Efecto de las propiedades del suelo, la materia orgánica y la actividad microbiana sobre la biodisponibilidad de zinc y otros micronutrientes y su interacción con el fósforo

Universidad de Sevilla Escuela Técnica Superior de Ingeniería Agronómica Programa de Doctorado en Ingeniería Agraria, Alimentaria, Forestal y del Desarrollo Rural Sostenible por la Universidad de Córdoba y la Universidad de Sevilla (RD. 99/2011) Línea de investigación: Tecnología de la producción vegetal TESIS DOCTORAL EFECTO DE LAS PROPIEDADES DEL SUELO, LA MATERIA ORGÁNICA Y LA ACTIVIDAD MICROBIANA SOBRE LA BIODISPONIBILIDAD DE ZINC Y OTROS MICRONUTRIENTES Y SU INTERACCIÓN CON EL FÓSFORO Autora: Aurora Moreno Lora Director y tutor: Dr. Antonio Delgado García Granada, 2022 Universidad de Sevilla Escuela Técnica Superior de Ingeniería Agronómica Programa de Doctorado en Ingeniería Agraria, Alimentaria, Forestal y del Desarrollo Rural Sostenible por la Universidad de Córdoba y la Universidad de Sevilla (RD. 99/2011) Línea de investigación: Tecnología de la producción vegetal Título de la tesis doctoral: “Efecto de las propiedades del suelo, la materia orgánica y la actividad microbiana sobre la biodisponibilidad de zinc y otros micronutrientes y su interacción con el fósforo” Doctoranda: Aurora Moreno Lora La presente Tesis Doctoral cumple con los requisitos establecidos por la Universidad de Sevilla para su presentación por compendio de publicaciones, al estar integrada por 3 artículos publicados en revistas científicas incluidas en los tres primeros cuartiles de la lista de revistas del ámbito de la especialidad y referenciadas en los últimos datos publicados por Journal Citations Report (JCR) y Scimago Journal & Country Rank (SJR): 1. Moreno-Lora, A., Recena, R., Delgado, A., 2019. Bacillus subtilis QST713 and cellulose amendment enhance phosphorus uptake while improving zinc biofortification in wheat. Applied Soil Ecology. 142, 81–89. DOI: 10.1016/j.apsoil.2019.04.013 Índice de impacto: 5.509 (JCR, 2021) (2º Q). Área: Soil Science - SCIE (2021). Posición: 10/39 Índice H: 127 (SJR, 2021) (1º Q). Área: Agricultural and Biological Sciences (2021) 2. Moreno-Lora, A., Delgado, A., 2020. Factors determining Zn availability and uptake by plants in soils developed under Mediterranean climate. Geoderma 376, 114509. DOI: 10.1016/j.geoderma.2020.114509 Índice de impacto: 7.422 (JCR, 2021) (1º Q). Área: Soil Science - SCIE (2021). Posición: 4/39 Índice H: 177 (SJR, 2021) (1º Q). Área: Soil Science (2021) 3. Moreno-Lora, A., Sousa-Ortega, C., Recena, R., Perea-Torres, F., Delgado, A., 2022. Microbial inoculants improve nutrients uptake and yield of durum wheat in calcareous soils under drought stress in the Mediterranean region. Archives of Agronomy and Soil Science, DOI:10.1080/03650340.2022.2146099 Índice de impacto: 2.242 (JCR, 2021) (2º Q). Área: Agronomy - SCIE (2021). Posición: 36/90 Índice H: 44 (SJR, 2021) (1º Q). Área: Agronomy and Crop Science (2021) A las mujeres de mi vida. A mi madre, a mi abuela. Agradecimientos A mi director, Antonio Delgado, por la confianza que depositó en mí para llevar a cabo este proyecto, por su apoyo durante estos años y por su ejemplo. Al Departamento de Agronomía y a los profesores y compañeros de la ETSIA que, de una forma u otra, han contribuido con este trabajo. Gracias al equipo de Sanidad Vegetal, por su disposición a ayudar y por compartir su experiencia y sus conocimientos. De manera especial, quiero agradecer a Oliva y a Puri su ayuda y su cariño. Gracias por enseñarme, por compartir conmigo vuestra experiencia, por vuestros consejos y vuestra implicación. Gracias por la paciencia que habéis tenido durante estos años. Sin vuestra ayuda este trabajo no hubiese sido posible. A mis compañeros y compañeras del despachito, por todos los momentos que hemos compartido. Gracias por vuestro tiempo, vuestra ayuda y vuestras palabras de ánimo, habéis sido un apoyo fundamental durante estos años. En especial, quiero agradecer a Carlos su esfuerzo y dedicación con este trabajo. Gracias por estar ahí siempre que lo he necesitado, y por no haber permitido que me rinda. También quiero mostrar mi agradecimiento al Dipartamento di Scienze Agrarie, a los profesores y compañeros de la Unipa. En especial, al proffesore Laudicina por acogerme y brindarme su laboratorio, y compartir conmigo su experiencia y sus conocimientos. A te, che sei stato con me nei momenti peggiori di questa lunga strada. Ti ringrazio per avermi accompagnato e per avere creduto sempre a me… anche quando io non ci credevo. A mis compañeros de IFAPA. A Milagros, por todo lo que he aprendido contigo y con tu ejemplo. A Marinetto, por tratar de entenderme y apoyarme, y por ser el soplo de aire fresco que necesité para continuar el camino. Gracias por dejar tus trazos en esta tesis. A mi familia. A mis padres y mi hermano. A mi tío Manuel, con quien compartí los primeros pasos de esta experiencia. A mi abuela, por enseñarme a ser valiente y a pensar en positivo. A mi madre, para quien no encuentro palabras de agradecimiento… gracias por tu paciencia infinita y tu amor incondicional. 9 Índice de contenido Resumen 13 Summary 17 Capítulo I. Introducción general y objetivos 21 1. El zinc como elemento esencial 25 2. La deficiencia de zinc en los cultivos 26 3. Factores que afectan la disponibilidad de zinc para las plantas 27 3.1. Las propiedades del suelo 27 3.2. Las interacciones con otros nutrientes 28 3.3. El antagonismo fósforo-zinc en la planta 29 3.4. La materia orgánica 30 3.5. La actividad microbiana de la rizosfera 30 4. El uso de inoculantes microbianos para mejorar la nutrición de las plantas 31 4.1. Principales mecanismos microbianos implicados en la mejora de la nutrición de la planta 32 5. Objetivos 34 Capítulo II. Factores que determinan la disponibilidad y absorción de zinc por las plantas en suelos y clima mediterráneos 35 Capítulo III. La disponibilidad de zinc y fósforo para el trigo se ve afectada por las sustancias húmicas en sustratos calcáreos y silíceos 49 Capítulo IV. Bacillus subtilis QST713 y una enmienda de celulosa mejoran la absorción de fósforo y favorecen la biofortificación de zinc en trigo 77 Capítulo V. Efectos de inoculantes microbianos y enmiendas orgánicas en la nutrición y desarrollo del trigo en un conjunto de suelos 91 16 17 Summary Zinc (Zn) is an essential element, required in minimal but critical amounts for the correct functioning of plants, animals and humans. Zinc deficiency in agricultural production causes significant losses of yield and nutritional quality in crops. This has a high impact on society since it is estimated that almost one third of the world's population suffers from Zn malnutrition, mainly ascribed to a deficient level of this micronutrient in diets. In Mediterranean region, calcareous soils used for cereal cultivation are inherently deficient in micronutrients and phosphorus (P). This has promoted the application of large amounts of phosphate fertilizer, which is a non-renewable resource and accentuates Zn deficiency due to the antagonistic relationship between both nutrients. Moreover, Zn availability to plants cannot be accurately predicted by the bioavailability tests commonly used, since this bioavailabilty is not only related to its total content or amounts chemically extracted in soil, but also to other soil properties, microbial activity, and the interaction with other nutrients. In this context, further knowledge on the factors involved in Zn availability to plants and the design of effective strategies for sustainable improvement of plant nutrition and crop production are needed. Agricultural practices increasing the content of organic matter in soil, as well as the application of organic amendments, could improve the availability of nutrients to plants. A portion of this soil organic matter are living microorganisms which are involved in processes related to nutrient mineralization and immobilization. Thus, this microbial activity plays a key role in biogeochemical nutrient cycles. Rhizosphere microorganisms are crucial in plant-soil interactions since their activity promotes the mobilization of nutrients and contributes to their uptake by plants, thus playing an important role in plant nutrition. The use of microbial inoculants able to solubilize nutrients has generated interest in the recent years, especially in the case of P. Nevertheless, the potential enhancement of P availability may induce a decreased Zn availability, and vice versa. Thus, it is important to consider these effects on plant nutrition in an integrated way. Microbial inoculants are also used as plant growth promoters and as biofortification agents, in order to increase the yield and to enrich the edible portion of the crop with essential minerals elements improving its nutritional quality. Therefore, it is also relevant to consider effect of inoculants on nutrient homeostasis and on the P-Zn balance within the plant. The effect on nutrient mobilization and the enhancement of plant nutrition derived from these practices affecting organic matter and rhizospheric microorganisms could be different depending on environmental factors, such as water availability, soil properties (physical, chemical and biological) and the application of fertilizers. The main objective of this work was to determine soil factors affecting Zn availability to plants and P-Zn interaction, and under this premise, to evaluate the effect of the addition of different sources of organic matter and the application of microbial inoculants on crop nutrition and biofortification. 18 The first experiment involved the growing of wheat in pots, using a collection of 40 soils that differed widely in their properties. The results obtained revealed that the relationship between soil properties and Zn uptake by plants was highly influenced by the carbonate content of soils. In calcareous soils, Zn associated with organic matter plays a key role in its availability to plants, meanwhile in non-calcareous iron oxide mineralogy was very relevant explaining Zn availability. The results obtained showed the importance of discriminating soils by their carbonate content for accurately estimation of Zn availability to crop. The study about the addition of organic matter to the soil in the form of humic substances showed a different effect on nutrient availability to plants depending on the type of growing medium. This difference may be ascribed to diverse nutrient adsorption dynamics, according to the main sorbing surfaces of each growing medium. Furthermore, the effect on the biology of growing media and plant physiology depended on the rate of applied humic substances and the type of growing medium, which also affected biomass production and grain quality. The experiments on the effect of the application of a labile carbon source (β-cellulose) and inoculation with microorganisms (Bacillus subtilis QST713 and Trichoderma asperellum strain T34) on P and Zn nutrition in wheat showed the benefits that the activity of rhizosphere microorganisms promotes in crop nutrition and development. In general, soil microbial activity is higher in soils where the nutrient availability is more restricted. The results obtained in the experiments revealed an altered pattern of nutrient accumulation in plant organs related to microbial activity in the growing medium. This promoted the preferential accumulation of Zn in wheat grains, which led to an improvement of the Zn harvest index (Zn HI) and a reduction of the P-Zn molar ratio in grains resulting in an enhancement of its nutritional quality. The first greenhouse experiment based on the application of β-cellulose and the inoculation of B. subtilis on two different soils and with phosphate fertilization shows that the improved of P nutrition did not necessarily lead to a worsening of Zn nutrition. It has been observed that this improvement resulting from inoculation with B. subtilis can be accompanied by an increased Zn accumulation in the grain and, consequently, a higher harvest index (ZnHI). In the case of βcellulose, this was the first time it has been shown that the addition of an organic amendment that does not affect the P cycle in the soil improves its uptake by plants. These results reveal the importance of considering environmental factors, soil properties and phosphate fertilization on the effect of microbial inoculation on Zn nutrition of wheat plants. Based on these results, the effect of β-cellulose application and inoculation with B. subtilis and T. asperellum was studied in different soil environments, using a collection of 10 soils that differed widely in properties. The experiment showed the activation of nutrient mobilization mechanisms derived from the microbial inoculation and the application of βcellulose. Although total plant nutrient uptake was not increased, Zn harvest index and grain P-Zn ratio were enhanced. These effects may be attributed to changes in hormone distribution in plant. 19 Mobilization mechanisms may differ according to soil type, inferring that soil factors are basic aspects determining the efficiency of microbial inoculants or organic amendments. Nevertheless, the results show an increase in biomass production and higher harvest index independently of soil. The last study was aimed at studying the effect of these microbial inoculants in Mediterranean climate conditions, where soils can lead to micronutrient and phosphorus deficiencies and where drought can promote low use efficiency of applied fertilisers. For this purpose, 3 field experiments were conducted in 2 growing seasons. In the first season, B. subtilis improved biomass production and tended to increase grain yield, as well as improving nitrogen, phosphorus and manganese uptake. Tirchoderma asperellum decreased the P concentration in the grain without affecting the Zn concentration, thus decreasing the P-Zn molar ratio and improving its nutritional quality. In the second season, the effect of the inoculants was less evident, probably due to more favourable environmental conditions for cultivation such as higher water availability or phosphate fertilization. Field experiments showed that B. subtilis can be effective improving wheat development and nutrition under adverse environmental conditions, such as drought stress. 20 21 Capítulo I. Introducción general y objetivos Chapter I. General introduction and objectives 22 23 Capítulo I. Introducción general y objetivos El impacto que el cambio climático y la degradación de los suelos tienen sobre la agricultura provoca pérdidas de biodiversidad, reducciones en el rendimiento de los cultivos y en la calidad de la cosecha y disminuye la rentabilidad de las explotaciones. La escasez de lluvias y el conflicto en Ucrania, unido al aumento progresivo de la población y la creciente demanda de alimentos, han puesto en riesgo la seguridad alimentaria a nivel global. En este contexto se hace necesario intensificar la producción de los cultivos de forma sostenible y hacer un uso eficiente de los recursos disponibles para asegurar las necesidades futuras (Ryan et al., 2013a; Schjoerring et al., 2019). En los últimos años, la búsqueda de métodos alternativos que mejoren el rendimiento de los cultivos y que sean efectivos y respetuosos con el medio ambiente, a la vez que rentables, ha representado un desafío clave para la sostenibilidad de la agricultura (Arora et al., 2020; de los Santos-Villalobos & Parra-Cota, 2020; Owen et al., 2015). La producción agrícola es altamente dependiente del uso de fertilizantes químicos, especialmente en países desarrollados. Su uso, particularmente en el caso del nitrógeno (N) y del fósforo (P), ha incrementado sustancialmente en las últimas décadas pero su eficiencia se ve limitada por las condiciones climáticas y las propiedades del suelo (Ryan et al., 2013a). En la mayor parte de los suelos agrícolas se aplican elevadas dosis de fertilizante fosfatado que no siempre están justificadas (Delgado & Scalenghe, 2008) y que pueden desequilibrar el suministro de otros nutrientes. Existen evidencias de que las aplicaciones excesivas de P acentúan las deficiencias de micronutrientes y repercuten sobre la producción y la calidad los cultivos, especialmente en el caso de los cereales (Chen et al., 2018; Ova et al., 2015; Rashid & Ryan, 2004; Sacristán et al., 2019). Del mismo modo, las aplicaciones de micronutrientes, como el hierro (Fe) y el zinc (Zn), pueden reducir la eficiencia del fertilizante fosfatado (Aboyeji et al., 2020; Ryan et al., 2013b). Por ello es fundamental encontrar un punto de equilibrio y optimizar el manejo de la fertilización. En general existe una conciencia limitada sobre la importancia de los micronutrientes en la agricultura y las implicaciones que un suministro limitado de estos tiene sobre la productividad de los cultivos, la calidad de la cosecha y la salud humana (Ryan et al., 2013b). En suelos mediterráneos la deficiencia de micronutrientes provoca importantes, y a menudo menospreciadas, pérdidas de productividad que repercuten de forma directa sobre la población, ya que además de provocar un detrimento de la producción también se reduce la concentración de micronutrientes en la cosecha y, por tanto, en los alimentos (Rashid & Ryan, 2004). Este problema se ha agravado considerablemente en los últimos años debido, principalmente, a la intensificación de la producción, a la pérdida de suelo por erosión, al mayor empleo de fertilizantes químicos respecto a orgánicos, a la utilización de tierras marginales para la producción de cultivos y a que los cultivares de variedades modernas son especialmente demandantes de micronutrientes (Fageria et al., 2002). Entre ellos, el Zn es considerado 24 el micronutriente más limitante, especialmente en suelos calcáreos típicos de la región mediterránea, que son propensos a la deficiencia de este nutriente (Alloway, 2009; Rashid & Ryan, 2004). Grandes extensiones de terreno con este tipo de suelo se destinan al cultivo de cereales, por lo que la deficiencia de Zn limita los rendimientos y reduce la calidad de la cosecha (Alloway, 2009). La malnutrición de Zn en humanos se considera un problema de salud grave, asociado a dietas basadas en el consumo de cereales, y se estima que casi un tercio de la población mundial tiene una dieta pobre en este micronutriente (Cakmak & Kutman, 2018). El problema ha ido en aumento durante los últimos 50 años, con la llegada de la “Revolución verde”, cuando comenzaron a cultivarse nuevas variedades de alto rendimiento (Alloway, 2009; Cakmak & Hoffland, 2012). El aumento del rendimiento en grano de los cultivos de cereales, como el trigo, también implicaba una mayor extracción de Zn (Rashid & Ryan, 2004). Sin embargo, y pese a los avances en mejora genética para obtener variedades de alto rendimiento, estas variedades suelen ser ineficientes en el uso de Zn (Alloway, 2009; Cakmak & Kutman, 2018; Rashid & Ryan, 2004). Es probable que la intensificación de la producción agrícola y la aplicación generalizada de fertilizantes acentúen las deficiencias de micronutrientes y, en especial, la de Zn, pero los avances en la agricultura de conservación y las prácticas sostenibles de manejo de la fertilización pueden contrarrestar esta tendencia. En los últimos años ha crecido el interés por la agricultura de bajos insumos, lo que ha fomentado la investigación y el desarrollo de técnicas de cultivo que mejoren la nutrición de las plantas y la calidad del producto cosechado, a la vez que permitan incrementar la producción de forma sostenible (Arora et al., 2020). 