Analysis of the microbial community of a suppressive soil from an avocado crop

TESIS DOCTORAL Analysis of the microbial community of a suppressive soil from an avocado crop Carmen Mª Vida Hinojosa Directores Dr. Francisco M. Cazorla López Dr. Antonio de Vicente Moreno P.D. Biología Celular y Molecular Facultad de Ciencias Universidad de Málaga MARZO 2017 AUTOR: Carmen María Vida Hinojosa http://orcid.org/0000-0002-6907-7255 EDITA: Publicaciones y Divulgación Científica. Universidad de Málaga Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercialSinObraDerivada 4.0 Internacional: http://creativecommons.org/licenses/by-nc-nd/4.0/legalcode Cualquier parte de esta obra se puede reproducir sin autorización pero con el reconocimiento y atribución de los autores. No se puede hacer uso comercial de la obra y no se puede alterar, transformar o hacer obras derivadas. Esta Tesis Doctoral está depositada en el Repositorio Institucional de la Universidad de Málaga (RIUMA): riuma.uma.es Departamento de Microbiología Facultad de Ciencias TESIS DOCTORAL ANALYSIS OF THE MICROBIAL COMMUNITY OF A SUPPRESSIVE SOIL FROM AN AVOCADO CROP Carmen Mª Vida Hinojosa Málaga, 10 marzo 2017 EVALUATION COMMITTEE President Dr. Rafael Rivilla Palma Departamento de Biología Universidad Autónoma de Madrid Secretary Dra. Emilia Antonia López Solanilla Centro de Biotecnología y Genómica de Plantas Universidad Politécnica de Madrid Vocal Dra. Sandra de Weert Process Development Microbiology Koppert Biological Systems (The Netherlands) Substitutes Dr. Cayo Juan Ramos Rodríaguez Departamento de Genética Universidad de Málaga Dr. Jesús Mercado Blanco Departamento Protección de Cultivos Instituto de Agricultura Sostenible (IAS-CSIC) Dra. Anastasia Lagopodi Department of Crop Production Aristotle University of Thessaloniki (Greece) Departamento de Microbiología Facultad de Ciencias ANALYSIS OF THE MICROBIAL COMMUNITY OF A SUPPRESSIVE SOIL FROM AN AVOCADO CROP Memoria presentada por Dña. Carmen Mª Vida Hinojosa Para optar al grado de Doctor por la Universidad de Málaga con mención Europea Departamento de Microbiología Facultad de Ciencias D. JUAN JOSÉ BORREGO GARCÍA, Director del Departamento de Microbiología de la Universidad de Málaga. INFORMA: Que, Dña. CARMEN Mª VIDA HINOJOSA ha realizado en los laboratorios de este departamento el trabajo experimental conducente a la elaboración de la presente Memoria de Tesis Doctoral. Y para que así conste, y tenga los efectos que correspondan, en cumplimiento de la legislación vigente, expedimos el presente informe, En Málaga, a 20 de octubre del 2016 Departamento de Microbiología Facultad de Ciencias D. ANTONIO DE VICENTE MORENO, Catedrático del Departamento de Microbiología de la Universidad de Málaga y D. FRANCISCO M. CAZORLA LÓPEZ, Profesor Titular del Departamento de Microbiología de la Universidad de Málaga. INFORMA: Que, Dña. CARMEN Mª VIDA HINOJOSA ha realizado bajo su dirección el trabajo experimental conducente a la elaboración de la presente Memoria de Tesis Doctoral. Y para que así conste, y tenga los efectos que correspondan, en cumplimiento de la legislación vigente, expide el presente informe, The present work was supported by the following funding sources: Plan Nacional de I+D+I. Project code: AGL2011-30354-C02-01. Title: “Estudio del control biológico de Rosellinia necatrix mediante el empleo de estrategias genómicas a la interacción en la rizosfera del aguacate”. Principal Investigator: Dr. Francisco M. Cazorla López. Plan Nacional de I+D+I. Project code: AGL2014-52518-C2-1-R. Title: “Aprendiendo de las interacciones multitroficas en la rizosfera de aguacate para avanzar en el control biológico contra Rosellinia necatrix”. Principal Investigator: Dr. Francisco M. Cazorla López. Dña. Carmen Mª Vida Hinojosa was supported by the following grant: Grant for “Personal investigador en formación (FPI)” from Ministerio de Economía y Competitividad (2012-2016). Grant from Ministerio de Economía y Competitividad for a short stay in Graz University of Technology in the Institute of environmental Biotechnology (2015). AGRADECIMIENTOS GRACIAS a todas aquellas personas que en algún momento se cruzaron en mi camino y me ayudaron a disfrutar de esta aventura llamada tesis. En especial me gustaría dar las gracias a mis directores de tesis, Antonio de Vicente y Francisco Cazorla. Gracias por confiar en mí desde el principio y dejarme libertad para crecer como investigadora. Gracias Antonio, por tus grandes consejos que siempre me hacen mantener los pies en la tierra, y gracias Francis, por aguantarme e inspirarme durante estos años de duro trabajo. Entre los dos habéis conseguido que aprenda a equilibrar mis decisiones. GRACIAS. Me gustaría agradecer al resto de Profesores e Investigadores que forman parte de este gran grupo, y que siempre están dispuestos a dar su opinión y consejo. Gracias Alejandro, JC Codina, JA Torés, Diego y Cayo. Ha sido un verdadero placer escucharos y aprender de vuestra experiencia. Gracias a todos los miembros del Departamento de Microbiología por mantener un ambiente de trabajo tan agradable. Especialmente, me gustaría agraceder al departamento la posibilidad de iniciarme en la docencia. Gracias a todos los profesores que me han ayudado en esa labor durante estos años. A la Estación Experimental “La Mayora”, en especial a José María, Emilio y Jorge, gracias por la ayuda prestada para poder llevar a cabo ensayos incluidos en este trabajo. INDEX 5 ABBREVIATIONS AND ACRONYMS LIST AS: soil amended with composted almond shells CT: soil under conventional management WRR: white root rot PGP: plant growth promotion QS: quorum sensing QQ: quorum quenching HCN: hydrogen cyanide PCA: phenazine-1-carboxylic acid HPR: 2-hexyl 5-propyl resorcinol PRN: pyrrolnitrin PLT: pyoluteorin DAPG: 2,4-diacetylphloroglucinol AHLs: acyl homoserine lactones VOCs: volatile organic compounds ACC: 1-aminocyclopropane-1-carboxylate ISR: induction of local and systemic resistance ARISA: automated ribosomal intergenic spacer analysis TRFLP: terminal restriction fragment polymorphism DGGE: denaturing gradient gel electrophoresis NGS: next generation sequencing FGAs: functional gene arrays EggNOG: public resource of orthologous groups of genes POGs: pan-genome orthologous groups CL/SM: confocal laser scanning microscopy GC/MS: gas chromatography-mass spectrophotometry BCAs: biological control agents PCL1601: Pseudomonas chlororaphis PCL1601 PCL1606: Pseudomonas chlororaphis PCL1606 AVO110: Pseudomonas pseudoalcaligenes AVO110 PCL1608: Bacillus subtilis PCL1608 CV026: Chromobacterium violaceum CV026 SUMMARY 7 SUMMARY SUMMARY 8 In this work, we focused in getting insight in the knowledge of soil Microbial Ecology of a suppressiveness-induced agricultural soil from an avocado (Persea americana Mill) crop after the application of composted almond shells as organic mulch. The study of the microbial interactions with their surroundings is essential in order to understand their involvement in plant growth and its general role in the global agricultural environment. It has been demonstrated that soil microbial community have a crucial role in the correct performance of biogeochemical cycling of nutrients, organic matter and also in the improvement of plant performance and soil quality, key issues for agroecosystem self-sustainability (Bulluck et al., 2002). In order to promote and maintain the soil qualities in agriculture, farmers have used different crop management practices along years based on ecological principles. Many of these practices are used in order to control plant diseases and improve crop yield, and include crop rotation, mulches incorporated as green manures, minimal tillage practices, soil solarization and/or applications of external organic inputs (Hadar and Papadopoulou, 2012). One of these practices is the application of soil organic amendments, which has been described as safe-environmental technique, used world-wide due to the positive effects performed in different agricultural crops (Bulluck et al., 2002). These positive effects on plants and soils have been associated with the maintenance of desirable soil properties, including physicochemical and microbial characteristics, such as soil aeration, structure, drainage, moisture, holding capacity, nutrient availability and microbial ecology, that often have been directly correlated with soil suppressive phenotype against different soilborne diseases (Bailey and Lazarovits, 2003). Thus, the incidence of different plant diseases caused by soilborne pathogen, could be controlled and/or reduced by the use of organic amendments from different sources (Liu et al., 2007; Mendes et al., 2011; Bonilla et al., 2012a; Pane et al., 2013; Wallisch et al., 2014). The generalized use of mulches in organic management of woody perennial crops, such as avocado, could be essential due to this amendment provides SUMMARY 9 several environmental and agronomical advantages, as a C source available and long lasting, improvement in top soil layer structure, and it is a low cost material (López e tal., 2014). Moreover, the presence of this type of decomposing litter layer in the top soil surface helps to proliferate feeder roots of avocado and reduce weeds growth, allowing an improvement in plant health and yield (Wolstenholme et al., 1997). One of this organic amendments is the composted almond shells (AS), which have been used to induce suppressive activity in avocado agricultural soils, leading to prevention and control of the avocado white root rot disease, causes by the soilborne phytophathogenic fungus Rosellinia necatrix Prill. The application of composted almond shells as organic amendments to the avocado crop soil, influences its physicochemical soil properties, such as ions concentration of Ca2+, K+, Mg2+ and Mn+, higher in soils amended with organic matter and sometimes correlated with its biocontrol ability (Bulluck et al., 2002), specially when linking this activity with the presence of specific C substrates and carbon:nitrogen (C:N) ratios (Hadar and Papadopoulou, 2012). Physicochemical analysis of amended soils with composted almond shells resulted in a high content of different cations (Na+, K,+ Ca2+, Mg2), organic matter, C:N ratio and some micronutrients, such as Fe, Cu, Mn and Zn, which could have an influence in the growth rate of different groups of microorganisms potentially implicated in the suppressive phenotype of this soil (Gupta et al., 2008). Previous studies showed that application of composted almond shells lead to changes in soil microbial properties (Bonilla et al., 2012a). Moreover, the role of microbial communities in soil suppressiveness had been widely described along years (Weller et al., 2002; Haas and Defágo, 2005; Mendes et al., 2011; Pane et al., 2013; Bonilla et al., 2015). For this reason, suppressiveness assays were performed in order to analyse the implication of soil microbiome of the agricultural avocado soil amended with composted almond shells in the biological control of the soilborne pathogen R. SUMMARY 10 necatrix. We used different soils from an experimental avocado field with adult avocado trees under two types of management: avocado trees only amended with composted almond shells and other group of trees under conventional management. The two types of soils were assayed using two types of experimental plant-pathogen systems, avocado and wheat (Triticum aestivum L.). The results showed that the soil mulched with composted almond shells displayed a better suppressive ability than conventional soil samples with a disease index significantly lower. The suppressive ability was reduced in amended almond shells soil samples when a moist-heat treatment was applied, and simultaneously, microbial population density decreased. However, no significant changes in suppressiveness were observed when the moistheat treatment was applied to conventional soil and remains disease-conducive. The suppressive phenotype was recovered when the heat-treated soils were complemented with amended soil in 9:1 (treated:untreated; weight:weight), revealing the essential role of the microbial community present in the soil influenced by the composted almond shells in suppressiveness against R. necatrix in both experimental plant model used. In particular, we observed that the microbiota induced in soils amended with composted almond shells resulted crucial for the suppressiveness (Weller et al., 2002). Similar studies have demonstrated this crucial role of the soil microbiome in suppressiveness, reducing the