampling, DHA, leucine-aminopeptidase activity and soil respiration rate were increased in BC compared to NA. N-acetyl-glucosaminidase and β-glucosidase activities were also positively affected by the organic amendment, while soil phosphatase activity was higher in NA than in amended soils. Biological activity was also highly influenced by the inoculation Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 110 treatment and the amendment × inoculum interaction (Table 1, Supplementary Table S5). In particular, the inoculum with the potentially highest levels of bacterial and fungal diversity (A) showed the highest respiration rates in both amended and unamended soils, and the highest dehydrogenase activity in NA soils (been the lowest in NA-D). Interestingly, soils inoculated with inoculums C (from the dilution-to extinction procedure) and D in the BC soils showed high values of N-acetyl-glucosaminidase, phosphatase and β-glucosidase activities. In addition, drought conditions significantly reduced soil N-acetylglucosaminidase (- 13 %) and phosphatase (- 12 %) activities in amended soils. However, no other effects of the drought treatment or the amendment × inoculum × drought interactions were found (Supplementary Table S5). Soil microbial community: bacterial and fungal abundance At the second soil sampling, one month after the inoculation and the beginning of the experiment, the amendment treatment increased the abundance of soil bacteria (from 146 ± 47 ng g-1 in NA to 265 ± 99 ng g-1 in BC) while the abundance of fungi was reduced (from 0.62 ± 0.25 ng g-1 in NA to 0.37 ± 0.15 ng g-1 in BC), significantly decreasing the ratio ITS:16S. However, the concentration of total DNA in BC (7.54 ± 1.44 µg g-1) was slightly lower than in NA soils (9.13 ± 4.19 µg g-1). At sampling two, the inoculation treatment also had a significant effect on soil DNA (being the highest in inoculum A with 12.24 ± 3.78 µg g-1). Bacterial abundance was higher in C and D (pooled mean of 254 ± 7 ng g-1) compared to A and B (pooled mean of 157 ± 19 ng g-1), while fungal abundance was higher in D than in C, been the ratio ITS:16S the lowest in C. The amendment × inoculum interaction also had a significant effect on total DNA (the highest in NA-A), the abundance of bacteria (higher in BC-C and BC-D than in the other treatments), and the ratio ITS:16S (Supplementary Tables S3 and S4). After the simulated drought event a significant increase in total DNA in BC (13.15 ± 2.54 µg g-1) in comparison to NA soils (9.71 ± 5.25 µg g-1) was observed, however, the abundances of bacteria and fungi were higher in the non-amended soil (114.31 ± 67.01 ng g-1 and 0.58 Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 117 Figure 5. Correlation analysis between soil multifunctionality and soil bacterial (a) and fungal (b) diversity (Shannon Diversity Index, H´) in amended (BC) and non-amended (NA) soils. Pearson correlation coefficients and p-values are indicated next to the lines. The structure of correlations among the studied variables was explored with structural equation modelling (Fig. 6). The SEM model indicated a positive correlation between soil bacterial and fungal diversity, and a positive and direct effect of the biosolid compost application on bacterial diversity. However, soil multifunctionality was controlled by soil total C content and positively affected by the organic amendment, instead of by soil microbial diversity. The amendment had a positive and significant effect on soil total C content and soil moisture. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 118 Figure 6. Structural equation model (SEM) exploring relationships between soil multifunctionality, soil properties and microbial diversity. Proposed model (a) and final full path diagram representing the hypothesized relationships among soil chemical fertility (indicated by soil C), soil moisture, fungal and bacterial diversity and soil multifunctionality (b). Regressions are indicated by simple arrows while double arrows represent correlations. Numbers next to arrows are the standardized coefficients of the significant relationships, and significance levels are indicated by the number of asterisks (*, ** and *** represent < 0.05, < 0.01 and < 0.001, respectively). R2 represents the proportion of variance explained. The Root Mean Square Error of Approximation (RMSEA) index value indicates a good fit of the model. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 119 Discussion Soil moisture, chemical properties and plant productivity As expected and due to the increase in soil organic matter content (Cooper and DeMarco, 2023), the application of the biosolid compost improved soil water retention capacity and helped to reduce water loss under the simulated drought conditions compared to the nonamended soil. Likewise, the positive effect of the inoculum A on soil moisture in both amended and non-amended soils could be partly explained by the supply of certain amount of organic matter, as inoculum A came from a long-term amended soil with a higher content of total C compared to inoculum B (Morales-Salmerón et al., 2024). The analysis of soil chemical properties also revealed a positive effect of the organic amendment on soil pH, total C and N, and the availability of macronutrients, that were patent since the biosolid compost was applied. The effect of the inoculum treatment was especially important in non-amended soils, where soil chemical properties were improved, especially by inoculum A. Besides, the simulated drought conditions did not affect soil chemical properties except for slight increases in soil pH and available Na content in amended soils. However, previous studies observed the opposite effect of drought on soil Na content, since the absorption of Na by plants is one of their strategies to face water stress in (Sardans et al., 2008; Huihui et al., 2021). Thanks to the improvement in soil water retention capacity and the higher availability of macronutrients, the organic amendment also had a positive effect on the plant cover. Although in both species germination rate was reduced due to the toxicity of the biosolid compost on the short-term, as found in other works (Zubillaga and Lavado, 2006), L. rigidum and M. polymorpha growth was promoted in amended soils. Drought conditions reduced L. rigidum biomass production in all cases, independently of the amendment or inoculum treatments. However, M. polymorpha showed a high tolerance to the simulated drought conditions and even had a higher growth under drought conditions in pots Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 120 belonging to the amendment treatment (as well as in soils inoculated with inoculum A), probably explained by the lower competition with L. rigidum and the higher content of organic matter and nutrients supplied by the biosolid compost. Other works have also showed the high tolerance of M. polymorpha to drought (Nichols et al., 2009). In particular, a previous study also showed that M. polymorpha is more resistant to water stress than L. rigidum (Morales-Salmerón et al., 2024). Soil microbial activity response to drought and compost addition Biosolid compost enhanced soil biological activity (enzyme activities and respiration rate), as it was observed in previous studies carried out with soils from the same area (Pérez de Mora et al., 2005; Montiel-Rozas et al., 2016). However, the higher content of inorganic P in amended soils, and especially in soils inoculated with inoculum A, resulted in the inhibition of soil phosphatase activity compared to the non-amended soils, as observed in other works (Janes-Bassett et al., 2022). The application of the organic amendment overshadowed the effect of the inoculation treatment on soil biological activity, explained by the higher relevance of soil chemical properties than of microbial diversity on soil functioning (discussed below). In nonamended soils there were some effects of the inoculation treatment on biological parameters, in particular dehydrogenase activity and soil respiration rates were the highest in soils where inoculum A, with the highest bacterial and fungal diversity, was applied, while soil leucine-aminopeptidase and N-acetyl-glucosaminidase activities were higher in inoculums C and D. The slight increases in organic C introduced with the A inoculum, rather than the highest levels microbial diversity, could be the reason for these observations, indeed, the SEM analysis confirmed that in the non-amended soils soil multifunctionality and bacterial/fungal diversity were disconnected (discussed below). In addition, the higher content of N and P in soils inoculated with A may be responsible of the observed reduction in soil enzyme activities (Xiao et al., 2018). Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 121 Regarding the impact of drought on biological activity, this experiment showed that under these experimental conditions soil functioning is quite resistant to decreases in water inputs, and that plant production is more sensible to the decline in soil moisture than microbial activity in these soils. Water stress often reduces the rates of enzyme activity due to limited substrate supply and diffusion or due to altered enzyme hydration and conformation (Henry, 2012). Indeed, reductions in soil enzyme activities have been reported in several field experiments with well-drained soils (Sardans and Peñuelas, 2005; Sardans et al., 2008; Steinweg et al., 2013). However, a meta-analysis of the effects of global change drivers of soil enzyme including data from 133 field experiments in natural ecosystems concluded that drought had a very limited effect on the activity of nutrientacquisition enzymes, in comparison to other global change drivers such as nutrient addition (Xiao et al., 2018), which is consistent with the results of this study, showing a much larger effect of compost addition than of drought on microbial activity. As with soil enzymes, soil respiratory was rather insensitive to the drought conditions simulated in this work. With our experimental design we aimed to simulate a reduction of water inputs during the plant growing season, which typically takes place over the Spring months in the area where soils were collected. Mediterranean soils are naturally exposed to more severe water stress over the summer months. Soils from semi-arid Mediterranean regions are adapted to drought, so that soil biological activity can be maintained even under extremely low moisture levels, for instance, in a previous work it was shown that biological activity was only reduced when soil moisture was below 2 % (Hueso et al., 2011). Our results are in line with other works showing that the responses of soil respiration or multifunctionality to changes in soil moisture are less pronounced in soils that receive a low annual precipitation or that have a legacy of exposition to drought events (Hawkes et al., 2017; Canarini et al., 2021), likely due to adjustments in the composition or the abundance of microbial taxa promoting a greater drought resistance (Dacal et al., 2022). Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 122 Effect of treatments on soil microbial diversity The application of the biosolid compost enhanced soil bacterial populations in the shortterm (one month after soil amendment), while fungal populations were significantly decreased. However, two months after the biosolid compost addition, this effect was not observed and bacterial and fungal abundances were slightly higher in non-amended soils. Despite the similar abundance of soil bacteria and fungi in both amended and nonamended soils, microbial richness and diversity were positively influenced by the organic amendment by the end of the experiment (two months after the amendment application). The application of the biosolid compost, as occurred with soil biological activity, masked the effect of the inoculation treatment on bacterial and fungal diversity. Despite the inoculation treatment did not have a clear effect on soil bacterial and fungal abundances on the short-term, within the non-amended soils those inoculated with inoculums A and B were characterized by higher bacterial and fungal richness and diversity, as expected given the diversity gradient established in the experimental design. It is noteworthy that the observed bacterial and fungal diversities accurately reflect the microbial diversity expected in an arid Mediterranean grassland (Castro et al., 2016; Gao et al., 2022; Bonanomi et al., 2022; Labouyrie et al., 2023). The NMDS analysis revealed the role of the organic amendment, represented in the axis NMDS1, as the main driver of the structure of soil both bacterial and fungal communities. The inoculation treatment also had a significant effect on soil microbial communities. In both amended and non-amended soils, bacterial and fungal communities in soils inoculated with A and B were the most clearly distinguished, while microbial communities in C and D soils were more similar and showed higher variability among samples. It is remarkable that soil bacterial communities, independently of the amendment and inoculation treatments, were dominated (around 67 %) by genera belonging to phyla Actinobacteriota (genera Nocardioides, Streptomyces and Kribbella) and Firmicutes (genera Bacillus, Pullulanibacillus and Alicyclobacillus), both of them composed of Gram Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 123 positive bacteria. Gram positive (monoderms) bacteria are characterized by a thick peptidoglycan cell wall layer, which contributes to enhance their tolerance to dessication (Schimel et al., 2007). This tolerance can also be related to the ability to form endospore of some Firmicutes groups (Schimel, 2018). A dominance of the Gram positive phylum Actinobacteria is often reported in arid, nutrient-poor soils (Fierer et al., 2012; Maestre et al., 2015; Delgado-Baquerizo et al., 2016). In addition, several experiments with soils from different biomes have shown that the relative abundances of Gram positive bacteria, including oligotrophic Actinomyces and Firmicutes, are increased in bulk and rhizospheric soils exposed to drought (Bastida et al., 2017; Canarini et al., 2021; Sun et al., 2023), as well in the root microbiome, which has been suggested to be an ubiquitous mechanism to promote drought resistance in plants (Naylor and Coleman-Derr, 2018; Xu and ColemanDerr, 2019). In particular, in Mediterranean soils it has been suggested that Gram positive bacteria are underrepresented under optimal conditions of soil moisture (during the spring season) in relation to Gram negative lineages (Curiel Yuste et al., 2014). In arid soils key microbial taxa, rather than the richness, abundance and the ratio of bacteria and fungi, can control the resistance of soil multifunctionality to climate change drivers (DelgadoBaquerizo et al., 2017). This dominance of Gram positive bacteria could explain the limited effects of soil moisture reduction on microbial activity and diversity in our work. Under our experimental conditions the simulated drought event had a very limited impact on soil bacterial and fungal abundance, taxa richness and diversity. The most relevant effect of drought conditions on soil microbial communities was limited to the increment of the relative abundance of the genus Trichoderma in NA-C-DR and NA-D-DR soils. Trichoderma, a filamentous fungi (Põlme et al., 2020), shows a high tolerance to water stress and several Trichoderma species have been recently used as bio-inoculant to increase the plant capacity to absorb nutrients and water, enhancing crop resistance to drought conditions (Boorboori and Zhang, 2023). This could explain the higher relative abundance of this genus under drought conditions. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 124 Soil multifunctionality Our results showed the positive effect of soil organic amendment on soil multifunctionality, supporting the initial hypothesis that the improvement in soil chemical properties due to the biosolid compost application (organic matter and macronutrients) results in an increase in soil multifunctionality. However, the inoculum treatment only had an effect in amended soils, where, in contrast with our expectations, a higher multifunctionality index was observed in BC-D than in BC-A and BC-B. Although differences among the amended soils in fungal abundance and diversity were not significantly different, the higher dominance of fungi in BC-D may explain the higher multifunctionality, supported by the positive correlation between the ITS:16S ratio and soil multifunctionality. In drylands, soil functionality is primarily influenced by the diversity of fungal taxa due to their higher tolerance to water stress when compared to bacteria (Delgado-Baquerizo et al., 2016). This highlights the significant role played by soil fungal diversity in Mediterranean grasslands. Although plant pathogens may negatively affect soil multifunctionality, it was demonstrated that soil fungal richness and saprotrophic fungi are the main biotic factors in regulating soil functioning in semiarid environments (Li et al., 2022). Under our experimental conditions, the results exhibited a high stability of soil multifunctionality to the simulated drought event, which could be explained by the high tolerance of the soil microbial community to water stress (discussed above). When inoculation and drought treatments were pooled together, the calculated soil multifunctionality index was positively correlated with bacterial and fungal diversity (estimated using the Shannon diversity index) and richness, what is consistent with the results obtained in previous studies (Delgado-Baquerizo et al., 2016, Delgado-Baquerizo et al., 2020). However, this positive relationship between microbial diversity and richness and soil multifunctionality was only observed in amended soils, while in non-amended soils this correlation, although it was not statistically significant, showed the opposite pattern (less multifunctionality in soils with higher bacterial and fungal diversity). This may respond to Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 125 the fact that higher levels of microbial diversity not always result in a higher soil multifunctionality, since the relationship is dependent of soil chemical properties and is affected by soil degradation (Xue et al., 2023). When the role of bacterial and fungal diversity, and the effects of the organic amendment application, soil chemical properties (represented by the content of total C) and soil moisture on soil multifunctionality where simultaneously analysed in the structural equation model (SEM), it was demonstrated that the observed relation between microbial diversity and soil multifunctionality was an indirect effect. While bacterial and fungal diversity showed a positive correlation, in our case of study the main driver of soil multifunctionality was the biosolid compost addition, followed by the total C content (also controlled by the organic amendment). The amendment application was also responsible for soil moisture and bacterial diversity, what proves the positive effect of organic amendments in degraded soils. Conclusions In this work we have explored several hypotheses related to the role of soil microbial diversity and of C inputs (in the form of an organic amendment) on soil multifunctionality and its resistance to a drought event in degraded Mediterranean soils. In summary, we could not confirm our initial hypothesis that a higher microbial diversity is linked to a higher stability of soil multifunctionality to drought in these degraded soils. It seems that microbial functioning in these soils is quite resistant to reduced water inputs, at least at the levels of moderate moisture reductions tested in this work, which could be related to the dominance of Gram positive bacteria. According to our expectations, the addition of an organic amendment conferred a higher stability of plant production to drought. This was due to the direct effect of organic C on soil water retention and chemical fertility, rather than due to the direct effect of organic Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 126 inputs on microbial richness or diversity. In these degraded soils physico-chemical limitations, rather than bacterial or fungal biodiversity, are the major drivers of soil multifunctionality. This was indicated by the lack of any significant relationship between biodiversity and multifunctionality in the most degraded, non-amended soils. These results highlight the importance of preventing C losses and increasing organic C levels in Mediterranean soils. Soil C was the main driver of soil multifunctionality and microbial diversity in these soils, and promoted a higher resistance of plant productivity to the reduction of water inputs. Given the climate change predictions for the Mediterranean region, where a large proportion of soils are threatened by C losses, it seems critical to increase the levels of organic C in these soils to promote a higher stability of soil productivity against droughts, even if microbial activity and diversity in these soils are highly resistant to fluctuations in soil moisture levels. However, it would be necessary to conduct further studies that replicate more severe drought conditions in order to better evaluate the resistance of Mediterranean soils to water stress. Due to the extremely dry conditions that Mediterranean soils are exposed to in the summer, it would be interesting to test the impact of drought conditions on soil microbial activity in situ trough a field experiment. Besides, an alternative way to inoculate soils must be considered in future experiments. The addition of organic matter through the solid inoculums, especially in the case of inoculum A, had an unexpected effect on soil chemical properties in these C-poor soils, and made it difficult to discern between the chemical and the biological effect of the treatment on soil functions. Acknowledgements This study was funded by the Spanish Ministry of Science and Innovation (projects CGL2017-85891-R DEGRAMED and PID2021-122628OB-I00 WASTE4DROUGHT) and Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 133 Steinweg, J., Dukes, J., Paul, E., Wallenstein, M., 2013. Microbial responses to multi-factor climate change: effects on soil enzymes. Frontiers in Microbiology 4. Sun, Y., Tao, C., Deng, X., Liu, H., Shen, Z., Liu, Y., Li, R., Shen, Q., Geisen, S., 2023. Organic fertilization enhances the resistance and resilience of soil microbial communities under extreme drought. Journal of Advanced Research 47, 1-12. WRB, 2014. World Reference Base for soil resources 2014: international soil classification system for naming soils and creating legends for soil maps. Food and Agriculture Organization of the United Nations, Rome. Xiao, W., Chen, X., Jing, X., Zhu, B., 2018. A meta-analysis of soil extracellular enzyme activities in response to global change. Soil Biology and Biochemistry 123, 21-32. Xu, L., Coleman-Derr, D., 2019. Causes and consequences of a conserved bacterial root microbiome response to drought stress. Current Opinion in Microbiology 49, 1-6. Xue, R., Wang, C., Zhao, L., Cao, J., Liu, M., Zhang, D., 2023. Agricultural intensification weakens soil multifunctionality by reducing fungal diversity. Applied Soil Ecology 189. Yilmaz, P., Parfrey, L.W., Yarza, P., Gerken, J., Pruesse, E., Quast, C., Schweer, T., Peplies, J., Ludwig, W., Glöckner, F.O., 2013. The SILVA and “All-species Living Tree Project (LTP)” taxonomic frameworks. Nucleic Acids Research 42, D643-D648. Zubillaga, M.S., Lavado, R.S., 2006. Phytotoxicity of biosolids compost at different degrees of maturity compared to biosolids and animal manures. Compost Science and Utilization 14, 267270. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 134 Supplementary material Figure S1. Mean (bars) and standard deviation (lines) of Lolium rigidum and Medicago polymorpha germination rate (a) and produced dry biomass (b) in each treatment. Different letters above bars indicate significant (p < 0.005) differences among treatments within amended (BC) and non-amended soils (NA). Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 135 Table S1. Initial characterization (mean values ± standard deviation) of the amended (BC) and non-amended (NA) soils before been inoculated. The results of the statistical analyses (F and p-value) are indicated. Soil type Statistics Parameters BC NA F p pH 4.68 ± 0.04 3.94 ± 0.03 616 < 0.001 EC (dS m-1) 2.96 ± 0.16 0.46 ± 0.06 653 < 0.001 Total C (g kg-1) 48.6 ± 1.0 34.6 ± 1.8 139 < 0.001 Total N (g kg-1) 3.41 ± 0.10 2.13 ± 0.13 144 < 0.001 Total S (g kg-1) 12.8 ± 0.2 15.0 ± 0.8 18.15 0.013 K (mg kg-1) 777 ± 7 653 ± 11 259 < 0.001 Na (mg kg -1) 278 ± 10 253 ± 7 11.63 0.027 Mg (mg kg-1) 35 ± 3 25 ± 3 15.86 0.016 Ca (mg kg-1) 1314 ± 80 412 ± 41 300 < 0.001 P (mg kg-1) 38.19 ± 2.02 21.03 ± 1.96 112 < 0.001 Dehydrogenase activity (µg INTF g dry soil-1 h-1) 1.902 ± 0.053 0.003 ± 0.005 3786 < 0.001 Aminopeptidase activity (nmol AMC g dry soil-1 h-1) 112 ± 2 88 ± 4 80.81 < 0.001 N-acetylglucosaminidase activity (nmol MUB g dry soil-1 h-1) 179 ± 7 207 ± 6 28.49 0.006 Phosphatase activity (nmol MUB g dry soil-1 h-1) 703 ± 43 616 ± 19 10.32 0.033 β-glucosidase activity (nmol MUB g dry soil-1 h-1) 634 ± 26 635 ± 20 0.005 0.949 Respiration rate (g C-CO2 g dry soil-1 day-1) 2.4 ± 0.6 1.2 ± 0.5 7.96 0.048 Table S2. Initial characterization (mean values ± standard deviation) of the inoculums A and B, and the sludge used to prepare inoculum C. The results of the statistical analyses comparing the three substrates (F and p-value) are indicated. Different letters indicate significant differences. Inoculums Statistics Parameters Inoculum A Inoculum B Sludge F p pH 6.85 ± 0.05 a 3.69 ± 0.02 b 2.53 ± 0.02 c 12379 < 0.001 EC (dS m-1) 0.68 ± 0.18 c 2.52 ± 0.03 b 2.95 ± 0.18 a 208 < 0.001 Total C (g kg-1) 54.0 ± 3.1 a 18.0 ± 2.0 b 12.2 ± 0.8 c 319 < 0.001 Total N (g kg-1) 4.03 ± 0.29 a 0.92 ± 0.17 b 1.10 ± 0.18 b 160 < 0.001 Total S (g kg-1) 0.42 ± 0.04 c 1.20 ± 0.17 b 5.51 ± 0.36 a 423 < 0.001 Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 136 Table S2. Continued. Inoculums Statistics Parameters Inoculum A Inoculum B Sludge F p K (mg kg-1) 776 ± 42 a 550 ± 10 b 477 ± 1 b 84.53 < 0.001 Na (mg kg -1) 282 ± 20 266 ± 8 246 ± 11 4.95 0.054 Mg (mg kg-1) 36 ± 1 30 ± 5 32 ± 7 1.21 0.360 Ca (mg kg-1) 1308 ± 63 b 1121 ± 88 b 4167 ± 346 a 200 < 0.001 P (mg kg-1) 105.3 ± 0.8 a 18.6 ± 1.3 b 3.7 ± 0.1 c 11976 < 0.001 Dehydrogenase activity (µg INTF g-1 h-1) 12.27 ± 0.32 a 0.11 ± 0.07 b 0.05 ± 0.07 b 4008 < 0.001 Aminopeptidase activity (nmol AMC g-1 h-1) 106 ± 4 a 89 ± 3 b 95 ± 7 ab 9.63 0.013 N-acetyl-glucosaminidase activity (nmol MUB g-1 h-1) 275 ± 6 a 213 ± 12 b 171 ± 1 c 132.8 < 0.001 Phosphatase activity (nmol MUB g-1 h-1) 584 ± 19 b 718 ± 16 a 398.20 ± 6.56 c 365.5 < 0.001 βglucosidase activity (nmol MUB g-1 h-1) 826 ± 30 a 608 ± 12 b 452 ± 1 c 299.3 < 0.001 Respiration rate (g C-CO2 g-1 day-1) 2.8 ± 0.6 a 0.9 ± 0.5 b 2.2 ± 0.9 ab 6.44 0.032 Table S3. Mean (± standard deviation) soil chemical properties, biological activity, total DNA, bacterial (16S) and fungal (ITS) abundance, and ITS:16S ratio at the second soil sampling (before drought). Different letters indicate significant differences among soils treated with the different inoculums within amended (BC) and non-amended soils (NA). Inoculum Parameters A B C D BC pH 5.86 ± 0.02 a 5.66 ± 0.01 a 5.82 ± 0.10 a 5.41 ± 0.13 b EC (dS m - 1 ) 3.63 ± 0.11 4.50 ± 0.59 4.11 ± 0.86 5.53 ± 1.92 Total C (g kg - 1 ) 46 ± 2 47 ± 3 48 ± 5 53 ± 4 Total N (g kg - 1 ) 4.6 ± 0.9 3.9 ± 0.3 3.8 ± 0.5 5.0 ± 0.6 Total S (g kg - 1 ) 7.6 ± 0.6 b 8.3 ± 0.8 b 8.5 ± 0.9 b 12.3 ± 1.4 a K (mg kg - 1 ) 998 ± 49 945 ± 64 1027 ± 53 1016 ± 60 Na (mg kg - 1 ) 314 ± 47 310 ± 29 283 ± 64 281 ± 51 Mg (mg kg - 1 ) 60 ± 8 b 112 ± 39 ab 133 ± 31 ab 154 ± 51 a Ca (mg kg - 1 ) 1287 ± 84 b 2267 ± 690 ab 2817 ± 295 a 3117 ± 362 a P (mg kg - 1 ) 58.1 ± 4.0 a 47.8 ± 1.2 b 54.2 ± 5.6 a 58.9 ± 10.0 a DHA (µg INTF g - 1 h - 1 ) 2.2 ± 0.5 1.8 ± 0.7 2.2 ± 0.4 2.2 ± 0.4 AMIN (nmol AMC g - 1 h - 1 ) 633 ± 76 508 ± 82 593 ± 128 570 ± 77 Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 137 Table S3. Continued. Inoculum Parameters A B C D BC NACT (nmol MUB g - 1 h - 1 ) 811 ± 214 b 1193 ± 249 a 1286 ± 349 a 1427 ± 252 a PHOS (nmol MUB g - 1 h - 1 ) 1595 ± 272 b 2130 ± 264 a 2375 ± 400 a 2204 ± 446 a BGLU (nmol MUB g - 1 h - 1 ) 1495 ± 447 b 1492 ± 624 b 2086 ± 1095 ab 2669 ± 962 a RESP (g C-CO2 g - 1 day - 1 ) 14.2 ± 6.1 a 8.7 ± 4.2 b 7.8 ± 2.4 b 5.1 ± 1.4 b Total DNA (µg g - 1 ) 8.9 ± 1.4 7.4 ± 0.9 7.0 ± 0.8 6.8 ± 2.0 16S abundance (ng g - 1 ) 186 ± 25 b 185 ± 15 b 303 ± 68 ab 386 ± 73 a ITS abundance (ng g - 1 ) 0.50 ± 0.05 a 0.22 ± 0.03 b 0.32 ± 0.24 ab 0.42 ± 0.04 a ITS:16S 0.0028 ± 0.0007 a 0.0012 ± 0.0001 b 0.0010 ± 0.0005 b 0.0011 ± 0.0001 b NA pH 5.26 ± 0.01 a 4.83 ± 0.12 b 5.13 ± 0.05 a 4.74 ± 0.17 b EC (dS m - 1 ) 2.64 ± 0.49 2.92 ± 0.05 2.08 ± 0.96 2.98 ± 0.57 Total C (g kg - 1 ) 38 ± 8 29 ± 2 35 ± 5 33 ± 5 Total N (g kg - 1 ) 3.4 ± 0.5 ab 2.4 ± 0.3 b 3.3 ± 0.6 ab 3.9 ± 0.6 a Total S (g kg - 1 ) 7.3 ± 0.9 10.6 ± 1.5 8.7 ± 3.4 11.1 ± 3.9 K (mg kg - 1 ) 859 ± 152 724 ± 29 825 ± 44 794 ± 13 Na (mg kg - 1 ) 247 ± 31 278 ± 59 222 ± 38 278 ± 73 Mg (mg kg - 1 ) 43 ± 13 40 ± 9 29 ± 8 50 ± 12 Ca (mg kg - 1 ) 802 ± 78 993 ± 52 772 ± 324 751 ± 146 P (mg kg - 1 ) 28.2 ± 1.0 a 20.2 ± 1.2 b 22.4 ± 0.6 b 21.0 ± 0.7 b DHA (µg INTF g - 1 h - 1 ) 2.0 ± 0.3 a 1.3 ± 0.2 c 1.7 ± 0.2 b 0.7 ± 0.2 d AMIN (nmol AMC g - 1 h - 1 ) 271 ± 64 b 257 ± 119 b 196 ± 93 b 402 ± 89 a NACT (nmol MUB g - 1 h - 1 ) 640 ± 133 b 653 ± 107 b 1103 ± 326 a 1061 ± 310 a PHOS (nmol MUB g - 1 h - 1 ) 2497 ± 720 bc 3253 ± 589 a 3069 ± 676 ab 1880 ± 335 c BGLU (nmol MUB g - 1 h - 1 ) 1268 ± 282 b 1867 ± 337 a 1431 ± 224 ab 1585 ± 739 ab RESP (g C-CO2 g - 1 day - 1 ) 7.0 ± 3.6 a 2.6 ± 0.3 b 3.2 ± 0.7 b 4.0 ± 1.3 b Total DNA (µg g - 1 ) 15.6 ± 0.9 a 8.2 ± 1.2 b 7.7 ± 0.6 bc 5.0 ± 1.6 c 16S abundance (ng g - 1 ) 155 ± 26 102 ± 29 195 ± 56 132 ± 27 ITS abundance (ng g - 1 ) 0.56 ± 0.06 0.62 ± 0.25 0.41 ± 0.14 0.91 ± 0.26 ITS:16S 0.0037 ± 0.0007 ab 0.0061 ± 0.0018 a 0.0021 ± 0.0002 b 0.0069 ± 0.0017 a DHA: dehydrogenase activity, AMIN: aminopeptidase activity, NACT: N-acetylglucosaminidase activity, PHOS: phosphatase activity, BGLU: β-glucosidase activity, RESP: respiration rate. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 138 Table S4. F statistic and p-value of the models used to study the effect of the amendment and inoculum treatments, and the interaction between them, on soil chemical properties, biological activity, total DNA, bacterial and fungal abundances, and fungi:bacteria ratio (ITS:16S) at the second soil sampling (before the establishment of the drought treatment). Significant effects (p < 0.05) are indicated in bold. Factors Parameters Amendment Inoculum Amendment × Inoculum pH 324.46, < 0.001 32.44, < 0.001 1.68, 0.212 EC 24.49, < 0.001 3.32, 0.114 0.84, 0.490 Total C 57.80, < 0.001 1.63, 0.223 2.02, 0.152 Total N 22.12, < 0.001 6.13, 0.006 0.94, 0.443 Total S 0.11, 0.746 4.66, 0.016 0.76, 0.532 K 47.40, < 0.001 2.39, 0.107 0.48, 0.699 Na 4.24, 0.057 0.72, 0.554 0.46, 0.715 Mg 48.16, < 0.001 3.70, 0.034 3.74, 0.033 Ca 136.60, < 0.001 8.79, 0.001 10.15, < 0.001 P 321.00, < 0.001 4.57, 0.017 1.55, 0.241 DHA 444.96, < 0.001 69.51, < 0.001 1.88, 0.187 AMIN 18.76, < 0.001 6.53, 0.007 5.71, 0.012 NACT 13.10, 0.006 25.59, < 0.001 17.06: < 0.001 PHOS 0.69, 0.419 3.25, 0.054 0.47, 0.710 BGLU 0.24, 0.633 5.44, 0.015 1.55, 0.258 Respiration rate 17.25, < 0.001 6.85, 0.004 1.71, 0.204 Total DNA 9.77, 0.007 28.81, < 0.001 12.39, < 0.001 Bacterial abundance 41.83, < 0.001 9.67, < 0.001 6.75, 0.004 Fungal abundance 14.66, 0.001 3.99, 0.027 2.66, 0.084 ITS:16S 42.38, < 0.001 6.33, 0.005 2.28, 0.048 DHA: dehydrogenase activity, AMIN: aminopeptidase activity, NACT: N-acetylglucosaminidase activity, PHOS: phosphatase activity, BGLU: β-glucosidase activity. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 139 Table S5. F statistic and p-value of the models used to study the effect of the amendment, inoculum and drought treatments, and the interaction among them, on soil chemical properties, biological activity, total DNA, bacterial and fungal abundances, richness and diversity, and fungi:bacteria ratio (ITS:16S) at the third soil sampling (one month after the establishment of the drought treatment). Significant effects (p < 0.05) are indicated in bold. Factors Parameters Amendment Inoculum Drought Amendment × Inoculum Amendment × Inoculum × Drought pH 348.78, < 0.001 12.79, < 0.001 7.57, 0.010 11.53, < 0.001 2.96, 0.047 EC 48.41, < 0.001 6.69, 0.001 0.44, 0.514 2.21, 0.106 0.35, 0.791 Total C 55.86, < 0.001 4.52, 0.009 0.26, 0.614 3.61, 0.024 0.70, 0.557 Total N 104.93, < 0.001 3.83, 0.019 0.63, 0.432 3.17, 0.038 0.48, 0.698 Total S 0.12, 0.731 1.05, 0.384 0.40, 0.534 6.56, 0.001 0.07, 0.978 K 1.99, 0.168 7.33, < 0.001 0.16, 0.695 4.35, 0.011 1.22, 0.318 Na 27.59, < 0.001 1.07, 0.377 5.61, 0.024 0.95, 0.429 4.90, 0.006 Mg 1.86, 0.183 5.19, 0.005 0.15, 0.703 11.32, < 0.001 0.36, 0.784 Ca 2.00, 0.167 7.72, < 0.001 0.01, 0.927 14.80, < 0.001 1.14, 0.349 P 2682.29, < 0.001 61.04, < 0.001 3.87, 0.058 35.04, < 0.001 2.65, 0.066 DHA 59.37, < 0.001 12.59, < 0.001 3.59, 0.063 10.54, < 0.001 1.86, 0.145 AMIN 175.96, < 0.001 5.23, 0.003 0.04, 0.836 5.63, 0.002 0.70, 0.559 NACT 32.29, < 0.001 18.93, < 0.001 5.01, 0.029 2.66, 0.057 1.28, 0.291 PHOS 32.22, < 0.001 14.30, < 0.001 6.42, 0.014 9.64, < 0.001 1.65, 0.188 BGLU 5.93, 0.018 3.74, 0.016 0.40, 0.529 4.05, 0.011 0.70, 0.554 Respiration rate 47.94, < 0.001 14.07, < 0.001 2.81, 0.099 3.13, 0.032 0.58, 0.633 Total DNA 30.48, < 0.001 25.59, < 0.001 2.78, 0.105 15.38, < 0.001 0.83, 0.486 Bacterial abundance 10.99, 0.002 61.18, < 0.001 2.68, 0.112 3.23, 0.035 0.31, 0.819 Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 140 Table S5. Continued. Factors Parameters Amendment Inoculum Drought Amendment × Inoculum Amendment × Inoculum × Drought Fungal abundance 9.11, 0.005 5.23, 0.005 1.84, 0.185 0.04, 0.989 0.37, 0.774 ITS:16S 0.15, 0.700 24.82, < 0.001 0.06, 0.809 2.30, 0.097 0.26, 0.851 Bacterial richness 85.94, < 0.001 11.95, < 0.001 1.04, 0.315 10.99, < 0.001 1.28, 0.299 Bacterial diversity 34.62, < 0.001 3.78, 0.020 0.001, 0.980 7.07, < 0.001 1.02, 0.398 Fungal richness 4.18, 0.049 4.87, 0.007 0.03, 0.873 8.21, < 0.001 0.09, 0.963 Fungal diversity 3.83, 0.059 2.79, 0.056 0.42, 0.520 6.42, 0.002 0.63, 0.599 DHA: dehydrogenase activity, AMIN: aminopeptidase activity, NACT: N-acetylglucosaminidase activity, PHOS: phosphatase activity, BGLU: β-glucosidase activity. Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? | Capítulo 2 141 Figure S2. Relative abundance of the 10 most abundant bacterial (a) and fungal (b) genera in each treatment. NA: non-amended soil, BC: amended soil, CT: control treatment (no water reduction), DR: drought treatment. Capítulo 2 | Does an enhanced microbial diversity promote the resistance of soil multifunctionality against drought events in amended soils? 142 Table S6. Correlation analysis between soil multifunctionality and soil microbial richness and abundance, and the fungi:bacteria ratio (ITS:16S) in the amended (BC) and nonamended (NA) soils. Pearson´s correlation coefficients and p-values are indicated. BC NA Parameters r p r p Bacterial richness 0.36 0.09 0.04 0.87 Fungal richness 0.41 0.05 -0.09 0.68 16S abundance -0.32 0.13 0.11 0.60 ITS abundance 0.35 0.09 0.32 0.13 ITS:16S 0.59 0.002 -0.20 0.36 Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought | Capítulo 4 245 Table S5. F statistic and p-value of the models used to study the effect of the tillage system, the rainfall exclusion treatment and the interactions among them on the analysed chemical properties of soil from a depth of 0-10 cm across the three sampling dates. Sampling Parameters Tillage Rainfall Tillage × Rainfall 8 th April 2021 SOC 11.102 (0.0186) 0.289 (0.601) 0.472 (0.635) SOM 11.102 (0.0186) 0.289 (0.601) 0.472 (0.635) N 6.506 (0.0122) 1.229 (0.289) 0.193 (0.827) P 1.352 (0.295) 6.123 (0.0292) 0.147 (0.865) K 0.254 (0.779) 3.416 (0.0894) 0.552 (0.589) Ca 0.217 (0.808) 0.419 (0.530) 0.611 (0.559) Mg 0.671 (0.530) 0.397 (0.541) 0.052 (0.950) Fe 3.345 (0.0701) 2.282 (0.156) 0.148 (0.864) Cu 2.235 (0.150) 0.00 (1.000) 0.037 (0.964) Mn 5.281 (0.0226) 0.081 (0.781) 0.021 (0.979) Zn 0.395 (0.682) 3.824 (0.0742) 1.176 (0.342) 18 th November 2021 pH 0.304 (0.744) 0.006 (0.939) 2.704 (0.105) CE 1.308 (0.306) 2.381 (0.149) 1.238 (0.324) CaCO3 1.823 (0.204) 4.762 (0.0497) 0.141 (0.870) SOC 0.390 (0.685) 0.567 (0.466) 0.069 (0.935) SOM 0.390 (0.685) 0.567 (0.466) 0.069 (0.935) N 1.224 (0.328) 0.278 (0.607) 0.789 (0.477) P 0.394 (0.682) 3.818 (0.0744) 0.095 (0.910) K 1.167 (0.344) 0.001 (0.979) 1.694 (0.225) Ca 0.018 (0.982) 0.405 (0.536) 0.297 (0.748) Mg 0.669 (0.530) 0.686 (0.424) 0.076 (0.927) Fe 0.415 (0.669) 0.048 (0.829) 1.036 (0.385) Cu 1.440 (0.275) 0.419 (0.530) 0.046 (0.955) Mn 0.218 (0.807) 2.073 (0.175) 0.211 (0.813) Zn 0.826 (0.461) 12.662 (0.00394) 1.380 (0.288) Na 0.057 (0.945) 0.071 (0.795) 1.092 (0.367) 4 th March 2022 pH 8.467 (0.00121) 0.114 (0.738) 0.273 (0.763) CE 1.260 (0.298) 0.116 (0.736) 1.015(0.375) CaCO3 0.398 (0.675) 0.295 (0.591) 0.325 (0.725) SOC 1.145 (0.332) 0.814 (0.374) 0.216 (0.807) SOM 1.145 (0.332) 0.814 (0.374) 0.216 (0.807) N 0.203 (0.818) 3.097 (0.0895) 1.224 (0.309) P 3.770 (0.0346) 1.133 (0.296) 0.874 (0.428) K 0.795 (0.461) 0.688 (0.414) 0.163 (0.851) Ca 0.485 (0.620) 0.012 (0.912) 0.823 (0.449) Mg 2.638 (0.0880) 0.262 (0.613) 4.208 (0.0245) Na 1.601 (0.218) 0.188 (0.738) 0,273 (0.518) Capítulo 4 | Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought 246 Table S6. F statistic and p-value of the models used to study the effect of the tillage system, the rainfall exclusion treatment and the interactions among them on the analysed chemical properties of soil from a depth of 10-20 cm across the two sampling dates. Tillage Rainfall Tillage × Rainfall Sampling Parameters 18 th November 2021 pH 2.592 (0.116) 0.525 (0.482) 1.953 (0.232) CE 2.439 (0.129) 0.191 (0.670) 0.351 (0.711) CaCO3 0.712 (0.510) 2.597 (0.133) 0.005 (0,995) SOC 0.239 (0.791) 1.194 (0.296) 1.653 (0.232) N 2.802 (0.100) 1.388 (0.262) 0.410 (0.673) P 4.435 (0.0361) 4.577 (0.0536) 3.556 (0.0613) K 0.994 (0.399) 0.201 (0.655) 0.835 (0.457) Ca 0.170 (0.845) 0.008 (0.931) 0.409 (0.673) Mg 2.189 (0.155) 0.846 (0.376) 0.343 (0.717) Fe 0.340 (0.719) 0.353 (0.564) 0.875 (0.442) Cu 0.842 (0.462) 0.274 (0.610) 0.475 (0.633) Mn 0.373 (0.696) 4.02 (0.0665) 0.065 (0.938) Zn 0.967 (0.408) 14.38 (0.00257) 0.417 (0.668) Na 0.122 (0.887) 0.314 (0.585) 0.277 (0.763) 4 th March 2022 SOC 13.19 (0.0009) 21.070 (0.0006) 2.154 (0.159) Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought | Capítulo 4 247 Figure S2. Soil respiration rate measured in situ in the different treatments at different sampling times. For each date, the effect of rainfall exclusion, if significant, is shown with red letters (results of the linear models). Green letters indicate significant differences among tillage systems. RT: reduced tillage; TT: traditional tillage, NT: no-tillage. E: rainfall exclusion treatment, C: control. Capítulo 4 | Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought 248 Table S7. OTUs richness and indices of alpha-diversity (mean ± standard deviaon) of bacteria in the soils from the different treatments. Phyl. distance: phylogenec distances. NT: no-llage; RT: reduced llage; TT: tradional llage. OTUs no. Shannon Simpson Chao1 Phyl. distance NT Exclusion 3920 ± 219 9.60 ± 0.08 0.995 ± 0.001 5216 ± 343 300 ± 16 Control 3953 ± 145 9.69 ± 0.06 0.996 ± 0.001 5006 ± 554 305 ± 23 RT Exclusion 4112 ± 280 9.75 ± 0.10 0.995 ± 0.001 5083 ± 902 300 ± 31 Control 4190 ± 186 9.76 ± 0.11 0.995 ± 0.001 5102 ± 658 314 ± 7 TT Exclusion 4244 ± 78 9.72 ± 0.08 0.995 ± 0.001 5552 ± 249 345 ± 73 Control 4108 ± 424 9.76 ± 0.08 0.996 ± 0.001 5225 ± 1016 330 ± 18 Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought | Capítulo 4 249 Capítulo 4 | Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought 250 Figure S3. Taxonomic tree with the most abundant bacterial taxa recorded for the different tillage systems (NT: no-tillage; RT: reduced tillage; TT: traditional tillage). The size of circles represents the relative abundance of the taxa. The first percentage below the taxonomic name represents the relative abundance within the global classification, while the second percentage represents the relative abundance within the upper taxonomic group. Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought | Capítulo 4 251 Table S9. OTUs richness and indices of alpha-diversity (mean ± standard deviaon) of fungi in the soils from the different treatments. Phyl. distance: phylogenec distances. NT: no- llage; RT: reduced llage; TT: tradional llage. No diversity index was significantly influence by either the llage or the rainfall treatment. Figure S4. Boxplot of the unweighted-unifrac distances, as an index of β-diversity, of soil bacterial (a) and fungal (b) communies, calculated for each pair of samples from each treatment. OTUs no. Shannon Simpson Chao1 Phyl. distance NT Exclusion 683 ± 11 5.6 ± 0.4 0.925 ± 0.031 789 ± 44 158 ± 3 Control 674 ± 112 5.4 ± 0.4 0.928 ± 0.018 725 ± 104 146 ± 16 RT Exclusion 615 ± 45 4.9 ± 0.4 0.891 ± 0.050 711 ± 103 142 ± 10 Control 607 ± 15 5.0 ± 0.4 0.913 ± 0.028 704 ± 75 139 ± 2 TT Exclusion 665 ± 59 5.2 ± 0.7 0.921 ± 0.035 767 ± 41 153 ± 15 Control 585 ± 41 4.6 ± 0.7 0.860 ± 0.077 724 ± 52 136 ± 8 Capítulo 4 | Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought 252 Figure S5. Taxonomic tree with the most abundant fungal taxa recorded for the different tillage systems (NT: no-tillage; RT: reduced tillage; TT: traditional tillage). The size of circles represents the relative abundance of the taxa. The first percentage below the taxonomic name represents the relative abundance within the global classification, while the second percentage represents the relative abundance within the upper taxonomic group. Potential benefits of conservation agriculture practices on the resistance of Mediterranean agrosystems to drought | Capítulo 4 253 254 DISCUSIÓN GENERAL 261 donde los factores abióticos del suelo pueden no ser tan limitantes para la actividad biológica, la funcionalidad de los suelos sometidos a factores de degradación está condicionada por la disponibilidad de agua y macro y micronutrientes (ambas propiedades muy influidas por el contenido de materia orgánica del suelo), y no tanto por la estructura de las comunidades microbianas. Los resultados obtenidos en el Capítulo 2 demuestran que la aplicación de una enmienda orgánica (en este caso compost de biosólidos) capaz de incrementar la disponibilidad de nutrientes y agua, fomentando asimismo la abundancia y diversidad de las comunidades de bacterias y hongos del suelo, es una práctica eficaz para mejorar la funcionalidad de los suelos mediterráneos degradados. Beneficios del laboreo de conservación para la funcionalidad de los suelos agrícolas Gracias al efecto positivo sobre la estructura del suelo y la estabilidad de los agregados (Nielsen et al., 2005; Li et al., 2019; Melman et al., 2019; Page et al., 2019), las técnicas de laboreo de conservación (especialmente el sistema de no-laboreo), en comparación con el laboreo tradicional, favorecieron la infiltración y la capacidad de retención de agua del suelo, aumentando su disponibilidad en la capa superficial del suelo (0-40 cm), donde se concentran las raíces de las plantas. El seguimiento de las cosechas durante dos años en este mismo experimento sugiere que en el sistema de no-laboreo la producción vegetal sufrió una menor reducción debido a la sequía en los otros sistemas de laboreo (Madejón et al., 2023; Madejón et al., 2024). Sin embargo, hay que tener en consideración que el nolaboreo puede aumentar la compactación del suelo a medio plazo (López-Garrido et al., 2014), revirtiendo su impacto positivo sobre la disponibilidad de agua. Este efecto es muy dependiente de las condiciones edáficas (principalmente de la textura) y suele atenuarse en ensayos de laboreo de muy larga duración (Blanco-Canqui y Ruis, 2018; Li et al., 2020). Junto a la mejora en las propiedades físicas del suelo, el sistema de no-laboreo promovió un aumento de los contenidos de materia orgánica, N y P biodisponible en el suelo, al igual que se había observado en otros estudios recientes (Soane et al., 2012; Shiwakoti et al., DISCUSIÓN GENERAL 262 2019; Nunes et al., 2020; Panettieri et al., 2020; Zhong et al., 2021). Gracias a estos cambios en las propiedades del suelo, se produjo una respuesta positiva en la actividad biológica de los suelos sometidos a laboreo de conservación, reflejada en los niveles de la actividad deshidrogenasa (considerada como una medida de la actividad biológica potencial). Sin embargo, la tasa de respiración del suelo medida in situ estuvo más influenciada por la estación del año, que determina la dinámica del agua del suelo, así como por el tratamiento de exclusión de lluvia. En relación a la comunidad microbiana del suelo, estudios previos han observado que las poblaciones de hongos son especialmente sensibles al laboreo debido a la destrucción del micelio (Kabir, 2005; Panettieri et al., 2020). Los datos obtenidos apoyaron esta teoría, observándose una tendencia a una mayor diversidad de hongos en los suelos bajo el sistema de no-laboreo, aunque el efecto no llegara a ser significativo. Por otro lado, la diversidad de bacterias mostró la tendencia contraria, siendo mayor bajo el sistema de laboreo tradicional, probablemente como respuesta a interacciones antagonistas entre bacterias y hongos. Finalmente, el laboreo de conservación también tuvo un efecto beneficioso sobre la comunidad de nematodos del suelo. Al igual que lo descrito en estudios previos (Bongers y Ferris, 1999; Ferris et al., 