25 1. El zinc como elemento esencial El zinc es un micronutriente esencial para el crecimiento y la reproducción normal de las plantas, los animales y los seres humanos. Está implicado en la mayoría de funciones fisiológicas de los seres vivos, como el mantenimiento estructural y funcional de las membranas biológicas, la síntesis de proteínas y la expresión génica (Alloway, 2009; Broadley et al., 2007; Cakmak & Hoffland, 2012; Cakmak & Kutman, 2018). En particular, la tolerancia a las condiciones ambientales de estrés tiene un alto requerimiento de Zn para regular y mantener la expresión de los genes que protegen a las células de los efectos perjudiciales del estrés (Sadeghzadeh & Rengel, 2011). Aunque se requiere en cantidades mínimas, su deficiencia es crítica para los seres vivos. En las plantas es un elemento clave como constituyente estructural o cofactor regulador de una amplia gama de enzimas y proteínas (Marschner, 1993). También participa en muchas rutas bioquímicas importantes, relacionadas principalmente con; el metabolismo de los carbohidratos, tanto en la fotosíntesis como en la conversión de azúcares al almidón, el metabolismo de las proteínas, el metabolismo de las auxinas, la formación del polen, el mantenimiento de la integridad de las membranas biológicas y la resistencia a la infección por ciertos patógenos (Alloway, 2009; Chattha et al., 2017; Mousavi, 2011). Un suministro insuficiente puede provocar que las funciones fisiológicas que dependen de este nutriente no se den con normalidad y que se produzcan cambios en los mecanismos de la planta que alteren su crecimiento y su desarrollo. Esto puede ocasionar que la planta desarrolle síntomas visibles de estrés, como una reducción de la altura, clorosis entre las nervaduras, bronceado de las hojas, desarrollo de hojas pequeñas y de forma anormal, formación de rosetas en las hojas, etc., que pueden variar en función de la especie y que, por lo general, aparecen con deficiencias severas (Broadley et al., 2007; Sadeghzadeh & Rengel, 2011). En humanos, un déficit de Zn provoca una amplia gama de problemas fisiológicos, incluyendo retraso en el crecimiento, deterioro del desarrollo cerebral, mayor susceptibilidad a enfermedades infecciosas, menor rendimiento físico y dificultades durante el parto (Cakmak & Hoffland, 2012; Cakmak & Kutman, 2018; Cu et al., 2020). Se estima que alrededor de un tercio de la población mundial está en riesgo de sufrirlo y resulta especialmente preocupante en niños menores de 5 años, puesto que se encuentran en etapa de crecimiento y desarrollo y su demanda es mayor. De hecho, casi medio millón de ellos muere cada año por causas relacionadas con esta deficiencia (Cu et al., 2020; Pfeiffer & McClafferty, 2007). Esto tiene un efecto considerable en la economía de los países en desarrollo, puesto que disminuye la productividad y aumenta los costes de atención médica. La principal causa de la deficiencia de Zn en humanos se debe a una ingesta insuficiente de este micronutriente (Cakmak & Kutman, 2018; White & Broadley, 2011). 32 2013; Viruel et al., 2014). En este sentido, la mejora de la nutrición de P podría empeorar la de Zn ya que el P interfiere en la absorción, el transporte de la raíz al brote y la acumulación de Zn en el grano (Cakmak & Hoffland, 2012; Zhang et al., 2016). En algunos casos, una elevada concentración de P en el grano llega incluso a inhibir la absorción de Zn durante la digestión (Gómez-Coronado et al., 2019; Khoshgoftarmanesh et al., 2017). Por ello es importante considerar el antagonismo P-Zn, tanto en el suelo como en la planta, y los inoculantes microbianos podrían ser cruciales para aumentar la fitodisponibilidad de ambos nutrientes manteniendo una proporción equilibrada entre ellos (Bouain et al., 2014; Hussain et al., 2017; Ova et al., 2015). Para utilizarlos de forma adecuada, potenciar su efecto y mejorar su eficacia, es necesario comprender los mecanismos involucrados en su actividad (Ahmad et al., 2008). 4.1. Principales mecanismos microbianos implicados en la mejora de la nutrición de la planta Muchos microorganismos tienen la capacidad de producir fitohormonas y alterar la fisiología de las plantas, ya que los cambios en la concentración y distribución de hormonas afectan el crecimiento y desarrollo de los vegetales. Sin embargo, las fitohormonas producidas por microorganismos no inducen cambios hormonales típicos, sino que estos cambios se ponen de manifiesto en la morfología de las raíces y en una promoción del crecimiento de las plantas. La fitohormona más determinante es el ácido indol-3-acético (IAA), cuya producción se ha relacionado con un incremento de la biomasa vegetal y con cambios en la raíz, como el acortamiento de las raíces primarias e incremento de las raíces laterales y los pelos radicales, que dan como resultado un aumento de la superficie radicular y permiten una mejor absorción de nutrientes (Nguyen et al., 2019; Spaepen, 2015). Estudios recientes han concluido que algunas cepas de Bacillus velezensis mejoran la absorción y la concentración de micronutrientes en las plantas de trigo (Nguyen et al., 2019a). Otros estudios señalan que la inoculación con Bacillus subtilis DS-178 favorece la translocación y acumulación de Zn en el grano, mejorando la eficiencia en el uso de nutrientes por parte de la planta (Singh et al., 2017). A pesar de que los resultados difieren según el estudio, y esto parece indicar que el efecto puede depender de la cepa de Bacillus inoculada (Khande et al., 2017), la mayoría atribuyen los beneficios a la producción de IAA de los microorganismos inoculados, que inducen una mayor superficie radicular y una mejor absorción de nutrientes. No obstante, la producción de IAA tiene un elevado coste energético y la biosíntesistes microbiana está estrictamente regulada (Owen et al., 2015; Spaepen, 2015). Tan solo el 20% de los aislados de Bacillus resultan productores de IAA según un estudio de Ahmad et al. (2008). A ello se suma que esta producción puede verse afectada por factores ambientales como el pH o limitaciones en la disponibilidad de nutrientes (Spaepen, 2015). Los microorganismos a menudo compiten entre sí por los nutrientes y ponen en marcha diferentes mecanismos de movilización, como la segregación de sideróforos que se menciona en el 33 apartado anterior y que resulta muy característica en el género Trichoderma spp. (Björkman, 2004; Martínez-Medina et al., 2011). Estos mecanismos liberan los nutrientes retenidos en el suelo y los ponen a disposición del microorganismo, pero frecuentemente también de la planta. El genero Bacillus spp. destaca por su potencial solubilizador, que se atribuye principalmente a la producción de ácidos orgánicos que reducen el pH (Mumtaz et al., 2019; Ramesh et al., 2014). Esto da como resultado la solubilización de compuestos insolubles de Zn y una redistribución de este en el suelo, incrementando la fracción intercambiable y, en consecuencia, su disponibilidad (Ramesh et al., 2014). Dado que la actividad de los microorganismos inoculados puede verse afectada por el pH o la disponibilidad de nutrientes, un factor importante a tener en cuenta es la aplicación de fertilizantes al cultivo (Nguyen et al., 2019; Singh et al., 2017; Spaepen, 2015). Hay estudios que evidencian que los fertilizantes nitrogenados pueden interaccionar con los microorganismos inoculados y modular su actividad (Nguyen et al., 2019). De igual forma, los fertilizantes fosfatados pueden provocar cambios en las comunidades microbianas del suelo y afectar el desempeño de los microorganismos inoculados (García-López & Delgado, 2016; Owen et al., 2015). Su efectividad también puede verse comprometida por la interacción con los microorganismos nativos del suelo, ya que su tasa de crecimiento y proliferación tras ser inoculados dependerá de su capacidad de supervivencia y su competitividad (Gouda et al., 2018; Khan et al., 2007; Spaepen, 2015). La climatología también puede limitar su establecimiento en el suelo tras su aplicación (Khan et al., 2007). Un factor crucial es la disponibilidad de agua, no sólo para la actividad de los microorganismos, sino para la solubilidad de los fertilizantes aplicados. Este, ante un escenario de cambio climático y en condiciones de escasez de agua, puede ser determinante sobre la eficacia de los microorganismos inoculados. Todos estos factores contribuyen a la alta variabilidad en el desempeño de los microorganismos inoculados, generando resultados contradictorios y, en consecuencia, incertidumbre sobre su uso. 5. Objetivos A tenor de todo lo expuesto, se puede asumir que sigue existiendo la necesidad de un mayor avance en el conocimiento de los factores del suelo que afectan la disponibilidad de Zn para las plantas para establecer modelos más precisos para predecir su deficiencia y que contribuyan a mejorar la eficiencia de los fertilizantes con Zn aplicados. Es necesario también avanzar en el conocimiento de los factores de suelo que condicionan la interacción entre el P y el Zn, como aspecto básico que afecta la disponibilidad de Zn para los cultivos. En este sentido, hay que considerar la nutrición de P y Zn de manera integrada para no generar desequilibrios nutricionales. Por último, se debe progresar en la utilización de métodos de manejo de la deficiencia de Zn que sean sostenibles y económicos, y en este sentido considerar el efecto de las adiciones de materia orgánica y de la inoculación con microorganismos capaces de mejorar la acumulación de Zn en las plantas. Resulta interesante que tanto el estudio de factores que afectan la disponibilidad de Zn para los cultivos, como los métodos de manejo 34 de su deficiencia, se estudien con plantas particularmente relevantes y sensibles al problema, como es el caso del trigo. Estos factores de manejo se deben estudiar tanto en condiciones controladas como de campo, para tratar de proporcionar una información que tenga la mayor relevancia científica y práctica posible. El principal objetivo de este trabajo fue esclarecer los factores implicados en la disponibilidad de Zn para las plantas y en el antagonismo P-Zn y, bajo esta premisa, evaluar el efecto de la adición de diferentes fuentes de materia orgánica y de inoculantes microbianos sobre la nutrición de la planta y la biofortificación de la cosecha. Para ello se establecieron los siguientes objetivos específicos: 1. Identificar los factores del suelo que afectan la disponibilidad de Zn para las plantas en el suelo 2. Estudiar el efecto de la materia orgánica sobre la disponibilidad de Zn y su interacción con el P 3. Evaluar el efecto de la actividad microbiana sobre la disponibilidad de Zn y su interacción con el P 4. Estudiar el efecto de inoculantes microbianos sobre la nutrición de las plantas y la biofortificación de la cosecha 5. Evaluar la eficacia de los inoculantes microbianos en la nutrición de plantas en condiciones de campo con restricción de agua 35 Capítulo II. Factores que determinan la disponibilidad y absorción de zinc por las plantas en suelos y clima mediterráneos Chapter II. Factors determining Zn availability and uptake by plants in soils developed under Mediterranean climate 36 37 Capítulo II. Factores que determinan la disponibilidad y absorción de zinc por las plantas en suelos y clima mediterráneos Constituído por la siguiente publicación científica: Moreno-Lora, A., Delgado, A., 2020. Factors determining Zn availability and uptake by plants in soils developed under Mediterranean climate. Geoderma 376, 114509. DOI: 10.1016/j.geoderma.2020.114509 Título en español: Factores que determinan la disponibilidad y absorción de Zn por las plantas en suelos y clima mediterráneos Autores: Aurora Moreno-Lora y Antonio Delgado Afiliación: Departamento de Ciencias Agroforestales, ETSIA, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain Revista: Geoderma Año de Publicación: 2020 Número y páginas: 376 (114509) DOI: 10.1016/j.geoderma.2020.114509 Índice de impacto: 7.422 (JCR, 2021) (1º Q) Área y posición: Soil Science - SCIE (2021). Posición 4/39 Resumen: La deficiencia de zinc (Zn) es un problema agronómico extendido, particularmente en cultivos básicos como los cereales. La disponibilidad de Zn para las plantas se rige por las propiedades del suelo, por factores biológicos de la rizosfera y por la interacción con otros nutrientes. Todos estos factores pueden limitar el valor predictivo de los índices de disponibilidad de Zn. Este trabajo tuvo como objetivo evaluar los factores de suelo que afectan la absorción de Zn por las plantas y mejorar el valor predictivo de los índices convencionales. Para ello, se realizó un experimento utilizando trigo duro (Triticum durum L.) cultivado en un conjunto de suelos, bajo clima mediterráneo. En los suelos calcáreos, la absorción de Zn por las plantas disminuyó con el aumento del contenido de arcilla y el fósforo Olsen (POlsen), mientras que en los suelos no calcáreos disminuyó con el aumento del contenido de óxidos de hierro (Fe) cristalinos. Los factores 38 biológicos como la actividad microbiana y la exudación de aniones orgánicos en la rizosfera contribuyeron a la absorción de Zn por las plantas. No se encontró relación entre la absorción de Zn por las plantas y el Zn extraíble por DTPA (ZnDTPA). El Zn extraíble por pirofosfato (ZnPyro) solo se relacionó con la absorción de Zn por las plantas en los suelos calcáreos (R2 = 0.29; P < 0.01). La mejor estimación de la absorción de Zn por las plantas en suelos calcáreos se obtuvo con un modelo que incluye la relación ZnPyro/POlsen y el contenido de arcillas (R2 = 0.57; P < 0.001). En los suelos no calcáreos, la absorción de Zn por las plantas se estimó con precisión con un modelo que incluye el Fe unido a óxidos cristalinos y la concentración de oxalato en la rizosfera (explica el 81% de la varianza). Los resultados revelan la necesidad de discriminar los suelos según su contenido de carbonatos y de considerar las propiedades del suelo relacionadas con la capacidad de adsorción de Zn, como el contenido de arcillas, óxidos de Fe y el P Olsen, para una estimación precisa de la absorción de Zn por las plantas. 39 40 41 48 49 Capítulo III. La disponibilidad de zinc y fósforo para el trigo se ve afectada por las sustancias húmicas en sustratos calcáreos y silíceos Chapter III. Zinc and phosphorus availability to wheat as affected by humic substances in calcareous and siliceous growing media 50 51 Capítulo III. La disponibilidad de zinc y fósforo para el trigo se ve afectada por las sustancias húmicas en sustratos calcáreos y silíceos Constituído por el siguiente trabajo científico: Moreno-Lora, A., Delgado, A. Zinc and phosphorus availability to wheat as affected by humic substances in calcareous and siliceous growing media Título en español: La disponibilidad de zinc y fósforo para el trigo se ve afectada por las sustancias húmicas en sustratos calcáreos y silíceos Autores: Aurora Moreno-Lora y Antonio Delgado Afiliación: Departamento de Agronomía, ETSIA, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain Publicación: En revisión Resumen: Las sustancias húmicas (SH) tienen un impacto en la dinámica del fósforo (P) y zinc (Zn) en el suelo y, en consecuencia, pueden afectar la disponibilidad de ambos nutrientes para las plantas. Este trabajo tuvo como objetivo estudiar el efecto de las sustancias húmicas sobre la disponibilidad de fósforo y zinc para el trigo dependiendo de las principales superficies adsorbentes en los medios de cultivo. Para ello, se realizó un experimento en maceta que involucró tres factores: i) dosis de SH, ii) fertilización con Zn y iii) medio de cultivo, calcáreo y no calcáreo (silíceo). La producción de biomasa y la absorción de Zn por las plantas disminuyeron con el aumento de las tasas de SH. La absorción de zinc disminuyó de forma más marcada en el medio silíceo. Los efectos negativos de las SH se pueden atribuir a un empeoramiento de la nutrición del cultivo y a la alta aromaticidad de las SH, que pueden promover efectos fitotóxicos. El efecto antagónico entre P y Zn fue menos evidente en el medio calcáreo que en el medio silíceo. Esto probablemente se explica por la menor disponibilidad de Zn y la consecuente disminución de su absorción por las plantas en el medio calcáreo, en comparación con el silíceo. Las diferencias observadas entre ambos medios se pueden atribuir a diferentes dinámicas de adsorción dependiendo de las principales superficies adsorbentes. La adición de SH, a las dosis intermedias estudiadas, tuvo un efecto positivo sobre la actividad microbiana de la rizosfera en medios calcáreos. Por tanto, no solo el desarrollo del cultivo, sino también la biología del suelo pueden verse afectados por la aplicación de SH. Este efecto puede ser diferente según la tasa de SH aplicada y el tipo de medio de cultivo. 