bacterial levels by using soil sterilization, autoclaving, steam pasteurization and irradiation, resulting those treated soils in conducive soils to the pathogen studied, allowing the advance of the disease (Malajczuk, 1983; Mendes et al., 2010; Weller et al., 2002). Once proved the crucial key of the microbial community in suppressiveness against R. necatrix, we performed massive DNA sequencing assays from the amended and unamended soil samples, in order to know the microbial community potentially involved in the induced suppressiveness of this amended soil. For this purpose, we performed independent sequencing analysis of 16S ribosomic RNA gene in order to SUMMARY 11 unravel the prokaryotic community, present on the amended soil and compared with those in the unamended soils. The obtained results of the microbial community present in the amended soil, showed an increase in the relative abundance of phylum Proteobacteria, specially a clear increase in relative abundance of Gammaand Betaproteobacteria. Gammaproteobacteria is a class of Proteobacteria containing bacterial representatives very well known for their plant protection abilities and their fungal interactions in previously studied suppressive soils (Mendes et al., 2011; Koyama et al., 2014). They included different fast-growing and easily cultivable genera from families such as Xanthomonadaceae, Enterobacteriaceae and Pseudomonadaceae. Nevertheless, special representation in this amended soil have the genus Steroidobacter, previously reported as a biodegrading bacteria. Only a few species for this genus are currently described, all of them isolated from soils with a high concentration of decomposing organic matter (Sakai et al., 2014; Gong et al., 2015) and involved in positive interactions with plants (Zarraonaindia et al., 2015). Simultaneously, sequencing analysis of internal transcribed spacer in ribosomal coding DNA (ITS regions) were performed in order to know the eukaryotic profile present in the amended soils. The results showed the importance of the fungal community, concretely an increase in the relative abundance of phylum Ascomycota was observed in the amended soil. One class of this phylum, Dothydeomycetes showed to be clearly increased in its relative abundance under the effect of the composted almond shells. This group of fungi have been previously reported to be abundantly present in soils with high hydrocarbon concentrations (Ferrari et al., 2011). Moreover, some genera belonging to this fungal class were reported in other suppressive soils able to harbour diverse endohyphal Gammaand Betaproteobacteria. Special importance had the order Pleosporales, fungi commonly isolated from plants environment (Shen et al., 2014) and directly involved in the degradation of lignin, which is considered the first step of biomass conversion of plant organic matter in soils SUMMARY 12 (Ortíz-Bermúdez et al., 2007). Interestingly, we observed in the soils under the influence of composted almond shells amendment, a reduction of the relative abundance of Xylariales order, which R. necatrix belong, thus revealing an antifungal effect on this group. In order to get insight on the functional profile of this suppressive microbial community present in amended soils, we used GeoChip®, a commercial microarray, that allowed the detection of hundreds of functional microbial genes involved in different soil process such as biogeochemical cycles, environmental adaptability and plant and microorganisms interactions (Tu et al., 2014). As expected, microbiome from samples of amended soil had higher hybridization to probes for C degradation (carbon cycle) related genes ranged from labile C to more recalcitrant C (e.g., starch, hemicelluloses, cellulose, chitin and lignin). These results suggest the important role of carbon degradation in the corresponding activities of the microbial community evolved in this soil. Other previous studies have observed this fact in other suppressive soils. For example, there is enough evidence to suggest a clear link between the abundance of chitins and chitin-derived C compounds in certain composts and the potential proliferation of chitinolytic microbial agents with the ability to degrade the fungal pathogen cell walls (Cretoiu et al., 2013). This functional analysis, also allowed us to analyse the specific putative activities from this amended soil. GeoChip® analysis showed that approximately 10% of the total probes analysed were unique for AS-amended samples (n= 2766 probes). When the sequence of these unique probes were analysed, we found, for example, genes for antibiotics biosynthesis such as phenazine (from Proteobacteria) an others related with some of the bacterial groups enhanced in the amended soil. These results suggest that a “microbe-specific suppressiveness” could been taking place in AS-amended soil. However, not only one specific group of microorganisms could be responsible of SUMMARY 13 biocontrol activity, but also multiple interactions between few groups of fungal and bacterial strains could finally result in the elicitation of suppressiveness in this soil. Thus, agricultural avocado soil amendment with composted almond shells, promoted the selection of several specific groups of microorganisms, stimulated by the efficient and sequential use of the compounds present in almond shells. This amendment is rich in lignin (36%; López et al., 2014) and lignin-degrading fungi such as Dothideomycetes can promote their growth, especially species from Pleosporales order. From lignin degradation, different aromatic compounds and C sources would be released, and they could be used then by fast-growing microorganisms such as Gammaproteobacteria, and Betaproteobacteria, where we can found the group of Steroidobacter spp. and other species with ability to use aromatic compounds, such as Pseudomonas spp., Serratia spp. and Burkholderia spp. Futhermore, these strains also produced a collection of exoenzymatic proteins (chitinases, proteases, etc), siderophores and some antifungal compounds with can lead to suppressiveness of some other group of microbes (Gross and Loper, 2009; Raaijmakers and Mazzola, 2012). In this case, after the amendment of composted almond shells, fungal order Xylariales, in which R. necatrix is included, was decreased. These results suggest, a suppressive effect against this avocado pathogen by the microbiome evolved after ASamendment. Because of the proposed key role of these specific members of Gammaproteobacteria, in this suppressive-induced soil, and due to our interest in bacterial biological control agents against R. necatrix, isolation and characterization of culturable members of this bacterial group, increased into the amended soil, were performed. For this purpose, we have used a selective medium described for the isolation of fluorescent Pseudomonads (Sands and Rovira, 1970). Finally, our results confirmed that this medium allowed selection of Pseudomonads and other Gram-negative bacteria from related groups. A collection of 246 Gram-negative bacteria were isolated and grouped according to their SUMMARY 14 metabolic patterns of glucose and other characteristics: Enterobacteriaceae-like (n= 148), fluorescent Pseudomonadaceae-like (n=26), nonfluorescent Pseudomonadaceae-like (n=11), Xanthomonadaceae-like (n=12) and 49 remained as unidentified isolates. Several approaches were carried out to characterize microbial activities potentially related with biological control of the diseases, including fungal antagonism, production of antimicrobial compounds and lytic exoenzymes or plantgrowth-promoting (PGP) related activities. Partial sequencing of the 16S rDNA were performed to help in the isolates characterization in order to futher select some representatives to be tested on biocontrol assays, and check their potential use as R.necatrix-biocontrol agents (BCAs). Antagonism was performed by the dual plate assays testing the antagonistic activity of the bacterial isolates against to 3 different soilborne fungal and oomycete pathogens, such as R. necatrix and Phytophthora cinnamomi (as avocado pathogens) and Fusarium oxymporum f. sp. radicis-lycopersici (as tomato fungal pathogen used as comparative model). The results showed that 22% of the bacterial isolates tested had antagonistic activity at least to one of the pathogens. At same time, we used colony blot assays to test the presence of biosynthetic genes of antimicrobial compounds production for the bacterial collection, such as, phenazine-1-carboxylic acid (PCA), 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN), pyoluteorin (PLT), 2-hexyl 5-propyl resorcinol (HPR) and hydrogen cyanide (HCN) (Cazorla et al., 2006; ChinA-Woeng et al., 1998; Castric, 1975; Howell and Stipanovic, 1979). These analyses showed that 11% of isolates could produce at least one of the antimicrobial compounds tested. Nevertheless, any of the 246 isolates analyzed produced DAPG or PRN. Its absence could due to that the production of these antimicrobial compounds have been described in strains with biocontrol activity only in herbaceous plant models (Hammer et al., 1997; de Souza et al., 2003; Barahona et al.,2010; Bankhead et al., 2016) but not from woody plants. Additionally, production of lytic exoenzymes such as lipases, SUMMARY 15 proteases, amilases, cellulases, β-glucanases and chitinases were tested. Seventy eight percent of the isolates showed lipases, proteases and/or chitinases activities being very often detected in those soil isolates. Amilases, cellulases and β-glucanases activities were not detected in any assayed strain. Regarding plant-growth-promotion (PGP) related activities, we analyzed both the ability of the isolates to degrade an insoluble P source, and the production of siderophores. Fifty five percent of the isolates showed at least one of the activities. At this point, and to get insight into these PGP activities, we selected 24 isolates representatives of all the diversity of results obtained, based in their characteristics evaluated in this first screening to perform in vivo PGP assays on tomato seedlings, showing that only 2 of the isolates showed such PGP activity. The overall results showed that Enterobacteriaceae-like group were mainly producers of lytic exoenzymes and PGP-related activities whereas putative antifungal producers were mainly allocated into the Pseudomonadaceae-like groups, although these results could be influenced by the types of antifungal compounds assayed, more of them described as products of Pseudomonas spp. and related groups. Moreover, partial sequencing of the 16S rDNA of 24 selected isolates allowed the identification of 9 of these strains, all of them from Serratia, Pseudomonas and Stenotrophomonas genera. In this point, a second selection step was performed based in the characteristics of isolates in order to perform biocontrol assays against R. necatrix using avocado as a susceptible plant-pathogen system (8 selected isolates). All of these strains showed biocontrol activity in avocado roots. These results confirmed that different representatives from Gammaproteobacteria class, after the increase of their relative abundance in suppressive amended soils, could perform a biological control activity against R. necatrix. The suggested specific suppression of AS-amended soil that could be caused by various groups of microorganisms, mainly culturable Pseudomonads, had led us to RESUMEN 23 RESUMEN RESUMEN 24 Este estudio se ha dirigido a profundizar en diferentes aspectos relacionados con la Ecología Microbiana y los distintos procesos que tienen lugar en un suelo agrícola de un cultivo de aguacate (Persea americana Mill) que desarrolla una actividad supresiva tras la aplicación de una enmienda orgánica. El estudio de las interacciones que tienen lugar dentro de la comunidad microbiana de un suelo, es esencial para poder entender la implicación de los distintos microorganismos en el crecimiento vegetal y su comportamiento general del ecosistema. Dicha comunidad microbiana tiene un papel primordial en el correcto funcionamiento de los ciclos biogeoquímicos de diferentes nutrientes, en la degradación de materia orgánica y mejora de la salud vegetal y la calidad del suelo, factores muy importantes para una agricultura sostenible (Bulluck et al., 2002). Existen diferentes prácticas de manejo agrícola basadas en principios ecológicos y que han sido utilizadas por los agricultores a lo largo de los años para promover y mantener la calidad del suelo. Muchas de estas técnicas se pueden utilizar