2001; Neher, 2010; Ito et al., 2015; Bongiorno et al., 2019; Wang et al., 2022), la reducción en la perturbación física del suelo en el sistema de no-laboreo favoreció una comunidad de nematodos más madura y estructurada, con mayor abundancia de los grupos de nematodos más sensibles a las perturbaciones, en comparación con los suelos manejados con laboreo tradicional. Efecto de la sequía en las emisiones de CO2 del suelo Aunque se ha comprobado que el contenido en materia orgánica tiene un efecto positivo (Montiel-Rozas et al., 2016), la tasa de respiración del suelo en ambientes mediterráneos está generalmente controlada por la humedad y la temperatura del suelo (Almagro et al., 2009; Morillas et al., 2017; Matías et al., 2021). En el experimento del Capítulo 4, la DISCUSIÓN GENERAL 263 exclusión de un 30 % de la precipitación redujo la respiración del suelo durante los meses secos (de abril a octubre). Sin embargo, las emisiones de CO2 tras los picos de máxima precipitación en marzo y septiembre, fueron mayores en las parcelas con exclusión de lluvia, pudiéndose comprobar como niveles altos de humedad en el suelo inhiben la tasa de respiración (Matías et al., 2021). Por lo tanto, incluso en sistemas como los mediterráneos, limitados por la baja disponibilidad de agua en el suelo durante buena parte del año, la actividad microbiana del suelo presenta un óptimo a ciertas condiciones de humedad, por el encima del cual esta actividad se ve inhibida. Estudios recientes han registrado un incremento en la tasa de respiración en suelos en los que se aplica laboreo de conservación, presumiblemente debido al mayor aporte de materia orgánica en comparación con el laboreo tradicional (Plaza-Bonilla et al., 2014; Nunes et al., 2020; Shakoor et al., 2021). Sin embargo, bajo nuestras condiciones experimentales, el sistema de laboreo, a pesar de su influencia sobre la comunidad microbiana y la actividad biológica del suelo, no tuvo un efecto claro sobre la respiración del suelo. El único efecto significativo que se observó fue un aumento de las emisiones de CO2 en las parcelas con laboreo tradicional y exclusión de lluvia durante la época de mayor precipitación (noviembre). Esto reveló la mayor influencia de los niveles de humedad del suelo sobre la respiración, coincidiendo con lo observado por Morell et al. (2011), que determinaron que en los años secos la respiración del suelo es mayor bajo el laboreo de conservación, mientras que en los años más lluviosos esta tendencia se invierte y la tasa de respiración se ve reducida por el no-laboreo. Elevada tolerancia de los suelos mediterráneos a la sequía En conjunto, los resultados obtenidos en los cuatro ensayos que componen esta Tesis han revelado la gran resistencia de los suelos mediterráneos a las condiciones de sequía. Tanto en condiciones controladas de invernadero (Capítulos 1, 2 y 3) como en condiciones de campo (Capítulo 4), la reducción en un 30 % del aporte de agua tuvo efectos muy limitados sobre la biomasa o la composición de las comunidades microbianas. Los niveles de DISCUSIÓN GENERAL 264 actividad biológica potencial (actividades enzimáticas y tasa de respiración) no se vieron limitados por la reducción de agua en los ensayos de invernadero (excepto por una leve reducción de las actividades deshidrogenasa y β-glucosidasa en el Capítulo 1; y de las actividades N-acetil-glucosaminidasa y fosfatasa en el Capítulo 2), mientras que en el ensayo de campo el tratamiento de exclusión de lluvia sí determinó en cierta medida la dinámica de las emisiones de CO2, como se ha comentado anteriormente. Sin embargo, cabe destacar que el rango de niveles de humedad del suelo en los diferentes experimentos fue muy contrastado. En los ensayos llevados a cabo en condiciones de invernadero se simuló una reducción del aporte hídrico a corto plazo durante la estación de crecimiento vegetal, mientras que el ensayo de campo permitió recoger la variabilidad estacional de la humedad del suelo a lo largo de dos años consecutivos. De esta manera, las condiciones de sequía contempladas en el ensayo del Capítulo 4 durante los meses de verano fueron considerablemente más extremas que las simuladas en los experimentos en macetas. Esta elevada tolerancia de las comunidades microbianas de los suelos mediterráneos a los periodos de sequía se ha observado igualmente en otros estudios recientes, en los que suelos expuestos de forma periódica a episodios de sequía son capaces de mantener su nivel de actividad biológica aún cuando la disponibilidad de agua es muy reducida (Hueso et al., 2011; Evans y Wallenstein, 2012; Curiel Yuste et al., 2014; Hawkes et al., 2017; Meisner et al., 2018; Xiao et al., 2018; Canarini et al., 2021). Ensayos anteriores han observado que las comunidades microbianas de los suelos de ambientes semiáridos están dominadas por organismos con una elevada tolerancia a la desecación, como es el caso de las bacterias Gram-positivas (Fierer et al., 2012; Maestre et al., 2015; Delgado-Baquerizo et al., 2016), y diferentes hongos de tipo filamentoso y micorrícicos, los cuales tienen además un efecto positivo sobre la capacidad de retención de agua del suelo (White et al., 2000; Querejeta, 2017). Los resultados observados en los Capítulos 1, 2 y 4 apoyan estas observaciones, ya que, aunque la abundancia de hongos micorrícicos arbusculares (AMF) estuvo probablemente infraestimada por las limitaciones en la metodología empleada, se observó una clara dominancia de taxones de bacterias DISCUSIÓN GENERAL 265 Gram-positivas (principalmente de los filos Actinobacteriota y Firmicutes); mientras que las condiciones de sequía simuladas incrementaron la abundancia de hongos filamentosos del género Trichoderma. Sin embargo, cabe mencionar que, aunque las comunidades microbianas de los suelos objeto de estudio han demostrado una alta tolerancia a las condiciones de sequía simuladas, la producción vegetal (especialmente de las especies más sensibles) presentó una mayor vulnerabilidad a la sequía. Este efecto se observó tanto en condiciones de invernadero (Capítulos 1, 2 y 3; discutido anteriormente) como en el experimento de campo (Madejón et al., 2023; Madejón et al., 2024). La menor producción de biomasa vegetal, así como los cambios en la composición de las comunidades vegetales, como consecuencia de la reducción en el aporte de agua, podría afectar a las comunidades microbianas del suelo a medio y largo plazo. Esta hipótesis se basa en la estrecha relación existente entre las comunidades microbianas (especialmente de hongos), y el contenido y naturaleza de la materia orgánica presente en suelo, como se ha demostrado en los Capítulos 1, 2 y 3. En el ensayo del Capítulo 4, posiblemente debido a la menor producción de biomasa de los cultivos bajo el tratamiento de exclusión de lluvia y por tanto del menor aporte de materia orgánica al suelo, se observó una reducción en la abundancia relativa de hongos saprófitos, lo cual se ha observado también en otros estudios como respuesta a una disminución de inputs vegetales al suelo (Clocchiatti et al., 2020; Ning et al., 2021). Dado que en los suelos de estudio las condiciones físico-químicas (en particular la disponibilidad de C orgánico) fueron más condicionantes para el grado de multifuncionalidad del suelo que los niveles de diversidad microbiana, y dada la elevada resistencia de las comunidades microbianas a la baja disponibilidad de agua, no se pudo demostrar que hubiera una relación positiva entre los niveles de diversidad de bacterias y hongos y la estabilidad del suelo frente a condiciones de sequía, al menos bajo las condiciones experimentales en las que se han desarrollado los ensayos de esta Tesis. A pesar de ello, el efecto positivo de la aplicación de enmiendas orgánicas en la capacidad de retención de agua del suelo y en la estabilidad de la producción vegetal frente a condiciones DISCUSIÓN GENERAL 266 de sequía (discutido previamente) podría favorecer igualmente la resistencia de las comunidades microbianas frente a los periodos de estrés hídrico. CONCLUSIONES 267 Conclusiones 1. Los aportes de C orgánico, especialmente mediante aquellos sustratos de baja relación C:N (compost de biosólidos y biomasa de Vicia faba) favorecen la producción vegetal en suelos degradados en comparación con los suelos no enmendados, a pesar de tener un impacto negativo a corto plazo sobre la tasa de germinación de las semillas. La aplicación de compost a largo plazo sí tuvo un efecto beneficioso sobre la resistencia a la sequía de la producción de Lolium (la especie más sensible al estrés hídrico). Sin embargo, no se observó un efecto positivo a corto plazo de la aplicación de enmiendas orgánicas sobre la estabilidad de la producción de biomasa vegetal frente a las condiciones de sequía simuladas. 