52 53 Zinc and phosphorus availability to wheat as affected by humic substances in calcareous and siliceous growing media Aurora Moreno-Lora* and Antonio Delgado Dpto. Agronomía, ETSIA, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain *Corresponding author: am[email protected] Abstract Humic substances (HS) have an impact on phosphorus (P) and zinc (Zn) dynamics in soil and consequently can affect the availability of both nutrients to plants. This work aimed at studying the effect of humic substances on phosphorus and zinc availability to wheat depending on main sorbent surfaces in growing media. To this end, a pot experiment was performed involving three factors: i) HS rates, ii) Zn fertilization, and iii) type of growing medium, calcareous and non-calcareous (siliceous). Biomass production and Zn uptake by plants decreased with increased HS rates. The uptake of zinc was more markedly decreased by HS in the siliceous medium. Negative effects of HS can be ascribed to an impaired crop nutrition and the high aromaticity of HS that can promote phytotoxic effects. The antagonistic effect between P and Zn was less evident in the calcareous medium than in the siliceous medium. This is likely explained by the reduced availability of zinc and the consequent decreased uptake by plants in the calcareous medium when compared with the siliceous one. These differences observed between both media can be ascribed to different adsorption dynamics depending on the main sorbent surfaces. The addition of HS, at the intermediate rates studied, had a positive effect on rhizosphere microbial activity in calcareous media. Thus, not only crop functioning, but also soil biology, can be affected by the application of HS. This effect can be different depending on the HS rates applied and the type of growing medium. Keywords: Fe oxides, carbonates, enzyme activity, micronutrients, bioavailability, oxalate 54 1. Introduction Zinc (Zn) is an essential micronutrient required by plants and animals (Alloway, 2009), whose deficiency in soils promotes yield losses in crops and reduce their quality for human consumption, especially in cereals (Rashid & Ryan, 2004; Ryan et al., 2013a). In Mediterranean regions, Zn is considered the most limiting micronutrient deficiency affecting crop production (Alloway, 2009; Rashid & Ryan, 2004). Since Zn has a strong affinity for calcium carbonate (CaCO3) and iron (Fe) oxides, the adsorption on these minerals is a crucial factor affecting its availability to plants. Basic pH promotes the formation of Zn-hydroxide forms (hydrozincite and Zn hydroxide), chemisorption on calcite (ZnCO3) and co-precipitation in Fe oxides (Alloway, 2009; Rengel, 2015; Ryan et al., 2013a). Iron oxides can adsorb Zn through surface complexation processes, which are considered irreversible at basic pH. Adsorption can also occur through a non-specific process depending on pH since the negative surface charges increases with increasing pH (Buekers et al., 2008, 2007; Stahl & James, 1991; Ryan et al., 2013a). Zinc adsorption increases with decreased crystallinity of oxides and with increased reaction time (Shuman, 1977; Buekers et al., 2008; Duffner, 2014). The main sorbing surfaces, carbonates or Fe oxides, in soils with basic pH needs to be considered separately to obtain accurate predictions on the risk of Zn deficiency in plants (Imtiaz et al., 2006; Moreno-Lora & Delgado, 2020). Phosphate can compete with Zn for adsorption sites (Madrid et al., 1991). On the other hand, Zn deficiency can be promoted in soils with elevated concentrations of P (Ryan et al., 2013b). There is an antagonistic effect between P and Zn that seems to vary depending on the minerals which absorb these nutrients (Sánchez-Rodríguez et al., 2013). In this regard, it has been suggested that P-induced Zn deficiency can increase with the ratio of Fe oxides to CaCO3 (Rahmatullah & Torrent, 2000). Phosphorus can contribute to decrease grain quality since an increased concentration of phythate in grains, which is the main P compound present in plant seeds, could bound to Zn inhibiting its absorption during digestion (Miller et al., 2007; Gharibzahedi & Jafari, 2017; Gómez-Coronado et al., 2019). Therefore, the equilibrium between these nutrients in soils and plants should be taken into account in order to overcome Zn deficiency and enrich cereal grains with Zn, the so called biofortification. In this regard, the effects of soil management practices affecting P or Zn availability should be considered in an integrated way since the increase in the availability of one nutrient may affect negatively the other. Organic matter is an important factor affecting the availability of P and Zn to plants. Thus, management factors affecting organic matter in soil can affect the availability to plants of both nutrients. As a dominant fraction of soil organic matter, the adsorption of humic substances (HS) on Fe oxides can alter that of P and Zn, affecting their concentration in solution and distribution in soil (De Santiago & Delgado, 2007, Rahmatullah & Torrent, 2000). Humic substances can increase Zn adsorption on oxides (Güngör & Bekbölet, 2010; Stietiya & Wang, 2014). They can also reduce the precipitation of 55 calcium phosphates (Delgado et al., 2002), increasing the concentration of P in the soil solution and, therefore, affecting that of Zn. Overall, the interaction between P and Zn can be affected by HS because these substances have complexing effect, capacity to compete for adsorption sites, and can induce coadsorption processes. Nevertheless, HS can promote negative effects on plants if they are applied at high rates due to phytotoxic effects (de Santiago et al., 2010). Thus, this work aimed at study the effect of HS on P and Zn availability to plants and their interaction in two different growing media, i. e. siliceous and calcareous, with a view of assessing how the potential effect of HS vary depending on the main sorbent surfaces in the plant growing media. This will provide new insight on the role of organic matter in the availability of both nutrients and in the efficiency of Zn fertilizer for overcoming its deficiency and for biofortication. In addition, the availability of Fe, Mn, and Cu will be assessed since these nutrients may interact with P and Zn and their availability can be also affected by HS. 2. Material and methods 2.1 Experimental design A pot experiment was carried out in a growing chamber using wheat plants involving three factors; i) growing media, calcareous or non-calcareous (siliceous) ii) Zn fertilizer rate (1, 5, and 10 mg Zn kg–1 medium) , and iii) HS rate (involving the application of 0, 0.05, 0.1, and 0.5 g C kg–1 medium). Four replicates of each combination of treatments were used in the experiment in a complete randomized design. 2.2 Growing media Two different growing media were prepared by mixing 2:3 siliceous sand coated with Fe oxides (ferrihydrite) with adsorbed P with: (i) 1:3 of siliceous sand (siliceous medium) or (ii) 1:3 of calcareous sand (calcareous medium). After homogenizing the mixture, 350 g of medium were placed in plastic pots (150 mm high and 55 mm in diameter). A wheat plant was placed in each pot. The siliceous sand was washed with distilled water and sodium carbonate, in order to disperse colloids, and dried in a forced air oven at 48 ºC. Siliceous sand coated by oxides were prepared following the procedure of Rahmatullah & Torrent (2000). Phosphorus was adsorbed to the oxides by immersing the coated sand into a solution containing 7 mg P L-1 (KH2PO4) at a ratio 1:1.5 (w:v) for 48 h. The composition of this solution, with the exception of P added, was the same that the solution using for irrigation and to supply nutrients (except Zn, Fe and P). The sand was carefully shaken every 6 h during this process. Then, the solution was removed and the sand was washed with distilled water to remove P in solution (not adsorbed), and dried in a forced-air oven. Calcareous sand was washed and sieved to < 2 mm by decantation. To this end, 100 g of calcareous sand were placed in a 30 cm high cylinder filled of distilled water and settle for 38 s. Immediately after that, the water with suspended sand was removed and the settled sand was washed again and dried in a forced-air oven. The siliceous 56 and calcareous medium were prepared by mixing the portions of each sand described above. Both media were analysed in triplicate for their characterization (Table 1). Nitrate concentration was determined using test strips (Nitracheck, Merk). Nutrient availability indices of Fe and Zn (DTPA-extractable) (Lindsay & Norvell, 1978) and P (Olsen et al., 1954) were determined. 2.3 Plant material and growing conditions Wheat seeds (Triticum aestivum) were disinfected by inmersion in 5% NaClO for 1 min (de Santiago et al., 2011). Then, seeds were germinated in petri dishes at 6 ºC for a few days to induce the germination. Plants were transferred to pots at Z1.2 stage of Zadoks scale (Zadoks et al., 1974). The experiment was carried out in a growing chamber under environmental controlled conditions, with 16 h of light at 20 ºC and 40% relative humidity during days and 8 h of darkness at 18 ºC and 60 % relative humidity during nights. Photosynthetic radiation was 300 µmol m-2s-2. Plants were irrigated daily with a modified Hoagland nutrient solution without P, Fe and Zn at pH 6.5, containing (mmol L-1): MgSO4 (2), Ca(NO3)2 (5), KNO3 (5), KCl (0.05), H3BO3 (0.009), MnCl2 (0.0023), CuSO4 (0.0005) and H2MoO4 (0.0005). Zn supply was one of the factors in the experiment, and P and Fe is supposed to be supplied by the growing media. 2.4 Treatments 2.4.1 Zn dose Three doses of ZnSO4 (low, medium and high) were dissolved in the nutrient solution and applied during the first three pot irrigations for applying a total dose of 1, 5, and 10 mg Zn kg-1 medium, respectively. 2.4.2 Humic substances Four rates of humic substances (HS) were applied based on the carbon (C) concentration to achieve 0, 0.05, 0.1, and 0.5 g C kg medium-1. Humic substances from a commercial product (Solfer Humicos, Lida Química) were dialysed before application to the growing medium. For dialysis, 50 mL of product was introduced in a dialysis membrane (Membra-cel, Serva), previously treated with 0.5 M Table 1. Properties of growing medium pH EC NO3 - Olsen P DTPA Medium 1:5 Zn Fe µS cm - 1 mg L - 1 mg kg - 1 Siliceous 5.58 63.55 14.33 12.15 0.03 6.67 Calcareous 8.60 111.17 20.67 17.08 0.03 4.71 EC, electrical conductivity; DTPA, diethylene-triaminepentaacetic acid 57 NaOH, and placed in a container filled with distilled water. The water was renewed every 2 h during the first 12 h of the dialysis process and, after that, every 48 h till reaching an electrical conductivity of the water lower than 100 µS cm-1. Then, dialysed HS were collected. The density of the suspension of dialyzed HS was 1,003 kg L–1, the pH 8.4, and the electrical conductivity 944 µS cm-1. The concentration of oxidable C was 0.358 % (Walkley & Black, 1934) and total C was 0.3704 % (Leco CNS analyser, LECO Instrumentos, Madrid, Spain). 2.5 Sample collection and analysis The pot experiment concluded 77 days after transplanting, when plants reached the stage Z9 (Zadoks et al., 1974). Plants were harvested, and shoots, roots, and grains separated. Roots were washed with distilled water and immersed in an ultrasonic bath for 1 min. Afterwards, all these plant material was placed in a forced-air oven at 65 ºC for at least 48 h to determine dry biomass. 2.5.1 Growing media analysis Rhizospheric medium was sampled as described Wang et al. (2009), considering only that bound to the roots (Nazih et al., 2001). A portion of that was used for the determination of organic anions and enzyme activities and the other portion was air-dried and used to determine chemical parameters. All samples were stored at < 6 °C for less than 48 h before preforming the organic anions and biochemical analysis. The concentration of organic anions was determined after extraction with 0.1 M NaOH (1:1) (Radersma & Grierson, 2004), following the chromatographic procedure of Gao et al. (2012). The βglucosidase (Eivazi & Tabatabai, 1988) and alkaline phosphatase (Eivazi & Tabatabai, 1977) were determined colorimetrically by measuring the amount of p-nitrophenyl from the hydrolysis of pnitrophenyl-β-glucopyranoside and p-nitrophenyl-phosphate, respectively. Dehydrogenase was determined by measuring the concentration of triphenyl formazan (TPF) produced by enzymes (Casida et al., 1964). To this end, 2.5 g of medium, 0.02 g of CaCO3, 0.8 mL of H2O and 0.3 mL of 3 % 2,3,5- triphenyl-tetrazolium chloride (TTC) were placed in a falcon tube. This was mixed and incubated in the dark for 24 h. Afterwards, a volume of 3.9 mL of ethanol was added to extract the TPF produced. The mix was centrifugated at 1258 g for 10 min. The amount of TPF in the supernatant was determined colorimetrically at 485 nm using a spectrophotometer. Nutrient availability indices of P (Olsen et al., 1954), Zn and Fe (DTPA extraction according to Lindsay & Norvell (1978) were determined after cultivation and compared to the initial values in order to estimate the depletion of these nutrients during plant growth. 2.5.2 Plant analysis The concentration and total amount of nutrients in each organ were determined. For this purpose, plant material was ground to < 1 mm, and 0.25 g of each sample were calcinated in a Mufla, at 550 ºC for 8 h. To ensure the recovery of all elements, ashes were dissolved in 10 mL of 1 N HCl at 100 ºC for 64 Figure 1. Effect of growing medium and Zn dose on P concentration in grain (a) and total P uptake by plants (b). HS rates: HS0, HS1, HS2, and HS3 = 0, 0.05, 0.1, and 0.5 g C kg medium-1, respectively. Zn rates: Z1, Z2, and Z3 = 1, 5, and 10 mg Zn kg–1 medium Error bars represent standar error (SE, n=4). 3.2. Effect on the growing media In the growing media, HS negatively affected the oxalate concentration in the rhizosphere at the highest rate (Table 4) and the phosphatase concentration in the calcareous medium (Table 5). In this medium, dehydrogenase activity increased at intermediates HS rates meanwhile it decreased at the highest HS rate in the siliceous medium (Table 5). Olsen P after crop decreased more in the calcareous than in the siliceous growing medium, and the effect of HS decreasing Olsen P was less marked in the siliceous medium (Table S2, [suppl]). In both media, the DTPA extractable Fe after crop increased at increased HS rates, although the trend was different between calcareous and siliceous growing media (Table 5). On the other hand, the DTPA extractable Zn increased with increased Zn doses, but only at the two highest doses. Intermediate HS rates promoted a decrease in the DTPA extractable Zn (Figure 2). Finally, the uptake of Zn by plants was related to DTPA extractable Zn in media; however, the relationship was different for calcareous and siliceous growing media (Figure 3). The pH of growing media increased with increased HS rate, although the trend was clearer in the calcareous growing medium (Table S2, [suppl]). 65 Figure 2. Effect of Zn doses and humic substances rate (HS) on Zn availability after crop cycle (Zn- DTPA). HS rates: HS0, HS1, HS2, and HS3 = 0, 0.05, 0.1, and 0.5 g C kg medium-1, respectively. Zn rates: Z1, Z2, and Z3 = 1, 5, and 10 mg Zn kg–1 medium Error bars represent standar error (SE, n=8). Figure 3. Relationship between Zn uptake and Zn availability after crop cycle (Zn-DTPA) in calcareous and siliceous growing medium. For the whole data set, the relationship was also significant (Y=0.046+0.031X; R2=0.36; p<0.000). 