para controlar enfermedades que pueden afectar al cultivo y para mejorar la producción de estos. Así, la rotación de cultivos, uso de cubiertas vegetales, uso controlado de la labranza, solarización de los suelos y/o aplicación de materia orgánica de diferentes orígenes (Hadar and Papadopoulou, 2012) son ejemplos de técnicas agrícolas utilizadas para este fin. La aplicación de enmiendas orgánicas ha sido descrita en diferentes trabajos como una técnica ambientalmente sostenible y utilizada en todo el mundo, debido al efecto positivo que generalmente causa en los cultivos (Bulluck et al., 2002). Este efecto ha sido asociado al mantenimiento de las propiedades del suelo, tanto fisicoquímicas como biológicas, que frecuentemente se han relacionado directamente con la actividad supresiva de algunos suelos frente a diferentes enfermedades de plantas causadas por patógenos de suelos. Diferentes trabajos recogen como la incidencia de diferentes enfermedades de plantas causadas por patógenos de suelo puede ser controlada y/o reducida por el uso de enmiendas orgánicas de diferente naturaleza (Liu et al., 2007; Mendes et al., 2011; Bonilla et al., RESUMEN 25 2012a; Pane et al., 2013; Wallisch et al., 2014). Un ejemplo de enmienda orgánica es la cáscara de almendra compostada, que induce actividad supresiva en suelos agrícolas de cultivos de aguacate, ya que previenen y controlan la podredumbre blanca radicular el aguacate, causada por el hongo fitopatogéno Rosellinia necatrix Prill. El uso de materia orgánica compostada, en el manejo de cultivos de leñosas de hoja perenne, como el aguacate, es una práctica esencial ya que puede conferir ventajas tanto ecológicas como agrícolas. La cáscara de almendra constituye un residuo orgánico de la industria almendrera, y que con su posible uso en agricultura, permitiría su reutilización y le aporta un nuevo valor añadido. Además, su aplicación supone la disponibilidad de una fuente de C de lenta degradación y larga duración, resistente y de bajo coste (López et al., 2014). La aplicación de capas de materia orgánica en degradación en la superficie del cultivo del aguacate, es una práctica habitual en este cultivo. La presencia de dicha capa orgáncia favorece la proliferación de las raíces alimenticias del aguacate en la parte superior del suelo, así como una reducción en el crecimiento de malas hierbas, causando una mejora en la salud de la planta y por lo tanto, en la producción de cultivo (Wolstenholme et al., 1997). En nuestro trabajo, se puso de manifiesto que la aplicación de cáscara de almendra compostada como enmienda orgánica influye en diferentes factores como la concentración de iones de Ca2+, K+, Mg2+ y Mn+, más abundantes en los suelos enmendados y correlacionados con la capacidad supresiva del suelo (Bulluck et al., 2002). También incrementa la presencia de fuentes de C específicas y de la razón carbono:nitrógeno (C:N; Hadar and Papadopoulou, 2012), indicador general de la fertilidad del suelo. Los análisis fisicoquímicos realizados al suelo enmendado con cáscara de almendra compostada también revelaron un alto contenido en algunos micronutrientes como Fe, Cu, Mn y Zn, y que pueden influir en la tasa de crecimiento de algunos grupos de microorganismos implicados en la actividad supresiva de este suelo (Gupta et al., 2008). RESUMEN 26 Ya en estudios previos se evidenció que la aplicación de la cáscara de almendra compostada también causaba cambios en las propiedades microbianas del suelo (Bonilla et al., 2012a). Además, el papel de la comunidad microbiana en la supresividad del suelo ha sido ampliamente descrito a lo largo de los años en otros trabajos (Weller et al., 2002; Haas and Defágo, 2005; Mendes et al., 2011; Pane et al., 2013; Bonilla et al., 2015). Por esta razón, se realizaron ensayos de supresividad para comprobar la implicación del microbioma que se desarrolla bajo la influencia del suelo enmendado, en el control del patógeno R. necatrix. Para llevar a cabo estos ensayos, se emplean muestras obtenidas de una parcela experimental de cultivo de aguacate con más de 100 árboles adultos sometidos a 2 tipos de manejo diferente: uno orgánico, con la aplicación masiva de cáscara de almendra compostada desde hace años, y otro sometido a un manejo convencional con los productos químicos recomendados. Además, en el laboratorio se emplearon dos tipos de modelos experimentales de plantas susceptibles al hongo fitopatógeno R. necatrix, aguacate y trigo (Triticum aestivum L.). Estos ensayos de supresividad mostraron que los suelos enmendados con cáscara de almendra tenían actividad supresiva frente a R. necatrix, mientras que los suelos de manejo convencional permanecían conductivos, con valores de índice de enfermedad significativamente mayores que los suelos enmendados. Por otro lado, la supresividad se veía reducida en las muestras de suelos enmendados tras la aplicación de un tratamiento térmico, que reducía la densidad microbiana. Además, observamos la recuperación del fenotipo supresivo de un suelo, cuando se complementaban con suelo enmendado original en una proporción 9:1 (tratado:no tratada; peso:peso). Estos resultados mostraron el importante papel de la comunidad microbiana de los suelos enmendados con cáscara de almendra compostada en la supresividad frente a R. necatrix, y que se puso de manifiesto empleando ambos modelos de planta ensayados. La recuperación de la supresividad cuando se complementan los suelos tratados térmicamente con una parte de suelo enmendado, indica una supresividad influenciada RESUMEN 27 por la actividad de grupos específicos de microorganimos que se desarrollan en el suelo enmendado (Weller et al., 2002). Nuestros resultados confirman lo ya observado en trabajos previos con otros sistemas, donde demuestran que el papel del microbioma del suelo puede tener en la supresividad, ya que tras tratamientos que reducen el número de microorganismos al suelo supresivo como la esterilización, pasteurización o irradiación, se conseguía perder la actividad supresiva de los suelos, favoreciendo así, el avance de la enfermedad (Malajczuk, 1983; Weller et al., 2002; Mendes et al., 2011). En todos los casos el papel de la microbiota del suelo era esencial para el control de la enfermedad, y se consideraban los dos tipos clásicos de supresividad descritos: una supresividad general dependiente de la comunidad microbiana completa y no transferible a otros suelos o una supresividad específica dependiente de algunos grupos de microorganismos y transferible a otros suelos (Weller et al., 2002). Una vez comprobado el importante papel de la comunidad microbiana en la supresividad del suelo enmendado con cáscara de almendra compostada contra R. necatrix, se inició una estrategia de análisis metagenómico para conocer los grupos microbianos presentes en el suelo. Para ello, llevamos a cabo la secuenciación masiva del ADN total de distintas muestras de suelo comparando suelo enmendado con suelo bajo manejo convencional, para conocer sus perfiles microbianos. Se realizaron secuenciaciones independientes del gen del ARN ribosómico de 16S para conocer el perfil procariótico de la comunidad microbiana. Los resultados mostraron el incremento en la abundancia relativa del phylum Proteobacteria, en concreto, el aumento de la abundancia relativa de las clases Gammay Betaproteobacteria. Hay que destacar que, Gammaproteobacteria es una clase de Proteobacteria ampliamente descritas en otros suelos supresivos por la capacidad de algunos de sus representantes para estimular la protección vegetal o para interaccionar contra patógenos fúngicos (Mendes et al., 2011; Koyama et al., 2014). En este grupo podemos encontrar diferentes grupos de bacterias cultivables en medios artificiales, de fácil manejo y RESUMEN 28 crecimiento rápido, como por ejemplo representantes de las familias Xanthomonadaceae, Enterobacteriaceae y Pseudomonadaceae. No obstante, destaca en el suelo modificado por la aplicación de cáscara de almendra compostada, la presencia el género Steroidobacter el cual tiene un interés especial como bacterias directamente implicadas en la degradación del material orgánico. Muy pocas especies de este género están descritas en la actividad, y algunas de ellas aisladas de suelos con una alta concentración en materia orgánica en descomposición (Sakai et al., 2014; Gong et al., 2015) e involucradas en interacciones beneficiosas con plantas (Zarraonaindia et al., 2015), lo que sugiere un papel relevante dentro de la microbiota que se desarrolla en los suelos enmendados con cáscara de almendra. Paralelamente, se analizaron las secuencias de las regiones intergénicas (ITS) del ARN ribosómico 18S, para conocer el perfil eucariótico del microbioma del suelo enmendado. Los resultados muestran la importancia de la comunidad fúngica en estos suelos. En concreto, se observó el incremento en la abundancia relativa del phylum Ascomycota, principalmente de la clase Dothideomycetes en las muestras de suelo enmendado. La abundancia de este grupo de hongos ha sido descrita anteriormente en suelos con altas concentraciones de compuetos hidrocarbonados (Ferrari et al., 2011). Se ha descrito la presencia de algunos representantes de esta clase en suelos supresivos, que además tendrían capacidad de portar especies de Gammay Betaproteobacteria como bacterias endohifales. Dentro de este grupo, mostró especial interés en este suelo el orden Pleosporales, grupo de hongos aislados de plantas (Shen et al., 2014) e implicados en los primeros pasos de degradación de la lignina (Ortíz-Bermúdez et al., 2007). Este análisis del perfil eucariótico de microorganismos, además mostró la reducción de la abundancia relativa del orden Xylariales, grupo al que pertenece el hongo patógeno R. necatrix, siendo este hecho la evidencia directa de la supresividad frente a este patógeno en suelos enmendados. RESUMEN 29 Para profundizar en el perfil funcional de esta comunidad microbiana, se empleó el análisis con GeoChip®, un microarray comercial que detecta cientos de genes microbianos funcionales y potencialmente implicados en diferentes procesos que se llevan a cabo en el suelo, bien como parte de los ciclos biogeoquímicos, actividades de adaptación ambiental e interacción con plantas otros microorganismos (Tu et al., 2014). En nuestro caso, las muestras de ADN extraido desde suelo enmendado, presentaron un mayor número de sondas relacionadas con la degradación de diferentes fuentes de C, desde formas más lábiles de C hasta más recalcitrantes (almidón, hemicelulosa, celulosa, quitina y lignina). Este efecto también se ha observado en otros suelos supresivos, donde las evidencias sugieren un claro vínculo entre la abundancia de quitinas y compuestos derivados en algunos compost con la proliferación de agentes microbianos quitinolíticos con actividad antifúngica (Cretoiu et al., 2013). Los resultados del análisis del GeoChip®, mostraron que aproximadamente el 10% del total de las sondas que hibridaron en el microarray solo estaban presentes en las muestras de suelos enmendados (n= 2766). Al analizar la secuencia de estas sondas específicas, se puso de manifiesto la hibridación con genes de biosíntesis de antibióticos como la fenazina (de miembros bacterianos pertenecientes al phylum Proteobacteria) y otros representantes pertenecientes a grupos de bacterias que habían aumentado su presencia en los suelos enmendados. Estos resultados sugieren que la supresividad inducida por la aplicación de cáscara de almendra compostada al suelo podría tener un carácter específico, es decir, se debería a las actividades desarrolladas por algunos grupos de microorganismos concretos de este suelo. Probablemente, no se deba a la actividad de un único grupo de microorganismos sino a las interacciones que tienen lugar entre diferentes grupos de hongos y bacterias presentes en la comunidad microbiana de este suelo. Así, el suelo enmendado con cáscara de almendra compostada promovería la selección de grupos concretos de microorganismos estimulados por su capacidad para la RESUMEN 30 degradación de este tipo de materia orgánica. Como esta enmienda orgánica es rica en lignina, algunos hongos degradadores de lignina como los Dothideomycetes pueden promover su crecimiento, especialmente los representantes del orden Pleosporales. Fruto de la degradación inicial de la lignina, se originarían diferentes compuestos aromáticos y otras fuentes de C, más accesibles para microorganismos de crecimiento rápido como son las Gammaproteobacteria y Betaproteobacteria, donde destacan las especias de Steroidobacter