2. El legado del suelo, entendido como el conjunto de procesos a los que ha estado históricamente sometido un suelo y que influyen en su contenido en materia orgánica, tiene una influencia importante sobre las propiedades del suelo y la composición y actividad de las comunidades microbianas. El efecto a largo plazo de la aplicación de compost de biosólidos, así como la exposición prolongada a ganadería extensiva, tuvieron un efecto igualmente positivo en la biomasa microbiana y en los valores de la tasa de respiración y actividades enzimáticas del suelo, presentando ambos suelos, además, comunidades microbianas muy similares. Además, el legado del manejo mecánico del suelo (sistema de laboreo) influyó tanto en la fertilidad química como en la capacidad de retención de agua y la actividad biológica de los suelos. 3. En los suelos mediterráneos degradados por la contaminación con elementos traza, el grado de multifuncionalidad, pese a estar positivamente correlacionado con los niveles de diversidad de bacterias y hongos, está principalmente controlado por las propiedades químicas del suelo y favorecido, por tanto, por la adición de C orgánico en forma de enmienda orgánica. CONCLUSIONES 268 4. En relación al laboreo tradicional, los sistemas de laboreo de conservación contribuyeron a mejorar las propiedades del suelo. Especialmente, el sistema de no-laboreo fue el más eficaz en la mejora de la capacidad de retención de agua del suelo, aumentando la disponibilidad de agua en la capa donde se concentran las raíces de las plantas. Por otra parte, el aumento en el contenido en materia orgánica y macronutrientes en los suelos sometidos a no-laboreo resultó en un incremento en la actividad biológica del suelo. Sin embargo, no se observaron diferencias en la composición de las comunidades microbianas entre sistemas de laboreo, que estuvieron principalmente influenciadas por las condiciones de sequía (especialmente las comunidades de hongos). Finalmente, los índices de ecología de nematodos, empleados como bioindicador del estado de conservación del suelo, revelaron igualmente un efecto positivo del sistema de no-laboreo sobre la comunidad de nematodos del suelo. 5. Las comunidades microbianas de los suelos mediterráneos estudiados, aún sometidos a factores de degradación, presentan una elevada resistencia a las condiciones de sequía, lo que implica que la reducción en un 30 % del aporte de agua a corto plazo tiene escasos impactos negativos sobre las actividades enzimáticas, la biomasa microbiana o la composición de las comunidades de hongos y bacterias del suelo. Esta tolerancia a la sequía se debe a las estrategias de adaptación de las comunidades microbianas de estos suelos, promovidas por la exposición prolongada a los periodos de sequía propios de las regiones de clima mediterráneo. Por otra parte, la producción vegetal estuvo más afectada por las condiciones de sequía que la biomasa o estructura de las comunidades microbianas. En los suelos agrícolas los efectos de la sequía en las comunidades de hongos estuvieron relacionados posiblemente con la disminución de la producción vegetal. CONCLUSIONES 269 6. Sin embargo, las emisiones de CO2 sí estuvieron condicionadas por la humedad del suelo, en especial en los suelos agrícolas. En estos suelos la exposición a corto plazo a una reducción del aporte hídrico redujo sensiblemente las tasas de respiración basal. En condiciones de campo, el tipo de manejo, al condicionar la capacidad de retención de agua del suelo, tuvo un efecto indirecto sobre la tasa de respiración del suelo, especialmente en los meses más húmedos. Durante los periodos de mayor precipitación, los mayores niveles de humedad del suelo en las parcelas con laboreo de conservación respecto al laboreo tradicional tienen un efecto negativo sobre la tasa de respiración del suelo; mientras que en los meses más secos cabría esperar el efecto contrario (aunque no se ha observado bajo las condiciones del ensayo). 7. Finalmente, a pesar de las relaciones positivas observadas entre los niveles de abundancia y diversidad microbianas y la funcionalidad de los suelos objetos de estudio, favorecidas por la adición de C orgánico al suelo, no pudo demostrarse la hipótesis de partida de que existe una relación positiva entre la diversidad microbiana y la resistencia del suelo frente a episodios de sequía. 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ANEXO I 277 Anexo I ANEXO I 278 ANEXO I 279 ANEXO I 280 ANEXO I 265 La región mediterránea es una de las zonas de mayor vulnerabilidad frente al cambio climático, donde se espera un incremento en la frecuencia y duración de episodios de olas de calor y sequía. Por ello, es de vital importancia conocer el impacto que la reducción de las precipitaciones tendrá sobre los ecosistemas mediterráneos y su sostenibilidad, incluyendo los efectos sobre la funcionalidad del suelo. Los suelos degradados, generalmente con un bajo contenido en materia orgánica y una menor biodiversidad edáfica, son los más vulnerables a las perturbaciones, incluidas las derivadas del cambio climático. En la Tesis se ha estudiado el efecto de la sequía sobre el funcionamiento de los suelos mediterráneos degradados, evaluando, además, la eficiencia de diferentes medidas destinadas a la recuperación del suelo en la mejora de la resistencia de estos suelos frente a la sequía. Especialmente, se ha tratado de analizar el papel específico de la biodiversidad del suelo en la estabilidad de los suelos frente a condiciones de sequía. Para ello se seleccionaron suelos afectados por dos de los factores de degradación más frecuentes en la Península Ibérica: la contaminación por elementos traza derivados de la actividad minera, y la pérdida de materia orgánica como resultado de un uso agrícola intensivo. Se han llevado a cabo cuatro ensayos, tres bajo condiciones controladas de invernadero y otro en condiciones de campo, en los que se han simulado condiciones de sequía mediante la reducción en un 30 % del aporte de agua, de acuerdo a las predicciones de cambio climático para la región mediterránea. En la evaluación del efecto de la sequía sobre la funcionalidad del suelo se tuvieron en consideración tanto los cambios en las propiedades químicas como el impacto sobre la actividad biológica y la comunidad microbiana del suelo. Hipotéticamente, aquellos suelos en los que las actuaciones realizadas hayan mejorado las propiedades químicas del suelo (especialmente el contenido en materia orgánica), derivando en una comunidad microbiana más diversa y estructurada, serán los más estables frente a la sequía.