66 4. Discussion Overall, biomass production and nutrient uptake, by except Mn, decreased with increased HS rates, independently of the growing media. Thus, it cannot be ascribed to particular reactions of applied HS nor nutrients in each growing media. These results agree with de Santiago et al. (2010) who observed adverse effects on lupines at HS concentrations higher than 0.1 g C kg–1, ascribed at least in part to the high aromaticity of HS that promoted phytotoxic effects. Root was the organ where the greatest inhibition in development was observed (by 30 %) with the highest HS rate. This likely reveal this phytotoxic effect. This reduction in root development explain the lower concentration of oxalate in the rhizosphere, since this production is partly due to root exudations that will decrease with less root development. The exudation of organic anions is a mechanism for nutrient mobilization in plants and microorganisms, in particular for metals and P (García-López & Delgado, 2016). Thus, reduced root development and exudation of organic anions contribute to explain the decreased nutrient uptake observed. The increased Zn and Mn concentration in roots with increasing HS rates could be explained by a concentration effect given the lower root development at this rate. On the contrary, the concentration of Fe in roots was markedly decreased with increased HS rates. In addition to a decreased oxalate exudation, potential adsorption of HS on Fe oxides and/or complexation of Fe by HS may negatively affect Fe mobilization from growing media and absorption by plants, explaining this negative effect on Fe nutrition. Without HS application, Zn uptake by plants was higher in the siliceous than in the calcareous growing medium. This reveals that carbonates may constraint Zn availability more the Fe oxides, or Zn adsorption on Fe oxides may be less reversible in the presence of carbonates. In calcareous soils, Zn precipitated or adsorbed on carbonates is not available to plants (Imtiaz et al., 2006). However, with increasing HS rates, there was not a significant effect decreasing Zn uptake by plants in calcareous growing media while this decrease was significant in the case of siliceous growing medium (Table 5). This reveal more negative effect of HS when the unique Zn sorbent surface is Fe oxides, perhaps due an increased adsorption of Zn on Fe oxides promoted by HS (Stietiya & Wang, 2014). Zinc complexed by HS can be a source of the nutrient for plants (García-López et al., 2022). Thus, it seems that the effect of HS decreasing Zn uptake may be ascribed to the interaction of the nutrient with sorbent surfaces decreasing its activity in solution. The Zn availability index, i.e. Zn extractable by DTPA, increased with increased Zn dose applied to the media. The addition of humic substances apparently impaired this index compared to the control at the two lowest Zn doses; however, there was no difference between the control and the highest HS rate (Figure 2). This is probably due to the interaction with sorbent surfaces and the formation of complexes between humic substances and Zn. At low HS rates, HS can induce a higher adsorption on oxides and carbonates, meanwhile at the highest HS rate, part may remain in solution as Zn complexes 67 that may be a source of available Zn to plants (García-López et al., 2022). However, the ratio between DTPA extractable Zn and Zn absorbed by the plant was different depending on the growing medium, with higher ratios found for the siliceous growing medium (Fig. 3). Thus, the accuracy of the Zn availability index should take into account the main reactions involved in Zn retention in soils. Humic substances had positive effects on Mn uptake by plants in agreement with Çelik et al. (2010). This could be associated with its complexation by humic substances. It should be highlighted that the negative effect of increasing HS rates on the concentration and total Fe uptake disagreed with the effect on DTPA extractable Fe which showed the opposite trend. This is not surprising since DTPA extractable Fe cannot be deemed an accurate index of Fe availability particularly in calcareous soils (de Santiago & Delgado, 2006). Thus, HS increase chemical extractability through complexation of Fe, but not its real availability to plants. Probably, the adsorption of HS on Fe oxides may decrease the efficiency of phytosiderophores produced by wheat roots by competing for adsorption sites on oxides decreasing the mobilization of the nutrient from oxides. The effect of HS on P nutrition varied depending on the growing media and Zn dose (Table 2 and table S1 [suppl]). There was a clear antagonistic effect of Zn on P uptake that was less marked in the calcareous growing medium (Figure 1). This contradicts results by Chen et al. (2018) which showed P-Zn antagonism with a P concentration 20 times higher in acid soil than in calcareous soil. In our case, the decreased availability of Zn in the calcareous medium not only led to a decreased Zn uptake by plants, but also to a decreased antagonistic effect on P. In addition, the calcareous medium had an initial Olsen P higher than that of siliceous medium. Thus, with a higher P availability the potential antagonism induced by Zn could be less marked. There is not any negative effect of HS on P uptake by except at the highest Zn rate in both media. Humic substances is known to increase P availability (Delgado et al., 2002). However, when the antagonism with Zn is more marked it seems that HS contribute negatively to P availability to plants. Plants grown in the siliceous medium showed a higher Zn concentration in grain than those grown in the calcareous medium. This contribute to explain that the P to Zn molar ratio tended to be lower in the siliceous medium than in the calcareous one (P < 0.053). Thus, the growing medium has a clear effect on grain quality in terms of Zn concentration and digestibility. On the other hand, HS reduced grain yield increasing the Zn concentration in the grain by concentration effect at the highest HS rate. Consequently, despite the decreased yield, the P to Zn molar ratio of grains were decreased. To cope with low Zn availability, especially in alkaline soils, the plant excretes organic acids, which lower the soil pH, thus increasing Zn availability (Bouain et al., 2014). However, no significant differences in oxalate production were found between Zn doses to support this hypothesis. Experiments carried out by Duffner et al. (2012) also did not show an increased root exudation of organic anions in response to low Zn availability. 68 The addition of the two intermediate rates of HS increased dehydrogenase activity in the calcareous medium by more than 65% compared to the control without HS; the highest HS rate did not show significant differences with control (Table 5). Since this enzyme activity has a microbial origin and it is intracellular, it can be assumed that HS were a source of C for microorganisms. In the siliceous medium, on the contrary, the intermediate levels did not improve the dehydrogenase activity, and a negative effect was observed at the highest rate compared to the control. In this medium, the stabilization of HS through adsorption on Fe oxides should make them less accessible for microbial degradation, explaining the lack of positive effect of HS on microbial enzymes. In the calcareous medium, the increased pH by effect of carbonates may lead to a decreased HS adsorption (Avena & Koopal, 1998) making HS more accessible for microorganisms. At the highest rate, HS may have negative effects on rhizospheric microorganisms. It is known that microbial activity in the rhizosphere, and consequently the dehydrogenase activity, increase with increased plant root development (Woli et al., 2012). Thus, phytotoxic effect on plants can lead to decreased microbial activity in the rhizosphere. Phosphatase activity showed a different pattern to dehydrogenase, with no clear negative effect of HS. It should be borne in mind that this activity is also associated with the plant and not only with microorganisms such as dehydrogenase. Increasing HS rates negatively affected Zn nutrition in the siliceous medium without significant effects in the calcareous medium, had little effect on P, and improved Mn nutrition. The different effect on Zn availability to plants depending on the growing medium can be ascribed to different adsorption dynamics depending on main sorbent surfaces. Impaired Zn and Fe nutrition contributes to explain, besides a phytotoxic effect, the negative effect on plant DM yield at the highest HS rate. Grain yield decreased with increased HS rates more markedly in the siliceous medium than in the calcareous medium. On the other hand, the addition of HS, at the intermediate rates studied, had a positive effect on rhizosphere microbial activity in calcareous media. Thus, not only crop functioning, but also soil biology, can be affected with the application of HS. This effect can be different depending on the HS rates applied and the type of growing medium. Acknowledgements This work was funded by the Spanish Ministry of Economy and Competitiveness of Spain and the European Regional Development Fund of the European Union through the National Research, Development and Innovation Programme (Plan Estatal I + d + i, Project PID2020-118503RB-C21). The authors thank the Agricultural Research Service of the University of Seville (SIA) for technical assistance and access to experimental facilities. We also appreciate the collaboration of Purificación Pajuelo and Oliva Polvillo, technicians of the SIA, for their invaluable help in some soil analysis. 69 References Acutis M, Scaglia B, Confalonieri R, 2012. Perfunctory analysis of variance in agronomy, and its consequences in experimental results interpretation. Eur J Agron 43:129–135. https://doi.org/10.1016/j.eja.2012.06.006 Alloway BJ, 2009. Soil factors associated with zinc deficiency in crops and humans. Environ Geochem Health 31(5):537–548. https://doi.org/10.1007/s10653-009-9255-4 Avena MJ, Koopal LK, 1998. Desorption of humic acids from an iron oxide surface. Environ Sci Technol 32(17):2572–2577. https://doi.org/10.1021/es980112e Borrill P, Connorton JM, Balk J, Miller AJ, Sanders D, Uauy C, 2014. Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops. Front Plant Sci 5:53. https://doi.org/10.3389/fpls.2014.00053 Bouain N, Shahzad Z, Rouached A, Khan GA, Berthomieu P, Abdelly C, Poirier Y, Rouached H, 2014. Phosphate and zinc transport and signalling in plants: Toward a better understanding of their homeostasis interaction. J Exp Bot 65(20):5725–5741. https://doi.org/10.1093/jxb/eru314 Buekers J, Amery F, Maes A, Smolders E, 2008. Long-term reactions of Ni, Zn and Cd with iron oxyhydroxides depend on crystallinity and structure and on metal concentrations. Eur J Soil Sci 59(4):706–715. https://doi.org/10.1111/j.1365-2389.2008.01028.x Buekers J, Van Laer L, Amery F, Van Buggenhout S, Maes A, Smolders E, 2007. Role of soil constituents in fixation of soluble Zn, Cu, Ni and Cd added to soils. Eur J Soil Sci 58(6):1514–1524. https://doi.org/10.1111/j.1365-2389.2007.00958.x Cakmak I, 2008. Enrichment of cereal grains with zinc: Agronomic or genetic biofortification? Plant Soil 302(1–2):1–17. https://doi.org/10.1007/s11104-007-9466-3 Casida LE, Klein DA, Santoro T, 1964. Soil dehydrogenase activity. Soil Sci 98(6):371–376. https://doi.org/10.1097/00010694-196412000-00004 Çelik H, Katkat AV, Aşık BB, Turan MA, 2010. Effect of foliar-applied humic acid to dry weight and mineral nutrient uptake of maize under calcareous soil conditions. Commun Soil Sci Plant Anal 42(1):29–38. https://doi.org/10.1080/00103624.2011.528490 Chen XX, Zhang W, Wang Q, Liu YM, Liu DY, Zou CQ, 2018. Zinc nutrition of wheat in response to application of phosphorus to a calcareous soil and an acid soil. Plant Soil 434(1–2):139–150. https://doi.org/10.1007/s11104-018-3820-5 de Santiago A, Delgado A, 2006. Predicting iron chlorosis of lupin in calcareous spanish soils from iron extracts. Soil Sci Soc Am J 70(6):1945. https://doi.org/10.2136/sssaj2005.0343 70 de Santiago A, Delgado A, 2007. Effects of humic substances on iron nutrition of lupin. Biol Fertil Soils 43(6):829–836. https://doi.org/10.1007/s00374-007-0191-0 de Santiago A, Expósito A, Quintero JM, Carmona E, Delgado A, 2010. Adverse effects of humic substances from different origin on lupin as related to iron sources. J Plant Nutr 33(2):143–156. https://doi.org/10.1080/01904160903434220 de Santiago A, Quintero JM, Avilés M, Delgado A, 2011. Effect of Trichoderma asperellum strain T34 on iron, copper, manganese, and zinc uptake by wheat grown on a calcareous medium. Plant Soil 342(1–2):97–104. https://doi.org/10.1007/s11104-010-0670-1 de Santiago A, Quintero JM, Carmona E, Delgado A, 2008. Humic substances increase the effectiveness of iron sulfate and vivianite preventing iron chlorosis in white lupin. Biol Fertil Soils 44(6):875–883. https://doi.org/10.1007/s00374-008-0272-8 Delgado A, Uceda I, Andreu L, Kassem S, Del Campillo MC, 2002. Fertilizer phosphorus recovery from gypsum-amended, reclaimed calcareous marsh soils. Arid L Res Manag 16(4):319–334. https://doi.org/10.1080/15324980290000421 Duffner A, Hoffland E, Temminghoff EJM, 2012. Bioavailability of zinc and phosphorus in calcareous soils as affected by citrate exudation. Plant Soil 361(1–2):165–175. https://doi.org/10.1007/s11104- 012-1273-9 Duffner A, 2014. Chemical and biological rhizosphere interactions in low zinc soils. Doctoral thesis. Wageningen University, The Netherlands. Eivazi F, Tabatabai MA, 1977. Phosphatases in soils. Soil Biol Biochem 9(3):167–172. https://doi.org/10.1016/0038-0717(77)90070-0 Eivazi F, Tabatabai MA, 1988. Glucosidases and galactosidases in soils. Soil Biol Biochem 20(5):601– 606. https://doi.org/10.1016/0038-0717(88)90141-1 Gao Z, Shao J, Sun H, Zhong W, Zhuang W, Zhang Z, 2012. Evaluation of different kinds of organic acids and their antibacterial activity in Japanese Apricot fruits. African J Agric Res 7(35):4911–4918. https://doi.org/10.5897/AJAR12.1347 García-López AM, Delgado A, 2016. Effect of Bacillus subtilis on phosphorus uptake by cucumber as affected by iron oxides and the solubility of the phosphorus source. Agric Food Sci 25(3):216–224. García-López AM, Recena R, Delgado A, 2022. Effect of Zn binding to phytate and humic substances on its uptake by wheat (Triticum durum L.) as affected by carbonates and Fe oxides. Pedosphere 32(6):823–832. https://doi.org/10.1016/J.PEDSPH.2022.06.022 71 Gharibzahedi SMT, Jafari SM, 2017. The importance of minerals in human nutrition: Bioavailability, food fortification, processing effects and nanoencapsulation. Trends Food Sci Technol 62:119–132. https://doi.org/10.1016/J.TIFS.2017.02.017 Gómez-Coronado F, Almeida AS, Santamaría O, Cakmak I, Poblaciones MJ, 2019. Potential of advanced breeding lines of bread-making wheat to accumulate grain minerals (Ca, Fe, Mg and Zn) and low phytates under Mediterranean conditions. J Agron Crop Sci 205(3):341–352. https://doi.org/10.1111/jac.12325 Güngör EBO, Bekbölet M, 2010. Zinc release by humic and fulvic acid as influenced by pH, complexation and DOC sorption. Geoderma 159(1–2):131–138. https://doi.org/10.1016/j.geoderma.2010.07.004 Imtiaz M, Alloway B, Aslam M, Memon M, Khan P, Siddiqui SUH, Shah S, 2006. Zinc sorption in selected soils. Commun Soil Sci Plant Anal 37(11–12):1675–1688. https://doi.org/10.1080/00103620600710330 Lindsay WL, Norvell WA, 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J 42:421–428. https://doi.org/10.2136/sssaj1978.03615995004200030009x Madrid L, Diaz-Earrientos E, Contreras MC, 1991. Relationships between zinc and phosphate adsorption on montmorillonite and an iron oxyhydroxide. Aust J Soil Res 29(2):239–247. https://doi.org/10.1071/SR9910239 Miller L V., Krebs NF, Hambidge KM, 2007. A mathematical model of zinc absorption in humans as a function of dietary zinc and phytate. J Nutr 137(1):135–141. https://doi.org/10.1093/jn/137.1.135 Moreno-Lora A, Delgado A, 2020. Factors determining Zn availability and uptake by plants in soils developed under Mediterranean climate. Geoderma 376:114509. https://doi.org/10.1016/j.geoderma.2020.114509 Murphy J, Riley JP. 1962. A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36. https://doi.org/10.1016/S0003-2670(00)88444-5 Nazih N, Finlay-Moore O, Hartel PG, Fuhrmann JJ, 2001. Whole soil fatty acid methyl ester (FAME) profiles of early soybean rhizosphere as affected by temperature and matric water potential. Soil Biol Biochem 33(4–5):693–696. https://doi.org/10.1016/S0038-0717(00)00197-8 Olsen SR, Cole CV, Watanabe FS, Dean LA, 1954. Estimation of available phosphorus in soils by extraction with sodium bicarbonate. Washingt United states Dep Agric USDA 939:1–19. https://doi.org/10.2307/302397 72 Radersma S, Grierson PF, 2004. Phosphorus mobilization in agroforestry: Organic anions, phosphatase activity and phosphorus fractions in the rhizosphere. Plant Soil 259(1–2):209–219. https://doi.org/10.1023/B:PLSO.0000020970.40167.40 Rahmatullah, Torrent J, 2000. Phosphorus dynamics and uptake by wheat in a model calcite-ferrihydrite system. Soil Sci 165(10):803–812. https://doi.org/10.1097/00010694-200010000-00005 Rashid A, Ryan J, 2004. Micronutrient constraints to crop production in soils with mediterranean-type characteristics: A review. J Plant Nutr 27(6):959–975. https://doi.org/10.1081/PLN-120037530 Rengel Z, 2015. Availability of Mn, Zn and Fe in the rhizosphere. J Soil Sci Plant Nutr 15(2):397–409. https://doi.org/10.4067/S0718-95162015005000036 Ryan J, Ibrikci H, Rashid A, Sommer R, 2013b. Phosphorus in Low-Input Dryland Agriculture: The Perspective from Syria. Commun Soil Sci Plant Anal 44(16):2378–2392. https://doi.org/10.1080/00103624.2013.794825 Ryan J, Rashid A, Torrent J, Yau SK, Ibrikci H, Sommer R, Erenoglu EB 2013a. Chapter one - Micronutrient constraints to crop production in the middle east-west Asia region: Significance, research, and management. Adv Agron. 