spp. con capacidad para utilizar diferentes compuestos aromáticos y Pseudomonas spp., Serratia spp. y Burkholderia spp. Además, se ha descrito que estas cepas portan una colección de exoenzimas (quitinasas, proteasas), sideróforos y compuestos antifúngicos, que pueden estar relacionados en la supresividad contra patógenos de estos microorganismos (Gross and Loper, 2009; Raaijmakers and Mazzola, 2012). Como resultados de todas estas interacciones, se observa una disminución en la abundancia del orden de los Xylariales, grupo al que pertenece la especie fitopatógena R. necatrix y causante de la podredumbre blanca radicular. Su disminución se puede interpretar como consecuencia del efecto supresivo del microbioma que se estimula en los suelos enmendados con cáscara de almendra contra este patógeno de aguacate. Debido al importante papel que las Gammaproteobacteria tienen en este suelo y a nuestro interés en agentes bacterianos de control biológico frente a R. necatrix, nuestro trabajo se centró en el aislamiento y caracterización de aquellos microorganismos cultivables pertenecientes a las gammaproteobacterias, y mas concretamente al género Pseudomonas, y que habían mostrado ser más abundantes en los suelos enmendados. Para llevar a cabo esta tarea, se utilizó un medio selectivo descrito para el aislamiento de pseudomonas fluorescentes (Sands y Rovia, 1970) pero que también permitía el aislamiento de otros grupos de bacterias Gram-negativas relacionados. Se obtuvo una colección de 246 bacterias Gram-negativas, que fueron agrupadas según distintas caracteristicas: enterobacterias (Enterobacteriaceae-like, n= 148), pseudomonas RESUMEN 31 fluorescentes (fluorescent Pseudomonadaceae-like, n= 26), pseudomonas no fluorescentes (nonfluorescent Pseudomonadaceae-like, n= 11), xanthomonas (Xanthomonadaceae-like, n= 12) y 49 aislados que quedaron sin clasificar. Además, llevamos a cabo diferentes experimentos para caracterizar actividades microbianas relacionadas con el control biológico de enfermedades como ensayos de antagonismo, producción de compuestos antimicrobianos o exoenzimas líticas, y ensayos de actividades relacionadas con la promoción del crecimiento vegetal (PGP). La secuenciación parcial del gen del RNA ribosómico de 16S también se realizó para facilitar la caracterización de algunos aislados y facilitar la selección de algunos aislados representativos para evaluar su potencial actividad como agentes de control biológico frente a R. necatrix. A continuación, se llevaron a cabo ensayos de antagonismo en placa para evaluar este fenotipo en la colección de aislados obtenidos frente a 3 patógenos diferentes: R. necatrix y Phytophthora cinnamomi (patógenos de aguacate) y Fusarium oxysporum f. sp. radicis-lycopersici (patógeno de tomate, y ampliamente usado como modelo de estudio). Los resultados de los experimentos mostraron que el 22% de los aislados bacterianos tenían alguna actividad antagonista, al menos frente a uno de los patógenos ensayados. Al mismo tiempo, se analizó la presencia de genes potencialmente implicados en la biosíntesis de compuestos antimicrobianos antifúngicos en la colección de aislados. Para ello, se realizaron ensayos de hibridación de sondas (mediante “colony blots”) para detectar la presencia de los genes biosintéticos para los compuestos fenazina-1ácido carboxílico (PCA), 2,4-diacetilfloroglucinol (DAPG), pirrolnitrina (PRN), pioluteorina (PLT), 2-hexil 5-propil resorcinol (HPR) y ácido cianhídrico (HCN) (Castric, 1975; Howell and Stipanovic, 1979; Chin-A-Woeng et al., 1998; Cazorla et al., 2006). Los resultados mostraron que el 11% de los aislados podría producir al menos uno de los antimicrobianos analizados. No obstante, ninguno de los 246 RESUMEN 32 aislados analizados podría producir DAPG o PRN. Este resultado está de acuerdo con las observaciones que sugieren que estos antibióticos son característicos de cepas con actividad de control biológico en modelos de plantas herbáceas (Hammer et al., 1997; de Souza et al., 2003; Barahona et al., 2010; Bankhead et al., 2016). Simultáneamente, analizamos la producción de exoenzimas líticas como las lipasas, proteasas, amilasas, celulasas, β-glucanasas y quitinasas. El 78% de los aislados presentaron actividad lipasa, proteasa y/o quitinasa, mientras que las actividades amilasa, celulasa y βglucanasa no fueron detectadas en ningún aislado. En cuanto a las actividades relacionadas con la promoción del crecimiento vegetal, la degradación de una fuente insoluble de fósforo, y la producción de sideróforos, el 55% de los aislados mostraron actividad en al menos una de estas pruebas. Teniendo en cuenta todos estos resultados, se seleccionaron 24 de los aislados que representaran la mayor diversidad posible, y se llevaron a cabo ensayos de PGP in vivo. Tras estos ensayos, solo 2 de los aislados mostraron una actividad de promoción del crecimiento vegetal de semillas de tomate. En general, estos análisis mostraron que dentro del grupo Enterobacteriaceae-like se encuentra un mayor número de aislados bacterianos productores de exoenzimas líticas y promotores del crecimiento vegetal mientras que los posibles productores de antibióticos pertenecían al grupo Pseudomonadaceae-like, aunque este resultado puede estar influenciado por la batería de antifúngicos ensayados, la mayoría descritos en Pseudomonas spp. y en grupos relacionados. Además, la secuenciación parcial del gen del ARN ribosómico 16S de estos 24 aislados seleccionados, junto con los resultados obtenidos en las pruebas bioquímicas de identificación, nos permitió asignar el género al que pertenecen 9 de estas cepas, todas ellas incluidas en Serratia sp., Pseudomonas sp. y Stenotrophomonas sp. Finalmente, se eligieron a 8 aislados diferentes para realizar los ensayos de control biológico frente a R. necatrix en aguacate en base a las características observadas RESUMEN 39 metabolismo entre las tres cepas de pseudomonas. Un tercer compuesto volátil producido solo por el consorcio microbiano fue el S-metil 3-metilbutanotioato, también de función desconocida. No obstante, un compuesto similar, el S-metil butanotioato, producido por diferentes cepas de Pseudomonas sp. ha sido descrito previamente como un compuesto volátil con efecto de inhibición de la germinación de esporangios, crecimiento miceliar y movilidad de zoosporas en algunos oomicetes como Phytophthora infestans (Vrieze et al., 2015). Así, el consorcio microbiano construido, podría utilizarse en estudios sobre las posibles interacciones que ocurren durante el control biológico de R. necatrix, y ayudar a diseñar futuros experimentos de transcriptómica, proteómica y/o metabolómica para profundizar en un futuro sobre el conocimiento a nivel molecular de las interacciones multitróficas que tienen lugar en un suelo supresivo enmendado con cáscara de almendra compostada. CHAPTER I: GENERAL INTRODUCTION 41 CHAPTER I GENERAL INTRODUCTION CHAPTER I: GENERAL INTRODUCTION 42 1. Soil general features Soil Microbial Ecology is the study of the microbial interactions in soil environments, including plants, animals and each other. Soil microbial communities (or soil microbiome) are large, diverse (>109 individual cells and >106 distinct taxa per gram of soil) and responsible for essential functions to plant growth and global environment. These soil microorganisms participate in the biogeochemical cycling of nutrients and organic matter, and can improve plant performance and soil quality, key issues for agroecosystem self-sustainability (Bulluck et al., 2002). Soil quality could be defined as the capacity of the soil to function within ecosystem boundaries to sustain biological productivity, maintain environmental quality, and promote plant and animal health. Indicators of soil quality are commonly classified in physical, chemical and biological parameters, which depend directly on soil management practices and maintenance of adequate soil organic matter (Doran and Parkin, 1994). The maintenance of soil quality is essential for crops yield. Several crop management practices could help to promote the right balance of soil quality measures based on ecological principles. Different examples of these techniques could be crop rotation, cover crops incorporated as green manures, minimal tillage practices, soil solarization and/or applications of external organic inputs (Hadar and Papadopoulou, 2012). Among them, the addition of organic matter to soils can improve soil quality by affecting many parameters, such as soil aeration, structure, drainage, moisture, nutrient availability (Bulluck et al., 2002), in summary, soil microbial ecology. Organic soil amendments or mulches, including composted or uncomposted plant residues and animal manures, differently affect on the balance of soil microbiome and plant diseases. Some types of mulches could have a negative effect in soils, as reported in several studies, where the impact of animal manures incorporation of soils can sometimes increase the incidence and severity of plant diseases (Aryantha et al., 2000). Nevertheless, numerous researches have been demonstrated the improvement of plant CHAPTER I: GENERAL INTRODUCTION 43 health and crop yield, as well as, the suppressive effect against soilborne diseases of composted materials used as organic amendments (Masry et al., 2002; Liu et al., 2007; Kyselková et al., 2009; Mendes et al., 2011; Pane et al., 2013; Bonilla et al., 2015). Several studies defend that the addition of plant residues to soil, in general, improves soil structure and soil health, improving plant growth and decreasing the disease incidence of plant pathogens (Garbeva et al., 2004; Bonilla et al., 2012a). Particularly, application of organic mulches could increase soil bacteria biomass, who normally have overlapping physiologies that promote the complexity of the soil food web. Different soil bacteria are critical to the maintenance of soil function displaying an important role in structure formation, decomposition of organic matter, toxin removal and biogeochemical cycling (Doran and Parkin, 1996). In addition, some groups of bacteria could play key roles in suppressing soilborne plant diseases and in promoting plant growth (Weller et al., 2002). 2. Suppressiveness-induced soils Following the classical definition of Cook and Baker (1983), a suppressive soil provides an environment in which plant disease development is reduced, even when the pathogen is favored by the presence of a susceptible host. Soil suppressiveness against soilborne plant pathogens, induced by the application of organic composts, is a widespread and ubiquitous phenomenon. Nevertheless, a single compost did not show a significant disease suppression against all pathogens and that pathogens were not affected similarly by all composts (Hadar and Papadopoulou, 2012). We can find several approaches with examples of suppressive soils to different pathogens. For example, soils naturally suppressive to Thielaviopsis basicola, causative agent of black root rot of tobacco, were described by Stutz et al., (1986). In this case, disease suppressiveness is a proven property conferred by specific microbial agents that are favored by amendment, specifically, fluorescent Pseudomonads isolates CHAPTER I: GENERAL INTRODUCTION 44 from these soils producers of antifungal compounds such as 2,4-diacetylphloroglucinol (DAPG) and/or hydrogen cyanide (HCN). Additionally, different examples of suppressiveness have been described in literature. The effect of suppressive soil against Rhizoctonia solani, a fungal pathogen of many crops including sugar beet, potato and rice was described by Mendes et al. (2011). Results suggest that the complex phenomenon of disease suppressiveness of soils cannot simply be ascribed to a single bacterial taxon or group, but is most likely governed by microbial consortia, suggesting that a general suppressiveness could be induced by a large metabolically active microbial community. Similarly, Pane et al., (2013), described the ability to control damping-off diseases caused by Rhizoctonia solani and Sclerotinia minor by different composted amendments. In this study, ecological relationships between organic carbon molecular distribution and microbial structure may contribute to discriminate a suppressive compost affecting to microbial community functions. Another example of soils harbouring microorganisms that can efficiently suppress pathogens has been also described by Latz et al. (2016). Their results indicate that plant communities performed soil-disease suppression via changes in abiotic soil properties and the abundance of bacterial groups including Actinobacteria, Bacillus and Pseudomonas. All these studies reflect that plant disease suppression could be considered frequently a direct result of the microbial activities (Hoitink et al., 1986). For this reason, studies focusing on microbial ecology in soil suppressiveness generated by amendments of organic matter could contribute to the identification of the sources of variability and models of action of the microbial communities. However, a lack of ecological theories to guide research in microbial ecology in complex environments has limited the progress in this field of study over the years (Hadar and Papadopoulou, 2012). CHAPTER I: GENERAL INTRODUCTION 45 2.1. Microbial community from suppressive soils The community structure of soil microbiome is influenced by many soil variables, among others, location, structure, particle size, mineral composition, temperature, humidity and agricultural practices (Bailey and Lazarovits, 2003). Moreover, depends on plant species and cultivar, developmental stage, plant growth substrate and stress factors. The composition of root exudates could vary, affecting directly to microbial biomass and activity around the roots. For this reason, plants are able actively selecting for their bacterial rhizosphere (Berg and Smalla, 2009; Berg et al., 2014). Nevertheless, bulk soil microbial composition is essential to rhizosphere community because directly influences the physical-chemical parameters of root zone and the available range of microorganisms which interact with the plant and each other (Bonilla et al., 2012a). In a soil community we can find different groups of microorganisms which may be beneficial or deleterious depending on its abundance such as putative human and plant pathogens and plant beneficial organisms (Mendes et al., 2013). Into the beneficial group of microbes we could found bacterial strains with beneficial effects on plant commonly known as plant growth-promoting rhizobacteria (PGPR) and biocontrol strains, who could use several mechanisms to suppress or reduce the severity of some plant diseases. The four most studied mechanisms used by soil microorganisms in order to induce plant protection by growth-promotion or disease control, are successful competition for C and nutrients, production of antimicrobial compounds toxic against pathogen, predation/parasitism by production of lytic enzymes and induction of plant resistance to disease (summarized in Figure 1). 2.1.1. Competition Disease suppression based on competition could be related to microbial metabolic activities and it is controlled by the availability and rate of utilization of nutrients and CHAPTER I: GENERAL INTRODUCTION 46 energy sources (Hadar and Papadopoulou, 2012). Several examples could be displayed. Mandelbaum and Hadar (1990) studied that competition for C source was suggested as a mechanism of suppression of Pythium aphanidermatum due to oospores could not germinate because of competition by microbial community. In this case, repetitive inputs of glucose and asparragine desequilibrated the microbial population and reduced the suppression phenomenon. Some studies showed Fusarium oxysporum was highly susceptible to competition for nutrients because application of organic matter favoring the increase of competitive microorganisms that could have an antagonistic activity against the pathogen (Alabouvette et al., 2006). In other cases, competition for other type of nutrients such as iron can occur. For example, competition for iron can suppress Fusarium wilt in radish (De Boer et al., 2003) because iron is important due to its extremely low solubility, making it often a limiting element in soil and rhizosphere. For this reason, some microorganism secrete siderophores that chelate iron harming the pathogens growth. CHAPTER I: GENERAL INTRODUCTION 47 Figure 1: Microbiome functions in the suppressive soil-plant system. Effects of bacterial mechanisms used to promote plant growth, control plant pathogens and maintenance of soil quality. P, phosphorus; N2, atmospheric nitrogen; ACC, 1-aminocyclopropane-1-carboxylate deaminase. CHAPTER I: GENERAL INTRODUCTION 48 2.1.2. Antimicrobial compounds The production of secondary metabolites such as antibiotics, biosurfactans or volatiles compounds have an antagonistic effect against pathogen. Antibiotics are lowmolecular weight compounds produced by microorganisms that are deleterious to the metabolism or growth of other microorganisms. For example, pyrrolnitrin (PRN) is a secondary metabolite produced by Pseudomonas and Burkholderia spp. strains with strong antifungal activity to control plant diseases caused by fungal pathogens (Hammer et al., 1997); 2,4-diacetylphloroglucinol (DAPG) is a secondary metabolite implicated in the primary mechanism of suppression of takeall of wheat by Pseudomonas fluorescens strain CHA0 (Weller et al., 2007). Bacillus spp. are able to produce more than two dozens of antibiotics with antimicrobial effects (Pérez-García et al., 2011). These antimicrobial compounds are peptides that are either ribosomally synthesized and post-translationally modified (lantibiotics and lantibiotic-like peptides) or non-ribosomally generated. All of these compounds have different actions: non-ribosomally produced lipopeptides are involved in biofilm and swarming development whereas lantibiotics function as pheromones in quorum sensing (QS; Stein 2005). In the other hand, biosurfactans are amphiphilic compounds that can damage cellular membranes, thereby causing leakage and cytolysis. A wide range of structurally different biosurfactants have been identified to date, including glycolipids, lipopeptides, polysaccharides, proteins and lipoproteins, or mixtures, implicated in interactions with coexisting organisms, including bacteria, fungi, oomycetes, protozoa, nematodes and plants (Raaijmakers et al., 2010). Furthermore, volatile compounds, with an inorganic or an organic (VOCs) chemistry, are commonly produced by bacteria as communication molecules (Effmert et al., 2012), but some of them have been described by its suppressive effects. For example, hydrogen cyanide (HCN) was an inorganic volatile compound produced by several CHAPTER I: GENERAL INTRODUCTION 49 Pseudomonas spp. strains with antagonistic activity against different pathogens first described by Castric et al. (1975). Different studies have described the key role of volatile organic compound in antagonistic events, as described Zhou et al., (2007) who isolated soil bacteria from different groups with VOCs production involves in soil fungistasis. The VOCs emitted from different bacterial antagonists (Pseudomonas spp., Serratia spp., Stenotrophomonas spp. and Bacillus spp.) negatively influence the mycelial growth of the soilborne phytopathogenic fungus Rhizoctonia solani (Kai et al., 2007). Moreover, the antifungal volatiles produced by Collimonas spp. could play an important role in performing its mycophagous lifestyle (Garbeva et al., 2014). 2.1.3. Lytic enzymes Production of lytic enzymes allows microorganism to degrade several components that are present in the cell walls of fungi and oomycetes. Wide variety of bacterial lytic enzymes are known, including cellulases, glucanases, proteases and chitinases. The parasitic activity of various microorganisms toward plant pathogens involves recognition of the pathogen by the antagonist and excretion of several cell-walldegrading enzymes to penetrate the hyphae of its pathogen (Hadar and Papadopoulou, 2012). This type of antagonism cause the death of the plant pathogen resulting in reduction of its inoculum density. Previous work based on isolated microbial community members from chitin-amended soils, showed the increase in bacterial isolates from Streptomyces spp., Stenotrophomonas spp. and Bacillus spp. strains and revealed the molecular diversity of the chitinases present in this soil (Cretoiu et al., 2013). CHAPTER I: GENERAL INTRODUCTION 56 use of high throughput next generation sequencing (NGS) methods (e.g.: Ilumina MiSeq / HiSeq, Ion Torrent PGM, Roche 454 FLX Junior, PacBio RS II) to directly sequence PCR amplicons, facilitate the knowledge of the microbial community faster (and cheaper) that previous methods involves in cloning and sequencing. This technology has also been applied to functional microbial community analysis, using functional gene arrays (FGAs). In this case, NGS technologies allow capturing sequences for targeted genes with available primers. For example, GeoChip® is a comprehensive functional gene array targeting hundreds to thousands genes of different families that play important roles in various biogeochemichal processes, enabling researchers to comprehensively analyze the functional diversity, composition and structure of microbial communities in various environments (Tu et al., 2014). GeoChip® was developed for broader applications in analyzing biogeochemical processes and microbial responses to environmental perturbations with gene families involved in carbon, nitrogen, sulphur and phosphorous cycles, organic remediation, metal reduction and antibiotics, environmental stress responses, bacteriophages and virulence processes (http://www.glomics.com/gch-tech.html). Metagenomics approaches can be complemented with metatranscriptomic, metaproteomic and metabolomic approaches. Metatranscriptomic facilitates insight into the potential expression of genes at the time of the sampling (Carvalhais et al., 2012), providing a real transcriptional profile that correspond to discrete populations within a microbial community. This information can indicate the potential activities of complex microbial community and mechanisms that regulate those, at the time of sampling. But there are some key limitations inherent to metatranscriptomics, such as the half-lives of mRNA molecules, additional amplification steps of starting material which can skew the amplification towards most repeated sequences, and the assigned identification sequence step with comparison CHAPTER I: GENERAL INTRODUCTION 57 with publically available databases instead of databases generated using metagenomics data from the same or highly similar communities (Carvalhais et al., 2012). In the other hand, metaproteomic approaches allow the study of the proteins recovered directly from environmental samples at a given point of time (Wang et al., 2011). These type of approaches complemented the metatranscriptomic studies, favouring the identification of proteins present in soil samples, and gaining insight in the role of them in soil ecological processes (Wang et al., 2011) (Figure 3). In this sense, recent studies suggest that knowledge of volatile compounds produced by microorganisms could help us to get insight in this purpose. Concretely, volatile organic compounds (VOCs) are compounds present in soils, characterized by low molecular weight and high vapor pressure that can act as mediator molecules of interand intraspecies relationships (Effmert et al., 2012). Bacterial VOCs can be considered as important components of the complex interactive mechanisms among bacteria and between bacteria and other organism, including eukaryotes, in their natural environments (Popova et al., 2014). In order to obtain a complete analysis, metabolomics experiment could be performed, capturing the complexity of metabolic networks via the comprehensive characterization of the small-molecule metabolites (such as aminoacids, sugars, and lipids) in biological systems and how they vary in response to a variety of stimuli. Some researches, as Jones et al., (2014), defend that metabolomics has a large practical advantage over other “omic” systems due to a fully annotated genome is not required for analysis, and analytical methods are transferable between species. They proposed the term “community metabolomics” for the application of metabolomics technique to the study of the entire community of a soil sample, obtaining the metabolic profiling that can be used to assess the changes in biochemical profiles of soil communities (Figure 3). CHAPTER I: GENERAL INTRODUCTION 58 4. Case of study: the avocado crop Avocado is an appreciate fruit around the world, due to its excellent nutritional value (https://www.avocadocentral.com). The main avocado-producing and exporting areas are located in South America, but in Europe, we could find avocado crops in the Mediterranean region, mainly restricted to the south of Spain and Portugal. In fact, Spain is the main European country producing avocado, with a production of 69400 tons in 2013 with around a 94% of the total Spanish production exported to the European Union (FAOSTAT, http://www.fao.org/faostat/en/#data). In the Iberian Peninsula, the avocado crop is mainly located in the Andalusian coast (Málaga and Granada) where the area of planting has reached 10000 Ha. In this zone, the production during 2014-2015 has been 47500 tons (40000 tons in 2013-2014), selling to national trade around a 19% and exporting to EU a 78% (mainly France, United Kingdom and Germany; ASAJA Málaga, http://www.asajamalaga.com/?n=1596). 