122:1–84. doi:10.1016/B978-0-12-417187-9.00001-2. Sánchez-Rodríguez AR, Cañasveras JC, del Campillo MC, Barrón V, Torrent J, 2013. Iron chlorosis in field grown olive as affected by phosphorus fertilization. Eur J Agron 51:101–107. https://doi.org/10.1016/J.EJA.2013.07.004 Shuman LM, 1977. Adsorption of Zn by Fe and Al Hydrous Oxides as Influenced by Aging and pH1. Soil Sci Soc Am J 41(4):703. https://doi.org/10.2136/sssaj1977.03615995004100040016x Stahl, R.S., James, B.R., 1991. Zinc sorption by B horizon soils as a function of pH. Soil Sci. Soc. Am. J. 55, 1592-1957. https://doi.org/10.2136/sssaj1991.03615995005500060015x Stietiya MH, Wang JJ, 2014. Zinc and cadmium adsorption to aluminum oxide nanoparticles affected by naturally occurring ligands. J Environ Qual 43(2):498–506. https://doi.org/10.2134/jeq2013.07.0263 Walkley A, Black IA, 1934. An examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method. Soil Sci 37(1):29–38. https://doi.org/10.1097/00010694-193401000-00003 Wang G, Xu Y, Jin J, Liu J, Zhang Q, Liu X, 2009. Effect of soil type and soybean genotype on fungal community in soybean rhizosphere during reproductive growth stages. Plant Soil 317(1–2):135–144. https://doi.org/10.1007/s11104-008-9794-y 73 Woli A, St Z, Wolińska A, Stępniewska Z, 2012. Dehydrogenase activity in the soil environment. Dehydrogenases:183–210. https://doi.org/10.5772/48294 Zadoks JC, Chang TT, Konzak CF, 1974. A decimal code for the growth stages of cereals. Weed Res 14(6):415–421. https://doi.org/10.1111/j.1365-3180.1974.tb01084.x 80 La adición de celulosa mejoró la absorción total de P por las plantas en ambos suelos, independientemente del suministro de P. En general, la inoculación de B. subtilis también proporcionó beneficios en la absorción de P, a pesar de que este efecto varió según los suelos y el suministro de P. B. subtilis mejoró la concentración de Zn en el grano en un 24 % respecto al control, mientras que el efecto sobre la absorción total de Zn por las plantas no fue significativo. Esto explica el incremento del índice de cosecha de Zn con la inoculación B. subtilis. La mejora de la absorción de P por B. subtilis y la adición de celulosa no condujo a un efecto antagónico sobre el Zn, cuya absorción por las plantas incrementó linealmente con el aumento de la absorción de P (R2=0,68; P < 0,001). Estos resultados evidencian los beneficios de las prácticas agrícolas que afectan las propiedades microbianas de la rizosfera sobre la nutrición de P de las plantas. B. subtilis incrementó la acumulación de Zn en el grano, promoviendo así un efecto de biofortificación. 81 82 83 84 85 86 87 88 89 96 1 Introduction Micronutrient deficiency is a relevant agronomic problem that restricts crop yields and quality in soils with basic pH (Alloway 2009; Mousavi 2011; Ryan et al. 2013; Rengel 2015; Moreno-Lora et al. 2020). Furthermore, the decrease in Fe and Zn concentration in cereals grown in that type of soils may cause a relevant health problem in regions with cereal-based diets (Cakmak et al. 2010; Borrill et al. 2014; Wang et al. 2014; Zhao et al. 2014; Cakmak and Kutman 2018). Overcoming micronutrient deficiency and biofortication of crops to increase their concentration in edible parts requires the application of fertilizers (McBeath and McLaughlin 2014; Moreno-Lora et al. 2019). These fertilizers are expensive and not always efficient due to their reactions in soil (Alloway 2009; White and Broadley 2009; Zhang et al. 2012). Phosphorus deficiency can also be frequent in soils prone to micronutrient deficiency, and this nutrient can negatively interact with Fe and Zn, decreasing its availability to plants (Moreno-Lora et al. 2020; Recena et al. 2021). Therefore, more cost-effective and sustainable practices are required to supply micronutrients to crops, such as those that involve the use of microbial inoculants capable of increasing the availability of micronutrients to plants (de Santiago et al. 2009; 2011; 2013; Khande et al. 2017; Moreno-Lora et al. 2019). Soil microorganisms can play a key role in the soil nutrient cycle, solubilizing insoluble compounds that lead to an increase in exchangeable micronutrient concentration; this increases its availability to plants (Ramesh et al. 2014). In response to nutrient deficiency, microorganisms trigger mobilization mechanisms that may increase the availability of nutrients to plants (García-López et al. 2021). These mechanisms involve acidification, the release of low-molecular weight organic acids, chelating agents (e.g. siderophores), and hydrolytic enzymes (Marschner et al. 2011; Zhao et al. 2011; Rengel 2015; García-López et al. 2021). As a particular case, the efficiency in mobilizing Zn by Bacillus genera has been mainly ascribed to the production of organic acids and decreased rhizosphere pH (Ramesh et al. 2014; Mumtaz et al. 2019). However, microbial mechanisms leading to improved micronutrient uptake by crops and the functioning of these mechanisms under different soil conditions are not fully elucidated. Many soil microorganisms influence plant nutrition and development by producing phytohormones or affecting their transport and signaling pathways (Spaepen 2015; Kudoyarova et al. 2019). Indole-3-acetic acid (IAA) produced by rizhospheric microorganisms promotes plant growth and morphological root changes, resulting in an increased root surface that allows for an enhanced uptake of nutrients (Sukumar et al. 2013; Owen et al. 2015; Gouda et al. 2018; Qiu et al. 2019; de los Santos-Villalobos and Parra-Cota 2020). Gibberellins (GA3), cytokinins and abscisic acid (ABA) produced by microorganisms such as B. subtilis may also affect root and plant development (Arkhipova et al. 2005; Singh et al. 2017). Therefore, the benefits of microorganisms can be ascribed not only to improved nutrients mobilization that increases their availability to plants, but also to the effects on plant 97 physiology that lead to increased development and/or capacity to absorb nutrients. Consequently, it is necessary to take into account the possible effects on phytohormone homeostasis as a relevant mechanism that explain the effect of microorganisms on plant development and nutrition. The activity of rhizospheric microorganisms is strongly affected by soil properties (Khan and Joergensen 2010; Shi et al. 2013; Owen et al. 2015; Ma et al. 2017; Nguyen et al. 2019). In particular, the composition, size, and activity of microbial communities in the rhizosphere can be modulated by the quantity and quality of carbon (C) sources in soil (Hobbie and Hobbie 2013). Thus, the application of a labile C source to soil can increase the activity of native soil microorganisms (Demoling et al. 2007). This may promote the mobilization of nutrients that induce benefits on plant nutrition (Kuzyakov 2002; de Santiago et al. 2013; Moreno-Lora et al. 2019). The properties of soil affect the establishment, activity, and potential benefits of microbial inoculants (García-López et al. 2018; Moreno-Lora et al. 2019; Nguyen et al. 2019). The effects of microbial inoculants on micronutrient nutrition and biofortification have been reported (Ramesh et al. 2014; Khande et al. 2017; Singh et al. 2017; Moreno-Lora et al. 2019; Nguyen et al. 2019). However, most of these studies have been developed with a reduced number of soils. Therefore, the influence of soil factors on the benefits of microbial inoculants has been poorly evaluated. This is a relevant aspect for fully understanding how microbial mechanisms may enhance the nutrition of plants. The benefits of microbial inoculants and the supply of organic C in plant nutrition and biofortification must be confirmed in a wide range of soils to provide solid recommendations in their use. We hypothesized that the application of organic amendment or microbial inoculations will have three positive effects on crops: (i) improve the uptake of micronutrients and P by plants, (ii) increase the portion of micronutrients accumulated in the edible part of the crop, that is, the harvest index of nutrients, and (iii) improve the growth and yield of the plant. This work aims to assess the effects of the application of a readily available source of C and two microbial inoculants (Trichoderma asperellum T34 and Bacillus subtilis QST713), with the ability to mobilize nutrients, on micronutrient and P nutrition and plant growth in different soils. To explain possible effects, we will study the effect of these treatments on mobilization mechanisms in the rhizosphere such as organic acid and hydrolytic enzymes and on the concentration of phytohormones in plants. The study will be conducted on a set of soils with a wide range of properties to better understand the microbial mechanisms involved in the supply of nutrients to plants and to assess the consistency of the results in different edaphic environments. 98 2 Material and methods 2.1 Experimental design A randomized block experiment with four replications was carried out using durum wheat plants (Triticum durum cv Amilcar). It was carried out in pots under controlled environmental conditions in a growing chamber, involving two factors: i) soil and ii) treatments that affect microbial activity in the rhizosphere: two microbial inoculants (Bacillus subtilis QST713 and Trichoderma asperellum strain T34), source of labile C, ß-cellulose (CF11, Whatman), and a control without treatment. 2.2 Soils Ten soils were selected for the study in different locations in Spain. Soil samples were collected from the soil surface (at a depth of 20 cm) defining an area of around 1000 m2 in each sampling site from which at least 12 soil cores were taken. The sampling sites were selected to achieve a wide range of soil properties, such as clay, Fe oxides, and carbonate content, that can affect the availability of P and micronutrients to plants. This selection was made on the basis of color and a proxy estimate of textural classes in the upper soil horizon. The soil samples were air dried and sieved to < 4 mm for the pot experiment and to < 2 mm for soil characterization (Table 1). The availability of micronutrients and phosphorus in soils was assessed by DTPA extraction (Lindsay and Norvell 1978) and Olsen P (Olsen et al. 1954), respectively. For DTPA extraction, soil samples were mixed with extractant (0.005 M diethylenetriaminepentaacetic acid, 0.1 M tricthanolamine and 0.01 M CaCl2 at pH 7.3) at a rate of 1:4 (w:v) and shaken in a falcon tube (2.7 s–1 for 120 min). Subsequently, suspensions were centrifuged (1260 g for 15 min) and micronutrient concentration in supernatants was determined by atomic absorption spectrometry in a Soolar M device (Thermo, Madrid, Spain). The Olsen P (Olsen et al. 1954) was determined by extraction with 0.5 M NaHCO3 at pH 8.5 in a soil: extractant ratio of 1:20 (w:v) shaking at 2.7 s–1 during 30 min. After extraction, suspensions were centrifuged at 1260 g for 10 min and the concentration of P in the extract was determined colorimetrically by the molybdate blue method (Murphy and Riley 1962) in a Unicam UV2 spectrophotometer (Thermo, Madrid, Spain). Zinc bound to the organic matter fraction weakly associated to mineral matrix (Lopez-Sangil and Rovira 2013), which can be indicative of available Zn to plants (Moreno-Lora et al. 2020), was determined by extraction of sodium pyrophosphate (Znpyr) following Reed and Martens (1996): 0.25 g of soil were mixed in a ratio of 1:100 (w:v) with 0.1 M Napyrophosphate and shaken at 4 s-1 for 16 h. The suspension was then centrifuged (1260 g, 15 min) and the collected supernatant was centrifuged again at 17900 g for 15 min to decant colloids. The concentration of Zn in the extract was determined by atomic absorption spectrometry using the same device mentioned above. 99 Tables and figures Table 1. Main properties and P and Zn availability indices of studied soils Soil Sand Silt Clay SOC ACCE CCE pH EC CEC DTPA extractable Zn pyr Olsen P Fe Cu Mn Zn –––––––––––––––––– g kg–1 –––––––––––––––––– µS cm-1 cmoclc kg–1 ––––––––––––––– mg kg–1 –––––––––––––– BLG2 450 220 108 4.9 72 231 8.01 397 14.4 3.6 3 5.3 0 .41 1 26 BLG3 475 253 116 5.1 56 153 8.27 132 14.8 3.1 1.2 4.4 0.27 1 7.5 LCV1 484 102 385 7.4 28 32 8.19 163 24.5 6.6 5.4 13.1 0.28 0.8 9.3 LCV4 426 135 387 7.4 16 44 8.14 178 24.6 6.6 2.4 11.5 0.40 1 16.7 OTR1 264 184 383 3.2 71 174 8.17 116 20.0 5.9 0.3 7.8 0.1 0.4 4.7 OTR2 311 191 355 10.8 54 150 8.19 197 18.1 6 0.5 12.7 0.24 1 7.5 RCN1 767 43 162 5.5 0 0 7.38 55 12.1 13.7 12.8 10 0.31 1.1 9.4 RCN2 794 41 170 5.5 0 0 7.24 132 13.9 10.9 15.7 14.7 0.25 1.5 21.7 VGT1 787 89 127 5.8 0 0 7.97 102 8.0 10.7 0.7 15.4 0.38 1.1 22.8 VGT2 810 100 92 5.8 0 0 8.03 119 7.7 10 0.6 15.9 0.49 0.5 23.1 SOC, soil organic carbon; ACCE, active calcium carbonate equivalent; CCE, calcium carbonate equivalent; EC, electrical conduc tivity in the saturated extract ; CEC , cation exchange capacity; Zn pyr , pyrophosphate - extractable Zn. 100 2.3 Plant growing conditions The seeds were disinfected by immersion in 5% NaClO according to de Santiago et al. (2011) and pre-germinated at 8 ºC under darkness in Petri dishes before transplanting to a perlite seedbed. Later, at stage Z1.3 of Zadoks scale (Zadoks et al. 1974), individual plants were relocated into 350 ml (15 cm high × 5.5 cm diameter polystyrene cylinders) containing 300 g of soil. The cultivation was carried out in a growing chamber with a photoperiod of 16 h (300 µmol m-2s-2 of light intensity), at 25/18 °C of temperature and 40/60% relative humidity day/night. The plants were daily irrigated using a modified Hoagland nutrient solution without P and Zn, and with a limited supply of other micronutrients, containing (mmol l-1): Ca(NO3)2 (5), KNO3 (5), MgSO4 (2), KCl (0.05), H3BO3 (0.024), MnCl2 (0.0023), Fe-EDDHA (0.02), CuSO4 (0.0005) and H2MoO4 (0.0005). A total volume of 800 ml per pot was applied during cultivation that ended when the plants reached the Z9 stage of the Zadoks scale. 2.4 Application of treatments Commercial microbial inoculants were applied as described García-López et al. (2016). Bacillus subtilis (Serenade Max, Bayer CrospScience) was inoculated at a rate of 2·104 colony forming units (CFU) per gram of soil applying 20 ml of an aqueous suspension containing 3·105 CFU ml-1 over the soil surface in each pot at four different points around the plants. The product was tested before inoculation in a nutrient agar medium following the procedure of Tuitert et al. (1998). Trichoderma asperellum (Biocontrol Technologies, Barcelona, Spain) was inoculated at a rate of 104 conidia per gram of soil, as described de Santiago et al. (2009); before being transplanted, plants were immersed in a water suspension with 103 conidia ml-1 and later 20 ml of an aqueous suspension (1.5·105 conidia mL-1) were applied over the soil surface in each pot at four different points around the plants. ß-cellulose treatment was applied at a rate of 200 mg C per kg of soil split in two applications: at the beginning (Z1.3) and in the middle (Z5.1) of the crop cycle, following the same procedure as in inoculation: applying a volume of 20 ml of water suspension (3.4 g of ß-cellulose per l) on the soil surface in each pot at four different points around the plants. The application of cellulose was split to avoid losses due to irrigation and to maintain a more constant effect throughout the crop cycle. The C content was 0.44 g per g of ß-cellulose. The pots without treatments (controls) were irrigated with 20 ml of water to balance the water content in all the pots. 2.5 Harvest and analysis of plants Immediately after harvest, roots, shoots, and grains were separated and dried (65 °C, 48 h) in a forced air oven to determine the dry matter (DM) in each organ. Roots were cleaned by immersion in distilled water and by ultrasonic treatment for 1 min. Subsequently, fresh weight was determined and a portion of fresh material was kept for hormone determination and other dried as described above and 101 weighed to determine DM. The harvest index (HI) was calculated as the ratio of grain DM to total in all aerial parts. The portion of fresh roots for hormone analysis was immediately weighed and frozen in liquid nitrogen (-70 °C) after sampling. The samples were then ground (< 1 mm) using liquid nitrogen and extracted overnight in the dark with 10 ml of 80 % cold aqueous methanol (< 0 °C). The extract was centrifuged (2470 g, 15 min at 4 °C) in a 15 ml flacon tube and the supernatant was collected. The remnant was extracted following the same procedure 3 additional times. The total volume of the supernatant was dried in a rotary evaporator and dissolved in 2.5 ml of methanol. Indolacetic acid (IAA), abcisic acid (ABA) and gibberellins 3 (GA3) in roots were determined by injection of the extract into a reverse phase HPLC Varian ProStar 410 instrument furnished with a column C18 (Varian, 250 mm × 34.6 mm, 8 µm particle size) using as mobile phase methanol-0.6% ethanoic acid, with gradient elution, column temperature 35 °C, injection volume of 10 µl, and a flow rate of 1 ml min-1. Detection was performed at 254 nm according to Tang et al. (2011). For mineral nutrient analysis, 0.25 g of dried and milled (<1 mm) plant material (1 mm) were mineralized in a Mufla furnace at 550 °C for 8 h. After calcination, the ashes were dissolved in 10 ml of 1 M HCl. This solution was heated (100 °C, 15 min) to ensure full recovery of nutrients. Micronutrient concentrations (Zn, Fe, Mn, and Cu) in the digest were determined by atomic absorption spectrometry and P concentration colorimetrically (Murphy and Riley 1962). The total P and micronutrients in the plants were calculated as the sum of the product of DM in each organ and its nutrient concentration. Nutrient harvest indices were calculated as the ratio of the total nutrient accumulated in the grain to that accumulated in all aerial parts of the plant. The P-to-Zn and P-to-Fe molar ratios were calculated as the quotients of P and Zn or Fe concentrations in grain. The phosphate in grains is present mainly as phytate, which inhibits the absorption of Zn and Fe during digestion, thus decreasing the grain value for the supply of Zn and Fe in diets. Therefore, these molar ratios are commonly used as an index of potential nutritional quality for humans (Miller et al. 2007; Gómez- Coronado et al. 2019). 2.6 Soil chemical analysis Soil samples for analysis were collected at harvest from rhizospheric soil by shaking it off from the roots in air as described Wang et al. (2009). Thus, only the soil adhered to the roots was considered (Nazih et al. 2001). Immediately after sampling, the wet soil samples were sieved to < 2 mm and homogenized for chemical, biochemical, and microbial analysis. A portion of them was air-dried and used to determine pH and CE (1:2.5 in water) and further chemical analysis. The population density of B. subtilis and T. asperellum in rhizospheric soil was measured by dilution plating according to Tuitert et al. (1998). To this end, a suspension was shaken with 5 g of soil 102 and 90 ml of 0.1 % Na-pyrophosphate was shaken (2.5 s–1, 30 min) to promote the disruption of soil aggregates. Then, five-fold dilution series were prepared from an original dilution of 1 ml of vortexed suspension in 9 ml of 0.1 % water agar. From each dilution, 0.1 ml were pipetted into 3 replicate plates, containing a modified semiselective culture media for T. asperellum (Chung and Hoitink 1990) prepared according to Borrero et al. (2012). The colony-forming units (CFU) were counted 4 days after plating and expressed as CFU per gram of soil. Similarly, B. subtilis was isolated in a semiselective culture medium (400 ml of filtered V-8 juice®, 40 g of NaCl, 1 g of dextrose, 20 g of agar and 600 ml of Millipore water, adjusted to pH 5.2 before autoclaving) as described by Turner and Backman (1991). The suspensions were heated (80 °C, 10 min) in a water bath before preparing the dilution series (Tuitert et al. 1998). The CFU were counted twice; 48 h after plating to determine Bacillus spp. and a week after plating to determine and confirm the presence of B. subtilis based on colony morphogenesis as described by Aguilar et al. (2007). The concentrations of low molecular weight organic anions in the rhizosphere were determined by high performance liquid chromatography (HPLC) according to Gao et al. (2012). Soil extractions with 0.1 M NaOH were carried out in a 1:1 ratio (w:v) by shaking the mix (4 s-1, 90 min) (Radersma and Grierson 2004) as described by García-López and Delgado (2016). The suspensions were centrifuged at 1260 g for 10 min and the collected supernatants were centrifuged again at 17900 g for 10 min. The extracts were then acidified to pH 2-3 with 0.1 M H2SO4 and filtered through a 0.20 μm membrane filter before being separated on an HPLC Varian ProStar 410 instrument furnished with a column C18 (Varian, 250 mm × 34.6 mm, particle size of 8 µm particle size), using an isocratic elution with 98 % 5 mM H2SO4 at pH 2 plus 2 % methanol at 0.8 ml min−1 as carrier solution and 20 μl injection volume. Detection of organic anions was performed at 215 nm, using a Varian 486 photodiode array detector. Individual standard solutions of acetic, oxalic, citric, malic, fumaric, and succinic acid, all from Sigma (Barcelona, Spain), were used for the identification of organic anions. Only oxalate was detected in the extracts. The siderophores in the rhizosphere were determined by measuring the amount of Fe3+ complexed by them. The colorimetrical method (Schwyn and Neilands 1987) uses the affinity of siderophores for Fe3+ and a ternary complex (chrome azurol S / iron (III) / hexadecyltrimethylammonium bromide –CAS–) as an indicator, with an extinction coefficient of 100,000 M-1 cm-1 at 630 nm. The chelating complex (siderophore) moves Fe from the indicator which changes from blue to orange at pH 5.6. To extract siderophores from the rhizosphere, the same soil extraction procedure was used as for organic anions and 0.5 ml of this extract was mixed with 0.5 mL of CAS assay solution (Schwyn and Neilands 1987). After reaching equilibrium (6 h), the absorbance was measured at 630 nm and compared with a reference without soil extract. 103 Enzyme activities in the rhizospheric soil were determined at harvest. Dehydrogenase activity was determined after soil incubation (37 ° C in darkness, 24 h) in falcon tubes containing: 1 g of soil, 0.01 g of CaCO3, 0.25 ml of 3% 2,3,5-triphenyl-tetrazolium chloride (TTC) and 0.875 ml of water. The triphenyl formazan (TPF) produced was sequentially extracted (three times) by adding ethanol (up to 15 ml of total volume) and centrifuging (1260 g, 10 min) to separate it from the soil. The concentration of TPF in the supernatant was determined colorimetrically at 485 nm as described Casida et al. (1964), using a Lambda 35 spectrophotometer (Perkin Elmer, USA). The ß-glucosidase activity was determined according to Eivazi and Tabatabai (1988) by determining the p-nitrophenol produced from soil incubation (37 ° C, 1 h) with 0.05 M 4-nitrophenyl-ß-D-glucopyranoside (PNG) as an enzymatic substrate. A similar procedure was used to determine alkaline phosphatase, using 0.05 M 4-nitrophenyl phosphate as substrate and measuring the amount of p-nitrophenol (PNP) produced (Tabatabai and Bremner 1969; Eivazi and Tabatabai 1977). 2.7 Statistical analysis The effect of soil and treatments affecting microbial activity in the rhizosphere was evaluated using a two-way analysis of variance. Previously, normality according to the Shapiro-Wilks test and homocedasticity according to the Levene test were checked. Power transformations were performed if necessary to fully meet both criteria. Analysis of variance was performed with the General Linear Model procedure in Statgraphics Plus 5.1 (StatPoint 2000). When the effect of a factor was significant, the means for each factor level were compared using the LSD test (P < 0.05), except when the interaction between factors was significant. In the case of significant interactions, the effect of main factors cannot be assessed, and only the interaction can be discussed, since the effect of one factor depends on the level of the other. In this case, this means that the effect of treatments depends on soils, and it was not possible to conduct a mean comparison between treatments for each main factor (de Santiago et al. 2013). Then, two analyses were performed; first, the combined effect of both factors was evaluated with one-way analysis of variance and mean comparison for the combination of both factors in order to see differences between particular soils; second, a general linear model (GLM) involving two factors, one categorical (treatment) and another quantitative (specific soil property) was performed in order to identify the soil property with particular interaction with treatments. This was done for the main physicochemical and biochemical properties of soils. Pearson's correlation coefficients and regressions were calculated using the same software mentioned above. 3. Results 3.1 Effect of soil on plant development and nutrition The soils studied encompassed a wide range of properties, in particular, textural classes and nutrient availability indices (Table 1). The initial extractable Zn of DTPA (ZnDTPA) was below the 104 threshold value for nutrient deficiency (0.5 mg kg–1) in all the soils. The extractable Zn of pyrophosphate (Znpyr) ranged from 0.4 to 1.5 mg kg–1 (Table 1). In the case of P, the soils showed a wide range of availability status, Olsen P ranging from 4.7 to 26 mg kg–1. The pH of the soils ranged between 7.2 and 8.3. The yield and nutritional status of the crop differed widely between soils (Table S1, Supplementary material). Initial Olsen P explained 83 % of the variation in dry matter (DM) production (Y = 1.4 – 3.7 X–1; P < 0.001). The total P in the plant was not related to the initial Olsen P in the soil (not shown). The total Zn in the plant or the Zn concentration in the grain were not related to ZnDTPA or Znpyr. However, the ratio of Znpyr to Olsen P explained 48 % of the variation in total Zn in the plant (Y = 6.07 + 211 X; P < 0.001), and 63 % of the variation in Zn concentration in grains (Y = 9.4 + 308 X; P < 0.001). In general, the concentration of Zn in grains was low relative to the standards of good nutritional quality: in 5 of the soils were below 32 mg kg–1, and in 3 below 20 mg kg–1. The dry matter yield was not related to the extractable Fe or Mn. These extractions did not relate to the total nutrient content of the plants. A wide range of enzyme activities was also observed in the rhizospheric soil after the crop (Table S2, supplemental material); the phosphatase activity (phosphatase) of the soil was not related to the initial Olsen P nor other soil properties (not shown). In general, soils with the highest crop DM yield were those that showed the lowest indolacetic acid (IAA) and abcisic acid (ABA) in roots (Table S1, Supplementary material). 3.2 Effect of microbial inoculants and cellulose on crop The two microbial inoculants and the cellulose amendment (treatments) increased total root DM and grain yield compared to the control (Table 2). When the aboveground biomass was considered, only cellulose increased it significantly compared to the control (Table 2). Dry matter in nonreproductive organs in aboveground biomass was not significantly affected by inoculants or cellulose (not shown). Overall, B. subtilis and cellulose promoted lower levels of indolacetic acid (IAA), abcisic acid (ABA) and gibberellins 3 (GA3) than the control, while the differences in IAA and ABA between B. subtilis and T. asperellum were not significant (Table 2). The effect of treatments on GA3 varied depending on the soil, as revealed by the significant interaction between both factors (Table 2). Although T. asperellum increased the GA3 content compared to the other treatments (Table 2), this effect was more marked with increasing soil pH (Figure 1). The microbial inoculants and cellulose did not affect the total P and Zn concentrations in plants, the concentration of P in the different organs, or the concentration of Zn in grains compared to the control. However, a trend of decreasing Zn concentration was observed in roots and above-ground biomass with these treatments (Table 3). Treatments increased the Zn harvest index (P < 0.05) and decreased the molar ratio of P to Zn in grains relative to the control (Table 3). The effect of treatments 105 pH 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 Gibberellins 1 2 3 4 5 6 7 8 9 Bacillus Cellulose Trichoderma Control on total Fe and Mn in plants differed between soils, as revealed by the significant interaction between factors (P = 0.04 and P = 0.001, respectively; Table S3). In 8 of the soils, microbial inoculants or cellulose increased total Fe or Mn in plants relative to the control (Table S3). According to the GLM, the effect of cellulose and microbial inoculants on total Fe in the plant was affected by the pH of the soil in such a way that the effect of B. subtilis and T. asperellum increasing total Fe in plants decreased with increased pH (Figure 2a). On the other hand, the effect of these microorganisms increasing total Fe was more marked with increased microbial activity in soil estimated as the β-glucosidase activity in controls without treatment after harvest (Figure 2b). The total Cu in the plants, the concentration of Fe, Cu, and Mn in the different organs, and their harvest indices were only affected by soil (not shown). The molar ratio of Fe to P in the grain was not affected by treatments, only by soils (not shown). Figure 1. Effect of the different treatments (microorganisms and organic amendment) on gibberellins concentration (µg kg–1) as affected by soil pH according to the General Linear Model (GLM); the lines represent the trend in the effect of each treatment as a function of soil pH; there are significant differences in the regression slopes between treatments P = 0.0011. The effect of the interaction between soil and treatment was significant at P = 0.0015. 112 plants. Furthermore, these benefits were also more relevant with increasing microbial activity in soils (as estimated by the average β-glucosidase activity in nontreated soils). This reveals that more suitable conditions for microbial activity likely enhance inoculant development and consequently their effects. Microbial inoculants and cellulose increased microbial activity in the rhizosphere as revealed by the increased β-glucosidase activity. This enzyme is retained in the soil and reflects long-term fluctuations in microbial activity, likely along the crop cycle (Stott et al. 2010; Moreno et al. 2015). Phosphatase is ascribed in part to microbial activity as revealed by its correlation with β-glucosidase. The correlation between β-glucosidase and phosphatase activity and plant DM may suggest that increased plant development may promote an increased microbial activity in the rhizosphere perhaps through increased root exudation. The production of siderophores was related to microbial activity at harvest, as revealed by the positive correlation with the dehydrogenase activity. Thus, increased microbial activity may contribute to increased nutrient mobilization potential from soil. Although no improvement in Zn uptake by plants was observed with microbial inoculation or cellulose amendment, these treatments resulted in increased Zn HI. This agrees with previous evidence by Moreno-Lora et al. (2019). The effect was independent of the soil, since no interaction was observed. This may be explained by the decrease in the Zn concentration in roots and shoots promoted by inoculants and cellulose, which has no significant effects on grain concentration. Thus, this confirms our hypothesis of an alteration of the Zn partitioning with an increased accumulation of Zn in grains relative to other organs. This change in the pattern of accumulation of nutrients within plants may be attributed to observed changes in auxin or ABA concentration, since these hormones can regulate transporters involved in metal homeostasis in plants (David-Assael et al. 2006; Barickman et al. 2019). Increased Zn HI with inoculants and cellulose compared to control led to a decreased molar P-to-Zn ratio in the grain with these treatments and consequently to an improved grain quality, as Zn digestibility is increased. 5 Conclusions Microorganisms and cellulose promoted an increase in DM and grain yield in wheat, and this effect was independent of soil. In general, neither microorganisms nor cellulose increased total P and Zn in plants. However, the Zn HI and the P to Zn molar ratio were enhanced independently of the soil. Therefore, our results provide evidence of the benefits of using microorganisms and organic amendments as a tool to improve the yield, the portion of absorbed Zn accumulated in grains, and the grain quality in wheat grown in a set of soils that vary widely in their properties. The effect on Fe nutrition and gibberellins varied depending on the soil. These effects were affected by soil pH, while the microbial activity of the soils seemed relevant explaining the effect of the inoculants on total Fe in the plant. 113 Acknowledgements This work was funded by the Spanish Ministry of Economy and Competitiveness of Spain and the European Regional Development Fund of the European Union through the National Research, Development and Innovation Programme (Plan Estatal I + d + i, Project AGL2014-57835-C2-1-R). The authors wish to thank Agricultural Research Service of the University of Sevilla for the support in the performance of some analysis, and in particular to the technicians Purificación Pajuelo and Oliva Polvillo. References Aguilar C, Vlamakis H, Losick R, Kolter R (2007) Thinking about Bacillus subtilis as a multicellular organism. Curr Opin Microbiol 10:638–643. https://doi.org/10.1016/j.mib.2007.09.006 Alloway BJ (2009) Soil factors associated with zinc deficiency in crops and humans. Environ Geochem Health 31:537–548. https://doi.org/10.1007/s10653-009-9255-4 Arkhipova TN, Veselov SU, Melentiev AI, Martynenko E V., Kudoyarova GR (2005) Ability of bacterium Bacillus subtilis to produce cytokinins and to influence the growth and endogenous hormone content of lettuce plants. Plant Soil 272:201–209. https://doi.org/10.1007/s11104-004-5047-x Belimov AA, Dodd IC, Safronova VI, Dumova VA, Shaposhnikov AI, Ladatko AG, Davies WJ (2014) Abscisic acid metabolizing rhizobacteria decrease ABA concentrations in planta and alter plant growth. Plant Physiol Biochem 74:84–91. https://doi.org/10.1016/j.plaphy.2013.10.032 Borrero C, Trillas MI, Delgado A, Avilés M (2012) Effect of ammonium/nitrate ratio in nutrient solution on control of Fusarium wilt of tomato by Trichoderma asperellum T34. Plant Pathol 61:132– 139. https://doi.org/10.1111/j.1365-3059.2011.02490.x Borrill P, Connorton JM, Balk J, Miller AJ, Sanders D, Uauy C (2014) Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops. Front Plant Sci 5:53. https://doi.org/10.3389/fpls.2014.00053 Cakmak I, Kutman UB (2018) Agronomic biofortification of cereals with zinc: a review. Eur J Soil Sci 69:172–180. https://doi.org/10.1111/ejss.12437 Cakmak I, Pfeiffer WH, McClafferty B (2010) Biofortification of durum wheat with zinc and iron. Cereal Chem J 87:10–20. https://doi.org/10.1094/CCHEM-87-1-0010 Barickman TC, Kopsell DA, Sams CE (2019) Applications of abscisic acid and increasing concentrations of calcium affect the partitioning of mineral nutrients between tomato leaf and fruit 114 tissue. Horticulturae 5:49. https://doi.org/10.3390/horticulturae5030049 Casida LE, Klein DA, Santoro T (1964) Soil dehydrogenase activity. Soil Sci. 98:371–376 Chung YR, Hoitink HAJ (1990) Interactions Between thermophilic fungi and Trichoderma hamatum in suppression of rhizoctonia damping-off in a bark compost-amended container medium. Phytopathology 80:73–77. https://doi.org/10.1094/Phyto-80-73 David-Assael O, Berezin I, Shoshani-Knaani N, Saul H, Mizrachy-Dagri T, Chen J, Brook E, Shaul O (2006) AtMHX is an auxin and ABA-regulated transporter whose expression pattern suggests a role in metal homeostasis in tissues with photosynthetic potential. Funct Plant Biol 33:661–672 de los Santos-Villalobos S, Parra-Cota FI (2020) Current Trends in Plant Growth-Promoting Microorganisms Research for Sustainable