4.1. Avocado root rot diseases Soilborne disease caused by phytopathogenic organisms are one of the main problems for avocado tree (Persea americana, Miller). In southern Spain, the most destructive avocado root rot diseases are caused by Rosellinia necatrix and the oomycete Phytophthora cinnamomi. Phytophthora root rot is the main disease affecting avocados across all continents around the world causing severe losses in fruit production (Pérez-Jiménez, 2008). White root rot caused by R. necatrix is the main root disease in the Mediterranean region, with occur favorable environmental conditions that has turned this disease into one of the main limiting factors for avocado production. Moreover the currently areas used for avocado production were previously occupied by other susceptible plant hosts such as vineyards and almond crops (PérezJiménez, 2008). CHAPTER I: GENERAL INTRODUCTION 59 Avocado trees affected by R. necatrix show aerial symptoms as wilting and drying of leaves and a decline in the general vigour of the tree. The evolution of aerial symptoms occur very quickly and death of an adult tree can occur in a few weeks after the appearance of the first foliar symptoms (Pérez-Jiménez, 2008). The integrated control of the disease have been studied since 90’s decade. First, control of white root rot caused by R. necatrix in other susceptible host as apple, tea or vineyards, was mainly conducted by the use of antifungal compounds. For this reason, fungicides benomyl, carbendazym, methyl thiophanate and fluazinam have been tested in avocado crops. Fluazinam is the most promising fungicide tested in vitro and in vivo (López-Herrera and Zea-Bonilla, 2007). However, R. necatrix have shown to be resistant to several fungicides. Additionally, there are not available tolerant rootstocks to this soilborne pathogen. As alternative to use of chemical compounds, the physical treatment based in soil solarization was implemented successfully in avocado crops in order to eliminate the high temperature sensitive inocula of R. necatrix in the top layers of soil (LópezHerrera et al., 1998). Moreover, biological control approaches based on the application of Trichoderma and/or bacterial strains have been also studied (RuanoRosa et al., 2014) but remain experimental. Bacterial isolates from the rhizosphere of healthy avocado trees were obtained (Cazorla et al., 2006, 2007; Pliego et al., 2007, 2008), and tested for antagonistic activity against R. necatrix due to antagonism is a prevalent trait in the biocontrol bacteria selected by a direct protection strategy of avocado plants (Gónzalez-Sánchez et al., 2010). Several strains belonging to the genera Bacillus and Pseudomonas have been selected. For example, Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium that produces lipases, proteases, siderophores and the antifungal metabolites HPR (2-hexyl 5-propyl resorcinol), PRN and HCN and can also control the avocado white root rot by competition for the same niche and presumably, also for CHAPTER I: GENERAL INTRODUCTION 60 root exudate nutrients (Calderón et al., 2014). Pseudomonas chlororaphis PCL1601 produced proteases, lipases, siderophores, HCN, phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN), compounds with antagonistic activities (Cazorla et al., 2006). A third strain, such as Pseudomonas pseudoalcaligenes AVO110, was isolated due to its efficiently colonization of avocado root tips (Pliego et al., 2008), displaying plant protection by competition for nutrients and occupying the same niche that the pathogen. For this reason, many works have been focused in the study of Pseudomonas spp. strains producer of different antimicrobial compounds, lytic exoenzymes and colonization patterns with implications in the biological control of the pathogen. Furthermore, studies to test the effectiveness of single and combined applications of Trichoderma and rhizobacterial strains to control white root rot were performed. Compatibility between the combined species was observed and these combinations significantly improved the control of R. necatrix in vitro experiments. The combinations of T. atroviride strains with the strains P.chlororaphis PCL1601 and PCL1606 and P. pseudoalcaligenes AVO110 showed the best biocontrol activity against avocado white root rot (Ruano-Rosa et al., 2014). Furthermore, other groups of bacteria have been isolated and identified as antagonistic strains against R. necatrix. Bacillus subtilis PCL1608 and PCL1612, isolated from healthy avocado rhizosphere, presented biological control activity against different soilborne phytophathogenic fungi, including R. necatrix. These bacterial isolates were able to produce antifungal compounds involved in their antagonistic activity such as glucanases or proteases and the antibiotic lipopeptides surfactin, fengycin and iturin (Cazorla et al., 2007). Other example is B. subtilis CB115, isolated from avocado soil, and with an excellent biological control activity against R. necatrix, and antagonistic ability against different soilborne phytopathogenic fungi. This strain also produced CHAPTER I: GENERAL INTRODUCTION 61 several exoenzymes and antibiotics, such as fengycin and surfactin, and was able to induce plant-growth promotion (González-Sánchez et al., 2010). 4.2. Organic amendments in avocado crop Nowadays, demand for organically produced food has increased and many consumers have expressed concern over pesticide residues. Food and environmental safety are often-cited reasons for the use of alternative soil management based on the prevention of the appeareance of the pathogen (Bulluck et al., 2002). For this reason, it is important the usage of preventive management practices such as the use of organic amendments that changes the microbial and physicochemical characteristics of soils. Historically, the use of organic amendments or mulches in avocado crops has produced beneficial effects such as increased root growth and health, reduced plant stress and increased avocado yield (Wolstenholme et al., 1997; Moore-Gordon et al., 1997; López et al., 2014). These phenomena are directly related with the effect of the organic matter input in physicochemical and biotic properties related to microbial diversity, structure and activities (Bonilla et al., 2012b). The use of an appropriate mulch is crucial in organic management of most woody perennial crops such as avocado, because it provides several environmental and agronomical advantages. Moreover, and from a sustainable perspective, residual organic materials from nearby crops or agro-industrial activities would be the best option as sources of mulch. For this reason, farmers in the area have applied composted almond shells to avocado crops, maintaining the C balance due to its richness in lignin and phenolic compounds and influencing in soil microbial enzyme activity (Jafari et al, 2012). Composted almond shells are obtained as a residue and its ligneous character makes this waste suitable to obtain activated C or biomass fuel (Fernández et al., 2012). A work on, the possible uses of the almond shells, made this waste highly recommended as mulch due to its long lasting, availability, low cost and resistance CHAPTER I: GENERAL INTRODUCTION 62 (Esfahlan et al., 2010). In our case of study, the avocado is a subtropical crop whose adaptation to the growth in non-subtropical climates is highly dependent to the presence of a decomposing litter layer, in which its feeder roots proliferate. In this sense, composted almond shells act as reinforcement to the natural dead leaf layer, which is left as a traditional agriculture practice (Wolstenholme et al., 1997). Previous studies by culture-dependent approaches analyzed the population size of several groups of microorganisms in conventional and organic orchards, which use different organic amendments. Bacterial community structure was studied by denaturing gradient gel electrophoresis (DGGE) showing that all of the amendments used affect the soil microbiome structure. The largest effects were shown by commercial composts, especially animal compost that enhance the population sizes of some microbial groups and affecting bacterial community structure in superficial and deep soil layers but stronger in the superficial layer of the avocado soil (Bonilla et al., 2012a). Mesocosms analysis performed with two-year-old avocado trees growing in soil treated with composted organic amendments and then used for inoculation assays, showed that all of the organic treatments reduced disease development in comparison to unamended control soil, especially the amendments of yard waste (YW) and composted almond shells (AS). The YW had a strong effect on microbial communities in bulk soil and produced larger population levels and diversity, higher hydrolytic activity and strong changes in the bacterial community composition of bulk soil, suggesting a mechanism of general suppression. Amendment with AS induced more subtle changes in bacterial community composition and specific enzymatic activities, with the strongest effects observed in the rhizosphere. Even if the effect was not strong, the changes caused by AS in bulk soil microbiota were related to the direct inhibition of R. necatrix by this amendment, most likely being connected to specific populations able to recolonize conducive soil after pasteurization (Bonilla et al., 2015) (Figure 4). CHAPTER I: GENERAL INTRODUCTION 63 Figure 4: State of the art. Soil suppressiveness induced by the application of composted almond shells as organic amendment against Rosellinia necatrix, in avocado crops. WRR, avocado white root rot. OBJECTIVES 65 OBJECTIVES OBJECTIVES 67 The application of composted almond shells induce soil suppressiveness against avocado white root rot, caused by the phytopathogenic fungus Rosellinia necatrix. In this work, we aim to analyze the composition and function of the microbial community in this suppressive-induced soil. Furthermore, the isolation and characterization of relevant group of microorganisms from this soil, could allow the selection of new biological control agents against this soilborne pathogen. Additionally, we will use bacterial strains previously described by their biological control activity against this fungus, in order to improve the knowledge of the community interactions that take place during biological control of this disease. 1.- Unravelling the avocado field soil microbiome composition, and its role in the soil suppressiveness against Rosellinia necatrix. 2.- Isolation and characterization of new biological control agents against Rosellinia necatrix, belonging to a relevant bacterial group from a suppressive-induced soil revealed in previous objective. 3.- Use an artificial bacterial consortium of selected biological control strains, in order to understand the multitrophic interactions occurring in avocado roots. CHAPTER II: Microbiome of a suppressive soil 76 provide information about the phylogenetic diversity of the soil microbial community (van Elsas et al., 2007, 2008; Hirsch et al., 2013; Koyama et al., 2014). Moreover, complementary techniques have arisen, such as microarrays, which have considerable potential in environmental microbial ecology, providing novel insights into how environmental factors affect microbial communities in various habitats (Hazen et al., 2010; He et al., 2012; Bai et al., 2013; Zhang et al., 2013; Tu et al., 2014). The GeoChip microarray is a comprehensive functional gene array (FGA) targeting hundreds to thousands of different gene families that play important roles in various biogeochemical processes, enabling researchers to comprehensively analyse the functional diversity, composition, and structure of microbial communities in various environments. It is a powerful FGA-based technology that can be used to survey the functional diversity, composition, structure, metabolic potential/activity, and dynamics of microbial communities, and then link them with ecosystem processes and functions (Xie et al., 2011; Xue et al., 2013; Cong et al., 2015). Our research interest is focused on the avocado (Persea americana Mill.), for which southern Spain is one of the most relevant zones in the Mediterranean area for this crop. In this part of the world, one of the most limiting soilborne diseases affecting avocado trees is white root rot, caused by the fungus R. necatrix Prill. White root rot is considered to be an emergent threat to many woody crop plants worldwide (Pliego et al., 2009, 2012). The role of soil microorganisms in the plant protection have been broadly reported. Thus, different microbes can contribute to the biocontrol of avocado white root rot using different weaponry such as antagonism (Pseudomonas chlororaphis PCL1606 or Bacillus subtilis PCL1608; Cazorla et al., 2006, 2007), competition for niches and nutrients (Calderón et al., 2014), or induction of systemic resistance or predation (Trichoderma spp.; RuanoRosa and López-Herrera, 2009). These microorganisms can act as single or combined with other biocontrol agents against R. necatrix (Ruano-Rosa CHAPTER II: Microbiome of a suppressive soil 77 et al., 2014). Other studies have reported the positive effect of the application of arbuscular mycorrhizal fungi to soil and the biocontrol activity on avocado (Hass and Menge, 1990; González-Cortés et al., 2012). During the past decades, several approaches have been implemented to achieve an integrated management of R.necatrix, including physical, chemical and biological control approaches (López-Herrera et al., 1998; López-Herrera and Zea-Bonilla, 2007; González-Sánchez et al., 2013). All of these approaches seem to be effective at the experimental level, and some of them have been proven to be effective under certain conditions. However, at the same time, traditional strategies of land management have improved, and some of these strategies could be considered useful approaches to fight against diseases in avocado management, thus increasing the weaponry available against white root rot (Bonilla et al., 2012b). One of these approaches is the use of organic amendments or mulches, which have produced beneficial effects for plants, including increasing health and yields in avocado crops (Moore-Gordon et al., 1997; Wolstenholme et al., 1997; Hermoso et al., 2011). It has been previously shown that the application of such organic matter to avocado agricultural soil can affect soil physicochemical properties and microbial communities (Bonilla et al., 2012a; López et al., 2014). Additionally, organic amendments could play a critical role in global biochemical cycles (Bonanomi et al., 2014) and could cause different effects, such as the improvement of soil fertility and the enhancement of natural suppressiveness of the soil against several phytopathogens (Cretoiu et al., 2013). Several organic amendments have shown an obvious suppressive effect against another important avocado soil-borne phytopathogen, Phytophthora cinnamomi (Bender et al., 1992; Downer et al., 2001). In a previous study, it was shown that different organic matter applied as a mulch to the avocado crop exhibited suppressive effects against white root rot (Bonilla et al., 2015). Composted almond shells were one type of organic matter tested. The CHAPTER II: Microbiome of a suppressive soil 78 application of composted almond shells as a mulch led to an enhancement of the bacterial composition and activities of the soil communities in relation to the observed suppressiveness (Bonilla et al., 2015). The objective of the present study was to gain insight into the microbial profiling present in the amended soils showing suppressive ability against the avocado soil-borne phytopathogen R. necatrix. The use of different microbial approaches should uncover the microbial communities potentially involved in the suppressive phenotype. 2. Materials and methods 2.1 Field of study Soil samples were obtained from an avocado crop field (cv. Hass avocado trees grafted onto cv. Topa-Topa seedling rootstocks) located at the Experimental Station ‘La Mayora’ (IHSM-UMA-CSIC, Málaga, Spain) on the coast of the Malaga Province (SE Spain). This experimental field of 2.5 km2 (36°75’N, 4°04’O) contains 195 40-yearold avocado trees planted at 8 x 8 m. Selected avocado trees were grouped in pairs to facilitate their management. Sixteen pairs of trees were under ecological management (massive application of composted almond shells in 2002, 2007 and 2012), and another 16 pairs of trees were under conventional management (addition of mineral nutrients twice per year, as well as the application of herbicides and pesticides when necessary, López et al., 2014) and without any organic amendment. CHAPTER II: Microbiome of a suppressive soil 79 2.2 Soil sampling Natural field soil samples allocated underneath of avocado trees unamended (CT) or amended with composted almond shells (AS) were taken to perform the different experiments. Soil samples were collected in April 2013, November 2013 and April 2014. Composite soil samples were taken from four different groups of paired trees with (AS) or without (CT) organic amendment and were randomly selected from throughout the avocado orchard. To obtain a composite soil sample, two sampling distal points at 1.5m around the trunk base for each tree of a pair of trees under the same treatment were selected; the upper layer of compost was carefully removed, and 5–10kg of soil samples (15cm depth) were collected per pair of trees and merged. Samples were placed in cold storage and transported to the laboratory. Samples of each type of soil were sieved through a 20mm mesh and immediately used for physicochemical and suppressivenessexperiments.ToperformDNAextractions,three soil samples (1 g each) from composite soil samples per each pair of trees were sieved again (2mm diameter) and processed independently. The remaining unused soil samples were stored at −80◦C. 2.3 Physicochemical analysis of soil samples Physicochemical analysis of both types of soil samples were performed at Laboratorio Caisur S.L. (Granada, Spain) using standardized methodologies. Four subsamples from each field soil sample (AS and CT) were analysed independently. Soil tests included: soil texture, pH, conductivity, total cation exchange capacity (CEC), organic matter, organic C, nitric and total N, C/N, and macroand micronutrients, including phosphorus, potassium, iron, manganese, copper and zinc (Table 1). CHAPTER II: Microbiome of a suppressive soil 80 2.4 Soil processing To test the potential role of soil microorganisms in suppressiveness, we prepared three types of processed soils using different treatments: Field soils (raw soils), heat-treated soils, and complemented soils (Table 2). We applied a moist heat treatment to the field soil samples as previously described (Weller et al., 2002), with slight modifications. Briefly, the heat treatment consisted of heating the soil in high moisture conditions at 100◦C for 20min in an autoclave. The soil was allowed to recover at 4◦C overnight. Then, we performed a second treatment step, heating the soil at 100◦C for 10min in high moisture conditions. After allowing it to cool, the soil was ready to be used Table 1: Physicochemical analysis of natural avocado field soils used in this study. Characteristics of amended with composted almond shells (AS) and unamended (CT) agricultural avocado field soils CHAPTER II: Microbiome of a suppressive soil 81 (Figure 5). Complemented soils were prepared with the purpose of observing the partial recovery of the microbial characteristics of the natural soil (Weller et al., 2002). The complemented soil consisted of heat-treated soil mixed with natural raw field soil in a 9:1 (w/w) ratio (Table 2). To evaluate changes in the culturable microbiota fraction during different times of the soil sample processing, counts of cultivable colony forming units (cfu) of bacteria and fungi per gram of soil were performed. For this, 2 g samples of soil obtained at the different key times during the process were suspended in 20 ml of sterile saline solution (0.85% NaCl) with 0.5 g of sterile gravel and mixed at 150 rpm for 30 min on an orbital shaker at room temperature. Ten-fold serial dilutions of the obtained suspensions were plated on Luria Bertani (LB) agar with 100 mg of cycloheximide per litre, to analyse the heterotrophic bacteria group, and on potato dextrose agar (PDA) with 50 mg of chlortetracycline and 1 ml of tergitol NP-10 (Sigma) per litre (Bonilla et al., 2012a). 2.5 Suppressiveness assays Suppressiveness assays against white root rot caused by the virulent strain Rosellinia necatrix CH53 (López-Herrera and Zea-Bonilla, 2007) were conducted using two different susceptible pathosystems, avocado (Cazorla et al., 2006) and wheat (Triticum aestivum). The R. necatrix inoculum was produced on wheat seeds (Freeman et al. 1986). The seeds were soaked for 12 h in 250-ml Erlenmeyer flasks filled with distilled water. The flasks were autoclaved after excess water had been drained off. After sterilization, fungal disks of a 1-week-old culture of R. necatrix grown on PDA were placed aseptically in each flask. Flasks were incubated at 25°C for 2-3 weeks and were shaken every 2 to 3 days to avoid clustering of the seeds. CHAPTER II: Microbiome of a suppressive soil 82 Table 2: Types of processed agricultural soils used in this study. A scheme of the processing is described in Figure 5. Figure 5: Processing scheme of the soil heat-treatment and complementation used in this study for the agricultural field soil samples. The same procedure was followed for both unamended soil and soil amended with composted almond shells. T0-3 indicates sampling points to perform bacterial and fungal plate counts. CHAPTER II: Microbiome of a suppressive soil 83 Avocado/R. necatrix test system Six-month-old commercial avocado plants were obtained from Brokaw nurseries (Brokaw España, S.L., Vélez-Málaga, Spain). The roots from the avocado plants were disinfected by immersion in 0.1% NaOCl for 20 min and then washed twice (20 min) with sterile distilled water. Then, avocado plants were placed into square plastic pots (10.5 x 10.5 x 10.5 cm) containing 0.64 L of the sieved CT and AS types of soils. Fungal infection with R. necatrix was performed using wheat grains (4 holes of 2 cm depth were made per pot, 3 infected wheat grains were placed per hole) as previously described (Freeman et al., 1986). Non-infected plants were used as controls. Three sets of fifteen avocado plants were tested per type of soil. The plants were grown in a chamber at 25°C with 70% relative humidity and 16 h of daylight and were watered twice per week. Aerial symptoms of avocado white root rot were recorded on a scale of 0 to 3, and a disease index (DI) was calculated after 5 weeks using the previously described formula (Cazorla et al., 2006). Wheat/R. necatrix test system Wheat seeds were disinfected by immersion in 0.05% NaOCl for 10 min, washed and then placed in darkness between pieces of moist filter paper in a growth chamber for 2-3 days at 25°C to induce germination. Then, germinated seedlings were disinfected again by immersion in 0.1% NaOCl for 20 min and washed (20 min) with sterile distilled water. Seedlings were placed into plastic seedling trays (5 cm diameter x 5.5 cm) containing 0.08 L of different types of soils and either infected with R. necatrix using wheat grains (three grains per slot) or not infected to be used as controls. Three sets of fifty wheat seedlings were tested per type of soil. The seedlings were grown in a chamber at 25°C with 70% relative humidity and 16 h of daylight and were watered twice per week. Aerial symptoms were evaluated, and the disease index percentage was calculated as previously described for the avocado/R. necatrix system (Cazorla et al., 2006). Disease index percentage was recorded after evaluation of symptoms, with CHAPTER II: Microbiome of a suppressive soil 84 values ranging between 0 (healthy plant), 1 (yellowing stem base), 2 (drying stem base), and 3 (dead plant). The number of diseased seedlings was determined 7 weeks after beginning the assay, and the disease index was calculated as previously described (Cazorla et al., 2006). 