Food Security. Curr Res Microb Sci 100016. https://doi.org/10.1016/j.crmicr.2020.100016 de Santiago A, García-López AM, Quintero JM, Avilés M, Delgado A (2013) Effect of Trichoderma asperellum strain T34 and glucose addition on iron nutrition in cucumber grown on calcareous soils. Soil Biol Biochem 57:598–605. https://doi.org/10.1016/j.soilbio.2012.06.020 de Santiago A, Quintero JM, Avilés M, Delgado A (2009) Effect of Trichoderma asperellum strain T34 on iron nutrition in white lupin. Soil Biol Biochem 41:2453–2459. https://doi.org/10.1016/j.soilbio.2009.07.033 de Santiago A, Quintero JM, Avilés M, Delgado A (2011) Effect of Trichoderma asperellum strain T34 on iron, copper, manganese, and zinc uptake by wheat grown on a calcareous medium. Plant Soil 342:97–104. https://doi.org/10.1007/s11104-010-0670-1 Demoling F, Figueroa D, Bååth E (2007) Comparison of factors limiting bacterial growth in different soils. Soil Biol Biochem 39:2485–2495. https://doi.org/10.1016/j.soilbio.2007.05.002 Dodd IC, Egea G, Watts CW, Whalley WR (2010) Root water potential integrates discrete soil physical properties to influence ABA signalling during partial rootzone drying. J Exp Bot 61:3543–3551. https://doi.org/10.1093/jxb/erq195 Eivazi F, Tabatabai MA (1988) Glucosidases and galactosidases in soils. Soil Biol Biochem 20:601– 606. https://doi.org/10.1016/0038-0717(88)90141-1 Eivazi F, Tabatabai MA (1977) Phosphatases in soils. Soil Biol Biochem 9:167–172. https://doi.org/10.1016/0038-0717(77)90070-0 115 Gao Z, Shao J, Sun H, Zhong W, Zhuang W, Zhang Z (2012) Evaluation of different kinds of organic acids and their antibacterial activity in Japanese Apricot fruits. African J Agric Res 7:4911–4918. https://doi.org/10.5897/AJAR12.1347 García-López AM, Avilés M, Delgado A (2016) Effect of various microorganisms on phosphorus uptake from insoluble Ca-phosphates by cucumber plants. J Plant Nutr Soil Sci 179:454–465. https://doi.org/10.1002/jpln.201500024 García-López AM, Delgado A (2016) Effect of Bacillus subtilis on phosphorus uptake by cucumber as affected by iron oxides and the solubility of the phosphorus source. Agric Food Sci 25:216–224 García-López AM, Recena R, Avilés M, Delgado A (2018) Effect of Bacillus subtilis QST713 and Trichoderma asperellum T34 on P uptake by wheat and how it is modulated by soil properties. J Soils Sediments 18:727–738. https://doi.org/10.1007/s11368-017-1829-7 García-López AM, Recena R, Delgado A (2021) The adsorbent capacity of growing media does not constrain myo-inositol hexakiphosphate hydrolysis but its use as a phosphorus source by plants. Plant Soil. 459:277-288. https://doi.org/10.1007/s11104-020-04764-1 Gómez-Coronado F, Almeida AS, Santamaría O, Cakmak I, Poblaciones MJ (2019) Potential of advanced breeding lines of bread-making wheat to accumulate grain minerals (Ca, Fe, Mg and Zn) and low phytates under Mediterranean conditions. J Agron Crop Sci 205:341–352. https://doi.org/10.1111/jac.12325 Gouda S, Kerry RG, Das G, Paramithiotis S, Shin HS, Patra JK (2018) Revitalization of plant growth promoting rhizobacteria for sustainable development in agriculture. Microbiol Res 206:131–140. https://doi.org/10.1016/j.micres.2017.08.016 Hedden P (2016) Gibberellin biosynthesis in higher plants. Annu Plant Rev Gibberellins 49:37–72. https://doi.org/10.1002/9781119210436.CH2 Hobbie JE, Hobbie EA (2013) Microbes in nature are limited by carbon and energy: The starvingsurvival lifestyle in soil and consequences for estimating microbial rates. Front. Microbiol. 4:324 Khan KS, Joergensen RG (2010) Effects of Zn and P addition on the microbial biomass in a Zn deficient calcareous soil amended with glucose. Plant Soil 335:493–499. https://doi.org/10.1007/s11104-010- 0442y Khande R, Sharma SK, Ramesh A, Sharma MP (2017) Zinc solubilizing Bacillus strains that modulate growth, yield and zinc biofortification of soybean and wheat. Rhizosphere 4:126–138. 116 https://doi.org/10.1016/j.rhisph.2017.09.002 Kudoyarova G, Arkhipova T, Korshunova T, Bakaeva M, Loginov O, Dodd IC (2019) Phytohormone mediation of interactions between plants and non-symbiotic growth promoting bacteria under edaphic stresses. Front Plant Sci 10:1–11. https://doi.org/10.3389/fpls.2019.01368 Kuzyakov Y (2002) Review: Factors affecting rhizosphere priming effects. J Plant Nutr Soil Sci 165:382–396. https://doi.org/10.1002/1522-2624(200208)165:4<382::AID-JPLN382>3.0.CO;2-# Lei GJ, Zhu XF, Wang ZW, Dong F, Dong NY, Zheng SJ (2014) Abscisic acid alleviates iron deficiency by promoting root iron reutilization and transport from root to shoot in Arabidopsis. Plant Cell Environ 37:852–863. https://doi.org/10.1111/PCE.12203 Lindsay WL, Norvell WA (1978) Development of a DTPA Soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J 42:421–428. https://doi.org/10.2136/sssaj1978.03615995004200030009x Lopez-Sangil L, Rovira P (2013) Sequential chemical extractions of the mineral-associated soil organic matter: An integrated approach for the fractionation of organo-mineral complexes. Soil Biol Biochem 62:57–67. https://doi.org/10.1016/j.soilbio.2013.03.004 Ma W, Peng D, Walker SL, Cao B, Gao CH, Huang Q, Cai P (2017) Bacillus subtilis biofilm development in the presence of soil clay minerals and iron oxides. npj Biofilms Microbiomes 3:4. https://doi.org/10.1038/s41522-017-0013-6 Marschner P, Crowley D, Rengel Z (2011) Rhizosphere interactions between microorganisms and plants govern iron and phosphorus acquisition along the root axis - model and research methods. Soil Biol Biochem 43:883–894. https://doi.org/10.1016/j.soilbio.2011.01.005 McBeath TM, McLaughlin MJ (2014) Efficacy of zinc oxides as fertilisers. Plant Soil 374:843–855. https://doi.org/10.1007/s11104-013-1919-2 Miller L V., Krebs NF, Hambidge KM (2007) A mathematical model of zinc absorption in humans as a function of dietary zinc and phytate. J Nutr 137:135–141. https://doi.org/10.1093/jn/137.1.135 Moreno-Lora A, Delgado A (2020) Factors determining Zn availability and uptake by plants in soils developed under Mediterranean climate. Geoderma 376:114509. https://doi.org/10.1016/j.geoderma.2020.114509 Moreno-Lora A, Recena R, Delgado A (2019) Bacillus subtilis QST713 and cellulose amendment enhance phosphorus uptake while improving zinc biofortification in wheat. Appl Soil Ecol 142:81–89. https://doi.org/10.1016/j.apsoil.2019.04.013 117 Moreno MT, Carmona E, Santiago A, Ordovás J, Delgado A (2015) Olive husk compost improves the quality of intensively cultivated agricultural soils. L Degrad Dev 27:449–459. https://doi.org/10.1002/ldr.2410 Mousavi SR (2011) Zinc in Crop Production and Interaction with Phosphorus. Aust J Basic Appl Sci 5:1503–1509 Mumtaz MZ, Barry KM, Baker AL, Nichols DS, Ahmad M, Zahir ZA, Britz ML (2019) Production of lactic and acetic acids by Bacillus sp. ZM20 and Bacillus cereus following exposure to zinc oxide: A possible mechanism for Zn solubilization. Rhizosphere 12:100170. https://doi.org/10.1016/j.rhisph.2019.100170 Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36. https://doi.org/10.1016/S0003-2670(00)88444-5 Nazih N, Finlay-Moore O, Hartel PG, Fuhrmann JJ (2001) Whole soil fatty acid methyl ester (FAME) profiles of early soybean rhizosphere as affected by temperature and matric water potential. Soil Biol Biochem 33:693–696. https://doi.org/10.1016/S0038-0717(00)00197-8 Nguyen ML, Glaes J, Spaepen S, Bodson B, du Jardin P, Delaplace P (2019) Biostimulant effects of Bacillus strains on wheat from in vitro towards field conditions are modulated by nitrogen supply. J Plant Nutr Soil Sci 182:325–334. https://doi.org/10.1002/jpln.201700610 Olsen SR, Cole CV, Watanabe FS, Dean LA. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. Washingt United States Dep Agric USDA 939:1–19. Owen D, Williams AP, Griffith GW, Withers PJA (2015) Use of commercial bio-inoculants to increase agricultural production through improved phosphrous acquisition. Appl Soil Ecol 86:41–54. https://doi.org/10.1016/j.apsoil.2014.09.012 Qiu Z, Egidi E, Liu H, Kaur S, Singh BK (2019) New frontiers in agriculture productivity: Optimised microbial inoculants and in situ microbiome engineering. Biotechnol Adv 37:107371. https://doi.org/10.1016/j.biotechadv.2019.03.010 Ramesh A, Sharma SK, Sharma MP, Yadav N, Joshi OP (2014) Inoculation of zinc solubilizing Bacillus aryabhattai strains for improved growth, mobilization and biofortification of zinc in soybean and wheat cultivated in Vertisols of central India. Appl Soil Ecol 73:87–96. https://doi.org/10.1016/j.apsoil.2013.08.009 Recena R, García-López AM, Delgado A (2021) Zinc uptake by plants as affected by fertilization with 118 zn sulfate, phosphorus availability, and soil properties. Agron 11:390. https://doi.org/10.3390/AGRONOMY11020390 Reed ST, Martens DC (1996) Copper and zinc. In: Sparks DL, Page PA, Helmke RH, Loeppert P, Soltanpour PN, Tabatabai MA, Jonhston CT, Sumner ME (eds)Methods of Soil Analysis, Part 3: Chemical Methods. John Wiley & Sons, Ltd, pp 703–722 Rengel Z (2015) Availability of Mn, Zn and Fe in the rhizosphere. J Soil Sci Plant Nutr 15:397–409. https://doi.org/10.4067/S0718-95162015005000036 Ryan J, Rashid A, Torrent J, Yau SK, Ibrikci H, Sommer R, Erenoglu EB (2013) Micronutrient constraints to crop production in the middle east-west Asia region: Significance, research, and management, 1st edn. Elsevier Inc. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56. https://doi.org/10.1016/0003-2697(87)90612-9 Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springerplus 2:587. https://doi.org/10.1186/2193-1801-2-587 Shi Y, Lalande R, Hamel C, Ziadi N, Gagnon B, Hu Z (2013) Seasonal variation of microbial biomass, activity, and community structure in soil under different tillage and phosphorus management practices. Biol Fertil Soils 49:803–818. https://doi.org/10.1007/s00374-013-0773-y Singh D, Rajawat MVS, Kaushik R, Prasanna R, Saxena AK (2017) Beneficial role of endophytes in biofortification of Zn in wheat genotypes varying in nutrient use efficiency grown in soils sufficient and deficient in Zn. Plant Soil 416:107–116. https://doi.org/10.1007/s11104-017-3189-x Spaepen S (2015) Plant Hormones Produced by Microbes. In: Lugtenberg B (ed) Principles of Plant- Microbe Interactions: Microbes for Sustainable Agriculture. Springer International Publishing, pp 247– 256 Stott DE, Andrews SS, Liebig MA, Wienhold BJ, Karlen DL (2010) Evaluation of β-Glucosidase Activity as a Soil Quality Indicator for the Soil Management Assessment Framework. Soil Sci Soc Am J 74:107–119. https://doi.org/10.2136/sssaj2009.0029 Sukumar P, Legué V, Vayssières A, Martin F, Tuskan GA, Kalluri UC (2013) Involvement of auxin pathways in modulating root architecture during beneficial plant-microorganism interactions. Plant, Cell Environ 36:909–919. https://doi.org/10.1111/pce.12036 119 Tabatabai MA, Bremner JM (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol Biochem 1:301–307. https://doi.org/10.1016/0038-0717(69)90012-1 Tang Y, Wang L, Ma C, Liu J, Liu B, Li H (2011) The use of HPLC in determination of endogenous hormones in anthers of Bitter melon. J Life Sci 5:139–142 Tuitert G, Szczech M, Bollen GJ (1998) Suppression of Rhizoctonia solani in potting mixtures amended with compost made from organic household waste. Phytopathology 88:764–773. https://doi.org/10.1094/PHYTO.1998.88.8.764 Turner JT, Backman PA (1991) Factors relating to peanut yield increases after seed treatment with Bacillus subtilis. Plant Dis. 75:347–353 Vysotskaya LB, Korobova A V., Kudoyarova GR (2008) Abscisic acid accumulation in the roots of nutrient-limited plants: Its impact on the differential growth of roots and shoots. J Plant Physiol 165:1274–1279. https://doi.org/10.1016/j.jplph.2007.08.014 Wang G, Xu Y, Jin J, Liu J, Zhang Q, Liu X (2009) Effect of soil type and soybean genotype on fungal community in soybean rhizosphere during reproductive growth stages. Plant Soil 317:135–144. https://doi.org/10.1007/s11104-008-9794-y Wang Q, Kong X-P, Zhang B-H, Wang J (2017) Adsorption of Zn (II) on the kaolinite surfaces in aqueous environment: A combined DFT and molecular dynamics study. Appl Surf Sci 414:405–412. https://doi.org/10.1016/J.APSUSC.2017.04.062 Wang Y, Li J, Gao X, Li X, Ren T, Cong R, Lu J (2014) Winter oilseed rape productivity and nutritional quality responses to zinc fertilization. Agron J 106:1349–1357. https://doi.org/10.2134/agronj14.0029 White PJ, Broadley MR (2009) Biofortification of crops with seven mineral elements often lacking in human diets – iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytol 182:49–84. https://doi.org/10.1111/j.1469-8137.2008.02738.x Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Res 14:415–421. https://doi.org/10.1111/j.1365-3180.1974.tb01084.x Zhang YQ, Sun YX, Ye YL, Karim MR, Xue YF, Yan P, Meng QF, Cui ZL, Cakmak I, Zhang FS, Zou CQ (2012) Zinc biofortification of wheat through fertilizer applications in different locations of China. F Crop Res 125:1–7. https://doi.org/10.1016/j.fcr.2011.08.003 Zhao A qing, Tian X hong, Cao Y xian, Lu X chun, Liu T (2014) Comparison of soil and foliar zinc application for enhancing grain zinc content of wheat when grown on potentially zinc-deficient 120 calcareous soils. J Sci Food Agric 94:2016–2022. https://doi.org/10.1002/jsfa.6518 Zhao Q, Shen Q, Ran W, Xiao T, Xu D, Xu Y (2011) Inoculation of soil by Bacillus subtilis Y-IVI improves plant growth and colonization of the rhizosphere and interior tissues of muskmelon (Cucumis melo L.). Biol Fertil Soils 47:507–514. https://doi.org/10.1007/s00374-011-0558-0 121 Table S1. Effect of soil on yield and nutritional traits of crop Factor Dry matter yield HI Hormones Zn HI P HI Zn in plant P in plant Zn concentration P concentration Molar ratio Total Grain Root Shoot IAA ABA GA3 Grain Root Shoot Grain Root Shoot P to Zn Soil –––––––– g pot-1 –––––––– ––––– µg kg-1––––– ––––– µg plant-1 –––– ––––––––––––––mg kg-1––––––––––––––– BLG2 1.31 0.54 0.11 1.21 0.44 0.39 0.11 2.93 0.61 0.90 24.9 782.3 28.2 28.2 5.3 1337 484 230 47.8 BLG3 1.05 0.44 0.08 0.96 0.46 0.48 0.13 4.14 0.51 0.87 39.2 726.3 47.9 31.0 16.9 1707 534 302 33.9 LCV1 0.88 0.29 0.08 0.80 0.37 0.50 0.14 4.29 0.60 0.86 15.3 798.9 31.7 24.2 5.2 2585 530 425 82.0 LCV4 1.09 0.37 0.09 1.0 0.37 0.43 0.13 4.30 0.59 0.87 11.3 596.8 18.3 18.9 2.9 1557 393 241 82.2 OTR1 0.60 0.22 0.05 0.55 0.41 0.49 0.77 16.3 963.0 36.0 28.9 13.0 3489 834 853 96.2 OTR2 1.00 0.42 0.08 0.92 0.46 0.61 0.82 32.7 960.2 48.8 25.1 12.2 2162 658 566 43.7 RCN1 1.05 0.38 0.14 0.92 0.42 0.34 0.10 3.38 0.54 0.88 37.9 741.2 53.2 23.2 15.7 1790 456 254 33.5 RCN2 1.41 0.51 0.22 1.19 0.42 0.22 0.06 3.01 0.59 0.83 32.2 1239.1 39.0 22.4 7.0 2149 831 574 53.3 VGT1 1.19 0.39 0.15 1.04 0.37 0.33 0.10 3.56 0.50 0.82 14.0 954.4 18.7 23.7 3.3 2307 822 529 121.7 VGT2 1.29 0.37 0.17 1.11 0.33 0.25 0.08 3.31 0.52 0.83 10.6 938.7 16.2 12.9 2.7 2237 686 409 142.7 LSD value 0.089 0.065 0.027 0.077 0.052 0.084 0.024 0.906 0.052 0.04 3.19 130 5.17 4.17 1.7 242 54 83 12.6 HI, harvest index; IAA, indol - acetic acid; ABA, abcisic acid; GA3, giberelic acid; Zn HI and P HI, proportion of total nutrient in plants that is accumulated in grains. Shoot DM includes grain DM. In the case of potential transformation to meet ANOVA requirements, back transformed results are shown. Cells without data correspond to soils where the amount of root DM did not allowed us to determine plant hormones. Supplemental material 128 subtilis aumentó la absorción de nitrógeno, fósforo y manganeso en relación con otros tratamientos en el experimento realizado durante la primera campaña. El aumento de la absorción de fósforo se atribuyó al aumento de la actividad de la fosfatasa alcalina. En esta primera campaña, T. asperellum incrementó la actividad de la fosfatasa ácida sin efecto sobre la absorción de fósforo, y disminuyó la relación molar entre fósforo y zinc en el grano, mejorando así su calidad nutricional. En la segunda campaña, los efectos de los inoculantes microbianos fueron menos evidentes posiblemente debido a las condiciones ambientales favorables para el cultivo (mayor disponibilidad de agua) o la fertilización con fosfato. Los resultados demostraron que B. subtilis puede ser eficaz para mejorar el desarrollo y la absorción de nutrientes del trigo duro en condiciones adversas, como estrés por sequía. 129 130 131 132 133 134 135 136 137 144 145 Table S1. Fertilizers applied Year 1 Year 2 Wheat stage N N P Field –––––– kg ha-1 –––––– Tomejil Pre-plant 23 23 Topdressing Z1.1-Z1.9 52 52 Z2.0-Z2.9 69 69 Total 144 144 Espejo Pre-plant 23 32 80 Topdressing Z1.1-Z1.9 78 101 Z2.0-Z2.9 55 69 Total 133 202 Wheat stage, phenological stage of wheat according to Zadok's scale; N, nitrogen applied as urea; P, phosphorus applied as P2O5 146 Table S2. Nutrient concentrations in plant tissues (year 1) Plant Straw Grain P Zn Cu Mn Fe P Zn Cu Mn Fe Cu Mn Fe Treatment g kg-1 –––––––––––––––– mg kg-1 –––––––––––––––– g kg-1 ––––––––––––––––––––––––––––––– mg kg-1 ––––––––––––––––––––––––––––––– B. subtilis 1.44±0.03 10.5±0.19 3.52±0.06 18.11±0.91 44.5±4.34 0.32±0.04 3.61±0.3 2.58±0.15 22.07±1.83 86.67±9.24 5.57±0.09 19.48±0.64 13.33±1.02 Control 1.42±0.03 10.38±0.5 3.49±0.15 18.5±1.02 55.24±10.77 0.38±0.05 4.25±0.71 2.62±0.14 23.96±1.71 111.44±23.25 5.63±0.2 18.69±0.86 12.28±0.79 T. asperellum 1.39±0.04 10.36±0.42 3.63±0.17 17.28±0.79 51.57±7.42 0.3±0.02 3.34±0.39 2.86±0.18 20.9±1.51 104.59±17.3 5.59±0.24 18.95±0.77 12.79±1.28 LSD Field Espejo 1.36±0.03 10.32±0.31 3.48±0.08 19.45±0.73 40.25±4.41 0.26±0.01 4.04±0.24 2.49±0.06 25.29±1.22 78.51±9.23 5.66±0.15 19.22±0.63 10.5±0.48 Tomejil 1.47±0.02 10.51±0.29 3.6±0.13 16.56±0.42 59.37±6.29 0.4±0.03 3.41±0.47 2.87±0.15 19.44±0.93 120.55±14.36 5.55±0.14 18.9±0.59 14.95±0.56 ANOVA Treatment NS NS NS NS NS NS NS NS NS NS NS NS 0.0943 Field 0.029 NS NS 0.0785 0.0373 0.0133 NS 0.0366 0.032 0.0381 NS NS 0.0032 Treatment*Field NS NS NS NS NS NS NS NS NS NS NS NS NS LSD, least significant difference Table S3. Nutrient concentrations in plant tissues (year 2) Plant Straw Grain P Zn Cu Mn Fe P Zn Cu Mn Fe Cu Mn Fe Field Treatment g kg - 1 ––––––––––––––– mg kg - 1 ––––––––––––––– g kg - 1 –––––––––––––––––––––––––––––––– mg kg - 1 –––––––––––––––––––––––––––––––– Tomejil B. subtilis 1.69 ± 0.06 10.64 ± 0.45 3.34 ± 0.14 10.43 ± 0.37 54.09 ± 2.14 0.38 ± 0.06 2.15 ± 0.69 2.39 ± 0.37 9.63 ± 0.52 109.47 ± 5.36 5.57 ± 0.07 15.13 ± 0.52 14.69 ± 0.67 Control 1.68 ± 0.07 10.42 ± 0.56 3.33 ± 0.05 11.52 ± 0.79 68.87 ± 4.02 0.33 ± 0.04 1.37 ± 0.15 2.46 ± 0.04 11.46 ± 0.38 144.77 ± 8.92 5.45 ± 0.15 15.82 ± 1.61 15.76 ± 0.39 T. asperellum 1.61 ± 0.05 9.92 ± 0.54 3.28 ± 0.15 10.96 ± 0.31 59.97 ± 6.15 0.27 ± 0.03 1.18 ± 0.42 2.13 ± 0.36 10.19 ± 0.36 125.24 ± 13.54 5.6 ± 0.1 15.68 ± 0.57 15.39 ± 0.34 LSD 1.36 ANOVA NS NS NS NS 0.0687 NS NS NS 0.0365 0.0564 NS NS NS Espejo B. subtilis 0.97 ± 0.06 12.3 ± 0.43 2.84 ± 0.03 12.84 ± 0.54 46.64 ± 3.68 0.16 ± 0.02 3.28 ± 0.51 1.84 ± 0.12 9.74 ± 0.61 102.18 ± 8.82 4.93 ± 0.14 20.88 ± 1.31 14.77 ± 0.21 Control 0.96 ± 0.1 12.25 ± 0.47 3.03 ± 0.12 12.26 ± 0.84 37.5 ± 4.13 0.15 ± 0.04 3.3 ± 0.25 2.52 ± 0.14 9.6 ± 0.36 78.32 ± 9.23 4.7 ± 0.07 19.62 ± 1.65 13.38 ± 0.68 T. asperellum 1.04 ± 0.01 12.96 ± 0.25 3 ± 0.15 13.35 ± 0.1 41.32 ± 3.94 0.16 ± 0.04 2.81 ± 0.45 2.13 ± 0.12 9.68 ± 0.41 90.69 ± 9.48 4.84 ± 0.3 21.42 ± 0.31 13.95 ± 0.4 LSD 0.4 ANOVA NS NS NS NS NS NS NS 0.0119 NS NS NS NS NS LSD, least significant difference 147 Capítulo VII. Discusión general Chapter VII. General discussion 148 149 Capítulo VII. Discusión general 1. Factores de suelo que determinan la disponibilidad de zinc para las plantas La relación entre las propiedades del suelo y el Zn extraído por el cultivo está determinada, en gran medida, por el contenido de carbonatos del suelo. Los resultados descritos en el Cap. II mostraron que el test de disponibilidad de Zn comúnmente utilizado para suelos neutros y calcáreos, el DTPA (Lindsay & Norvell, 1978), no se relacionó con la absorción de Zn de la planta, lo que evidencia su escaso valor predictivo. Por su parte, el Zn extraíble por pirofosfato (Znpyro), asociado a coloides y a la fracción orgánica del suelo (Lopez-Sangil & Rovira, 2013), muestra mejor relación con el Zn absorbido por la planta que el Zn extraíble por DTPA. La correlación entre el Znpyro y el Zn absorbido por la planta es más significativa si se estudia por separado para suelos calcáreos y no calcáreos, y especialmente en el caso de los calcáreos. Esto demuestra que el Zn asociado a la materia orgánica juega un papel clave en la fitodisponibilidad de este nutriente, ya que es liberado a la solución del suelo con mayor facilidad que el Zn unido a los carbonatos (Rashid & Ryan, 2004). Los resultados obtenidos evidenciaron la importancia de discriminar los suelos en base a su contenido de carbonatos para estimar la disponibilidad de Zn para el cultivo. En base a lo anterior, el Zn extraíble por el cultivo en suelos calcáreos puede estimarse a partir de la concentración de Zn asociado a materia orgánica. Además, la correlación entre el Znpyro y el Zn extraído por las plantas puede mejorarse si se tiene en cuenta la disponibilidad de P en el suelo, estimada como P Olsen (Olsen et al., 1954), y el contenido de arcillas. Una mayor disponibilidad de P en relación a esta fracción de Zn (menores relaciones Znpyro/POlsen) hace que disminuya su absorción, lo que puede atribuirse a un efecto antagónico entre ambos nutrientes (Mousavi, 2011). Por otro lado, los minerales de arcilla son una de las principales fuentes de adsorción del Zn en el suelo (González-Costa et al., 2017), en procesos mayormente irreversibles a pH básico (Zhao & Selim, 2010), y esto explica que las plantas absorban menos Zn en los suelos calcáreos con mayor contenido de arcilla. Los resultados descritos en el Cap. II muestran la relevancia de la mineralogía de los óxidos de Fe en la disponibilidad de Zn para las plantas, especialmente en los suelos no calcáreos. Por un lado, los óxidos de Fe cristalinos se relacionaron de forma negativa con la absorción de Zn por las plantas. A pH neutro y alcalino cabe suponer que la adsorción de Zn en éstos óxidos es irreversible (Buekers et al., 2007), lo que reduce su disponibilidad. Sin embargo, esto puede verse afectado por los ácidos orgánicos liberados por las raíces de las plantas y los microorganismos bajo condiciones de deficiencia de nutrientes. Así, se observó una relación positiva entre la concentración de oxalato en la rizosfera y la absorción de Zn por las plantas. Esto podría explicarse porque este anión, además de competir por los sitios de adsorción en el suelo, puede acidificar el medio y disolver los óxidos de Fe poco cristalinos (de Santiago & Delgado, 2006), liberando el Zn retenido en éstos óxidos y en otras superficies de 150 adsorción. Cabe suponer que el oxalato sea, en parte, producto de la actividad microbiana por la correlación positiva entre su concentración y la actividad enzimática β-glucosidasa y deshidrogenasa del suelo. Esta correlación no se observó en los suelos calcáreos, probablemente por la precipitación del oxalato con el calcio. 2. Efecto de la materia orgánica sobre la disponibilidad de zinc y su interacción con el fósforo La adición de sustancias húmicas (SH) no mejoró la absorción de Zn de las plantas y la concentración de Zn disponible (Zn DTPA) al final del ciclo de cultivo fue menor en el medio donde fueron aplicadas. Esto podría explicarse por los complejos formados entre las sustancias húmicas y el Zn, que reducen la concentración de Zn en la solución del suelo y, por tanto, su disponibilidad para la planta (Güngör & Bekbölet, 2010). La relación entre el Zn disponible (extraíble por DTPA) tras el cultivo y el Zn absorbido por la planta fue diferente según el medio de cultivo (silíceo o calcáreo). Este resultado volvió a evidenciar la relevancia de los carbonatos en la disponibilidad de Zn para el cultivo. Las SH empeoraron la absorción de Zn respecto al testigo en el medio silíceo, mientras que no hubo diferencias en el calcáreo. En éste último la producción de biomasa y el Zn absorbido por la planta fue menor que en medio silíceo, probablemente debido a que el Zn tiene una fuerte afinidad por los carbonatos y puede precipitar como ZnCO3, reduciendo su disponibilidad. Esto explica que la concentración de oxalato fuera mayor en el medio calcáreo (más del doble respecto al silíceo), ya que su producción se atribuye a un mecanismo de la planta para movilizar nutrientes bajo condiciones de baja disponibilidad. Las SH, aplicadas a una dosis elevada (0,5 g C kg-1), limitaron la producción total de biomasa (casi un 20% respecto al testigo). Estos resultados coinciden con los obtenidos por de Santiago et al. (2010), que mostraron unos efectos adversos parecidos en plantas de altramuz con concentraciones superiores a 0,1 g C kg-1, y lo relacionaron con el elevado índice de aromaticidad de las SH y la presencia de compuestos fitotóxicos. En lo que respecta a la nutrición de P, el efecto de las SH fue diferente dependiendo del medio y de su concentración en Zn. En general, la absorción de P fue mayor en el medio silíceo respecto al calcáreo, al igual que la de Zn. En el calcáreo se mejoró la absorción de P con la dosis más baja de SH (0,05 g C kg-1) y a concentraciones moderadas de Zn (1 mg kg-1 y 4 mg kg-1). Esto revela un efecto positivo derivado la adición de SH sobre la absorción de nutrientes en medios calcáreos, coincidiendo con los resultados de Çelik et al. (2010). No obstante, a una mayor concentración de Zn (10 mg Zn kg- 1), las SH disminuyeron la absorción de P, acentuando el antagonismo entre estos nutrientes. Este mismo efecto se observó en el silíceo, mientras que a menor concentración de Zn (4 mg kg-1) no hubo diferencias significativas. 151 3. Efecto de la actividad microbiana de la rizosfera sobre la nutrición de las plantas De forma general, en los suelos calcáreos donde la disponibilidad de P y Zn suele ser más limitada, la actividad enzimática es mayor que en los no calcáreos. Los resultados obtenidos indican que la actividad enzimática asociada a microorganismos se relaciona con un mayor índice de cosecha de Zn (Zn HI) y una reducción del ratio molar P-Zn en el grano, lo que se traduce en una mejora de la calidad de la cosecha (Bohn et al., 2008; Gómez-Coronado et al., 2019; Miller et al., 2007). Esto se explica porque la actividad enzimática no se relacionó con la concentración de Zn en el grano, pero se correlacionó negativamente con la absorción de P y Zn y la concentración de P en el grano. Este resultado revela un patrón alterado de acumulación de Zn en los órganos de la planta relacionado con la actividad microbiana en el medio de cultivo. Con ello, se intuye el efecto de la actividad microbiana sobre el transporte de Zn dentro de la planta. 4. Efecto de la inoculación con microorganismos y la aplicación de una fuente lábil de carbono sobre el desarrollo y la nutrición de las plantas Los resultados obtenidos con la aplicación de una fuente lábil de carbono (β-celulosa) y la inoculación con microorganismos (Bacillus subtilis QST713 y Trichoderma asperellum strain T34) evidencian los beneficios que la actividad de los microorganismos de la rizosfera promueve en la nutrición y el desarrollo de las plantas. En general, los tratamientos mejoraron la nutrición de P (Cap. VI) e incrementaron la producción del trigo (Cap. V). Esto concuerda con la literatura publicada, en la que se le atribuyen efectos positivos a los inoculantes microbianos estudiados sobre la nutrición de P y sobre la promoción del crecimiento. No obstante, estos beneficios no parecen estar completamente relacionados con una mejora de la absorción de nutrientes por las plantas. En el Cap. IV se estudió el efecto de la aplicación de β-celulosa y la inoculación de Bacillus subtilis QST713 sobre la nutrición de P y Zn en plantas de trigo. En el caso de la β-celulosa, es la primera vez que se demuestra que la adición de una enmienda orgánica que no afecta el ciclo del P en el suelo mejora su absorción por las plantas. B. subtilis incrementó la absorción y la concentración de P en los tejidos de la planta, aunque su efecto parece depender de la fertilización fosfatada y del tipo de suelo. Los beneficios sobre la nutrición de P podrían estar relacionados con la capacidad de movilizar P de los microorganismos (García-López & Delgado, 2016). Esta mejora de la nutrición de P no se tradujo en un efecto antagónico con el Zn, sino que la absorción de ambos nutrientes se correlacionó de forma positiva. La ausencia de antagonismo entre P y Zn podría explicarse por la limitada disponibilidad de ambos nutrientes en los suelos estudiados. Los resultados revelaron la importancia de considerar los factores ambientales, las propiedades del suelo y el manejo de la fertilización fosfatada a la hora de evaluar el efecto de la inoculación microbiana en la nutrición de Zn y P. 152 En el Cap. V se estudió el efecto de la aplicación de β-celulosa y la inoculación de Bacillus subtilis QST713 y Trichoderma asperellum strain T34 sobre la nutrición y el desarrollo de plantas de trigo en diferentes ambientes edáficos. Se observaron evidencias de la activación de mecanismos de movilización de nutrientes, como el aumento de la actividad fosfatasa con T. asperellum y β-celulosa, o la tendencia de B. subtilis a incrementar la concentración de oxalato en la rizosfera. La actividad fosfatasa se relacionó con la absorción de P por las plantas, aunque el efecto de los tratamientos sobre la absorción de P, Zn y Cu no fue significativo. Esto se explica porque las propiedades del suelo, como la proporción de Znpyr/P Olsen en el caso de Zn, pueden afectar la absorción de nutrientes (ver Cap. II). Por otro lado, la producción de sideróforos se relacionó con la actividad microbiana a final de ciclo, como revela la correlación positiva con la actividad deshidrogenasa. En este caso el efecto positivo de B. subtilis y T. asperellum sobre la absorción de Fe fue mayor cuanto menor era el pH y mayor la actividad microbiana del suelo, entendida como la actividad β-glucosidasa. Por ello, se puede inferir que el pH y la actividad microbiana del suelo son aspectos básicos que determinan la eficiencia de los inoculantes microbianos o las enmiendas orgánicas en la mejora de la nutrición férrica de los cultivos, y que explican el diferente efecto observado dependiendo del suelo. Es probable que las condiciones más adecuadas para la actividad microbiana mejoren el desarrollo del inoculante y, en consecuencia, su efecto sea más evidente. La aplicación de celulosa y la inoculación con B. subtilis y T. asperellum promovieron un incremento de la producción de biomasa y una mejora del índice de cosecha (HI). El incremento de la producción se correlacionó con la actividad enzimática del suelo, tanto fosfatasa como β-glucosidasa. Esta actividad enzimática fue generalmente mayor en las macetas tratadas, aunque el efecto fue diferente dependiendo del suelo. El aumento de la fosfatasa se atribuyó en parte a la actividad microbiana, dada su correlación positiva con la β-glucosidasa, que se considera un indicador de la actividad microbiana del suelo relacionada con el ciclo del C (Moreno et al., 2015). Esto sugiere que un mayor desarrollo de la planta puede promover, a su vez, una mayor actividad microbiana en la rizosfera, probablemente a través de una mayor exudación radicular. Por otra parte, la concentración de hormonas en la raíz, ácido indolacético (IAA), ácido abcísico (ABA) y giberelinas (GA3) fue menor con la inoculación de B. subtilis y la aplicación de β-celulosa. La acumulación de ABA en la raíz puede atribuirse a una respuesta a la deficiencia de nutrientes en trigo (Vysotskaya et al., 2008), especialmente de Fe (Lei et al., 2014). En los suelos dónde los tratamientos mejoraron la absorción de Fe y Mn se puede explicar la disminución en la concentración de ABA en raíz. Cabe suponer que esta menor concentración de ABA esté relacionada con el aumento de la producción de biomasa, inducida por los tratamientos, y con un efecto de promoción del crecimiento (Belimov et al., 2014) que ya ha sido descrito para los cereales por Dodd et al. (2010). Por otro lado, T. asperellum incrementó la concentración de GA3 en la raíz respecto a B. subtilis. Este aumento de la concentración de GA3, que estimula el crecimiento de las plantas (Hedden, 2016), fue mayor al incrementarse el pH del suelo. 153 Generalmente los suelos con pH elevado presentan condiciones más restrictivas para la nutrición y el desarrollo de las plantas, y en estos suelos el efecto de los microorganismos inoculados puede ser más evidente. En los casos en los que los tratamientos no afectaron la actividad enzimática cabe suponer que las propiedades del suelo, como el ratio C/N (Thébault et al., 2014) y la disponibilidad de nutrientes (Marschner et al., 2011), tuvieron una mayor influencia sobre la actividad microbiana y su potencial efecto en la nutrición de las plantas, como se ha mencionado anteriormente. Además, hay evidencias de que no sólo la actividad microbiana, sino el perfil de las comunidades microbianas, pueden afectar la absorción de nutrientes de las plantas. En el Cap. IV se ha observado que una mayor actividad microbiana, que se refleja en una mayor actividad enzimática, en el suelo tipo Vertisol puede contribuir a explicar una menor absorción de Zn con respecto al Alfisol. Esto concuerda con trabajos recientes que señalan que el empeoramiento de la absorción de micronutrientes puede atribuirse a cambios en la estructura de la comunidad microbiana (de Santiago et al., 2019), quizás relacionados con cambios en la relación C/N en el suelo (Thébault et al., 2014) y, en este caso, inducidos por la aplicación de βcelulosa y la inoculación con microorganismos. 5. Efecto de la inoculación con microorganismos y la aplicación de una fuente lábil de carbono sobre la biofortificación de zinc y la calidad de la cosecha La inoculación con microorganismos (Bacillus subtilis QST713 y Trichoderma asperellum T34) y la aplicación β-celulosa mejoraron el Zn HI en todos los suelos estudiados, independientemente de su efecto sobre la absorción total de nutrientes. En el Cap. IV se evaluó el potencial efecto biofortificante de Zn derivado de la aplicación de βcelulosa y la inoculación de Bacillus subtilis QST713. La inoculación con B. subtilis no incrementó la absorción de Zn por las plantas pero mejoró la concentración de Zn en el grano y el Zn HI respecto al control, un 24 y 18% respectivamente. Esto, unido al detrimento de la concentración de Zn en el tallo, revela un efecto de movilización del nutriente dentro de la planta. La alteración podría atribuirse a la producción de auxinas por parte de B. subtilis (García-López & Delgado, 2016), que afectaría la regulación de los transportadores involucrados en la homeostasis de los metales en los tejidos vegetales. En el Cap. V se evaluó el potencial biofortificante de Zn derivado de la aplicación de β-celulosa y la inoculación de Bacillus subtilis QST713 y Trichoderma asperellum T34 en diez suelos con diferentes propiedades. Los inoculantes microbianos y la β-celulosa, que no afectaron la absorción total de P y Zn, aumentaron el Zn HI y disminuyeron la relación molar P-Zn en el grano, independientemente del tipo de suelo. Esto puede explicarse por una disminución de la concentración de Zn en raíces y tallos promovida por los tratamientos, sin efectos significativos sobre la concentración en el grano. Los