2.6 Soil DNA extraction Soil DNA extraction was performed using 1.0 g of soil samples and a PowerSoil® DNA Isolation Kit (MOBIO Laboratories, Inc, Carlsbad, CA, USA). DNA was extracted from three independent soil samples per pair of trees for amended and unamended soil (AS and CT) and checked for quality. To test the DNA quality we performed a DNA digestion using the restriction enzyme EcoRI (New England BioLabs®, Inc, Ipswich, MA, UK) and PCR amplification of the variable region of the bacterial 16S rDNA with the universal bacterial primers 341F and 907R as described by Muyzer et al. (2004). Digestion and PCR products were analysed for size by agarose gel electrophoresis and ethidium bromide staining. Suitable subsamples were mixed and DNA quantity and quality (A260/A230 >1.8 and A260/A280 >1.7) were evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). Three independent DNA extractions were performed per each pair of trees, and then merged to create a composite DNA sample. Three of these composite DNA extractions were independently analyzed for each type of field soil (AS and CT). DNA was stored at −20◦C for further analyses. 2.7 Analysis of 16S rRNA and ITS gene sequence Two composite DNA samples from each soil type were sent for sequencing by STAB VIDA (NGS Laboratories, Caparica, Portugal) and sent to ChunLab (Seoul, Korea) to obtain the microbial DNA sequences of the 16S rRNA gene and ITS hypervariable CHAPTER II: Microbiome of a suppressive soil 85 regions. Sequences were analyzed using QIIME software (Caporaso et al., 2010) and CLcommunity™ software (ChunLab). Sequences of a length less than 200 nt were excluded from the analysis. The data were filtered for noisy sequences, checked for the presence of chimeras, and binned into OTUs (Peiffer et al., 2013) at the 97% sequence similarity level. A representative sequence of each OTU was taxonomically classified. The relative abundance of microbial clades at different taxonomic levels was calculated as the average value from two independent analyses and was used to perform the comparative distribution analysis. 2.8 GeoChip® analysis Three of the composite samples of purified test DNA (800 ng per sample) from the two different types of soils studied (AS and CT) were sent to Glomics Inc (Norman, Oklahoma) for the sequencing analysis (Tu et al., 2014). Briefly, after the hybridization steps, the arrays were washing, dried and then scanned. The images obtained were analyzed by NimbleScan software (Roche NimbleGen Inc., Madison, WI) using the gridding file containing GeoChip® 4.6 probes and NimbleGen control probes to determine the intensity of each spot and to identify low quality spots, which were removed prior to statistical analysis (probe spots with coefficient of variance > 0.8 were removed). Extracted data were then loaded into the GeoChip data analysis pipeline at the Institute for Environmental Genomics (Microarray Data Manager, http://ieg.ou.edu/microarray/; Liang et al., 2010; Deng and Zhou, 2013). First, the average signal intensity of the common oligo reference standard (CORS) was calculated for each array, and the maximum average value was applied to normalize the signal intensity of samples in each array. Second, the sum of the signal intensity of the samples was calculated for each array, and the maximum sum value was applied to normalize the signal intensity of all of the spots on an array, which produced a normalized value for each spot in each array. Spots were scored as positive based on a CHAPTER II: Microbiome of a suppressive soil 92 Figure 8: Microbial community analysis of most represented phyla in each samples. (A) Relative abundance (percentage) of different Proteobacteria classes (internal circle) and orders (external circle) detected by 16S rRNA gene sequence analysis of soil DNA isolate from amended soil (AS) or conventional soil (CT); (B) Relative abundance (percentage) of different Ascomycota classes (internal circle) and orders (external circle) detected by ITS region sequence analysis of soil DNA isolate from amended soil (AS) or conventionally managed soil (CT). CHAPTER II: Microbiome of a suppressive soil 93 In both soil samples, the phylum Proteobacteria is the most abundant (50.08% and 45.48%). Differences in this group have been shown between the two soil samples. In general, diversity is higher in AS soil samples that exhibit a predominance of the classes Gammaproteobacteria (36%) and Alphaproteobacteria (30%) and a low percentage of Deltaproteobacteria. In CT soil samples, a clear predominance of Alphaproteobacteria can be observed (39%). Remarkably, we observed an increase in AS soil samples (almost 2x) of the orders Steroidobacter (28%) and Burkholderiales (13%) and the decrease of Rhodospirales (from 18% in CT to 8% in AS) (Figure 8A). We observed 76 different classes in AS soil samples and 65 classes in CT soil samples. We detected 24 and 13 specific bacterial classes in AS and CT, respectively, and a slightly higher richness in AS samples (Figure 9A). The analysis of ITS sequences to reveal the abundance of eukaryotic microbes allowed us to identify a high abundance of fungal microbes. Eukaryotic microbes different from fungi ranged from 7.97% (AS) to 9.52 (CT). Among the fungi detected, the unclassified fungi comprises 8.04% (AS) and 4.28% (CT), and those below 1% represent 2.9% in CT soil samples and 3.4% in AS soil samples. The most abundant fungal groups (approximately 70%) that are in both soil samples are of the phyla Ascomycota and Basidiomycota and of the group Mortierellales. In AS soil samples, an increase in the relative abundance of Ascomycota can be observed (Figure 10), (35.37% in CT and 45.79% in AS), as well as a reduction in the group of Mortierellales (18.37 in CT and 9.92% in AS). CHAPTER II: Microbiome of a suppressive soil 94 Figure 9: Quality indixes, alpha-diversity and rarefaction curve of sequencing analysis. (A) Quality indixes (valid reads, OTUs, Chao1 and coverage %) richness (Shannon) , eveness (Simpson) and rarefaction curve obtained of 16S rRNA gene sequence analysis of soil DNA isolate from amended soil (AS) or conventional soil (CT); (B) Quality indixes (valid reads, OTUs, Chao1 and coverage%) richness (Shannon) , eveness (Simpson) and rarefaction curve obtained of ITS region sequence analysis of soil DNA isolate from amended soil (AS) or conventionally managed soil (CT). CHAPTER II: Microbiome of a suppressive soil 95 Figure 10: Analysis of microbial communities present in field soil samples unamended (CT) and amended with composted almond shells (AS). Relative abundance (percentage) of different eukaryotic groups detected by ITS region sequence analysis of soil DNA. Analysis of microbial groups are marked at the class level (thick bars) and at phylum level (boxed thin bars). < 1%, sum of all detected groups with a relative abundance less than 1%. *: taxonomic characteristics of these groups are uncertain. CHAPTER II: Microbiome of a suppressive soil 96 The analysis of the most abundant group of microorganisms (Ascomycota) revealed that in AS soil samples an increase of the class of Dothideomycetes (from 40% in CT to 54% in AS) was observed (Figure 10). Additionally, a reduction of the class of Sordariomycetes (from 38% in CT to 29% in AS) was observed. Also of note in reference to fungal order in AS soil samples, a huge increase of Pleosporales (from 16% in CT to 48% in AS) was observed. Remarkably, one of the fungal order that decreased in AS soil samples was the order Xylariales (from 8% in CT to 3% in AS), where the pathogen R. necatrix is allocated (Figure 8B). We observed 39 different classes in AS soil and 50 classes in CT soil. We detected 7 and 18 specific bacterial classes in AS and CT soil, respectively, and observed a slightly higher richness in CT samples (Figure 9B). 3.4 GeoChip® analysis in soil samples The number of total genes detected by GeoChip analysis and overlapping genes between treatments were measured to understand the functional diversity and structure of the microbial communities. The number of total genes detected ranged from 27348 to 28491 and from 29311 to 33526 in AS and CT samples, respectively. An unpaired Student’s t-test showed that these values were significantly different. The percentage of overlapping genes between samples ranged from 77.18% for AS (77.41%, 75.25%, and 78.88%) to 73.16% for CT (76.25%, 65.70%, and 77.52%) (Figure 11A). This value fell to 65.43% when we compared overlapping genes between treatments (AS13 and CT1-3). DCA (detrended correspondence analysis) and hierarchical clustering (with Bray-Curtis distance) were performed (Figure 11B-C) using all of the detected genes, showing that functional structure of the microbial community was similar in the replicates but different among the soils (AS and CT). CHAPTER II: Microbiome of a suppressive soil 97 Figure 11: Unique and overlapped genes, diversity indexes and beta-diversity from GeoChip analysis. (A) Summarized data of GeoChip analysis: italicized values indicates the number of overlapping genes between samples; values in parentheses shown percentages of overlapping genes between samples; (*) significantly different. (B) Bray-Curtis average distance cluster dendogram. (C) Detrended correspondance analysis (DCA) of samples from amended soil and convetionally managed soil treatments. CHAPTER II: Microbiome of a suppressive soil 98 To understand the effects of composted almond shells on the microbial communities and the acquired suppressive capacity, microbial functional genes categorized as participating in biogeochemical cycles and other important soil processes were examined (Figure 12). Gene functions related to the carbon cycle were the gene category most represented in all samples. C cycling probes were significantly more abundant than other categories in AS samples (36.65% in AS and 34.54% in CT), whereas genes related to organic contaminant degradation (12.42% in AS and 12.81% in CT), metal resistance (14.58% in AS and 16.32 in CT) and virulence (1.59% in AS and 1.61% in CT) were significantly more abundant in CT samples. There were no significant differences in N, P and S cycle genes and other gene categories such as stress, fungi functions, soil benefit and soilborne pathogens (Figure 12). Key genes for acetogenesis, C degradation, C fixation, methane metabolism and other genes related to the C cycle were detected in the two types of soils (Figure 13A). The relative abundance of genes related to the C degradation category were the highest and exhibited significant differences between the AS samples and the CT samples. In this category, we found the presence of degradative genes of the most abundant C sources derived from plant and animal sources that could be present in soil ecosystems, such as starch, hemicellulose, cellulose, chitin and lignin. There were few significant differences between samples in these categories of detected genes (Figure 13A). Of the nitrogen cycle category, only the ammonification subcategory had a higher significant difference for amended soil (Figure 13B). In this subcategory, there are genes that function in the decomposition of organic matter and cycling of accumulated N source. Related to the sulphur cycle, the analyses performed exhibited a higher significant difference (p<0.1) in only the sulphite reductase genes of AS samples compared to CT samples. These genes encode enzymes that catalyse the reduction of sulphite to sulphide, using iron as cofactor, and provide a source of S to microbiota. CHAPTER II: Microbiome of a suppressive soil 99 . Figure 12: GeoChip analysis of functional gene categories. Relative abundance of all detected genes from different gene categories analysed in this study. * indicates significant statistical differences (p<0.1) between the two types of soil samples, amended soil (AS) and conventionally managed soil (CT). Standard deviation bars are shown. CHAPTER II: Microbiome of a suppressive soil 100 The CT samples exhibited a higher significant difference in sulphate reductase, a protein involved in sulphur reduction by anaerobic respiration (Figure 13C). Statistical analyses showed no significant differences in the relative abundance of genes involved in the phosphorous cycle for these samples. The analysis of genes in the category of environmental adaptability showed significant differences (p<0.1) in the subcategories, as shown in Figure 13D-F. Genes involved in the organic degradation of aromatics, such as chlorinated and pesticide-related compounds, had a higher significant relative abundance for amended soil than conventional managed soil. Similar results were obtained for genes related to osmotic and oxygen stress, from the stress category, and metal resistance to cobalt and lead, which had slightly higher significant relative abundance for AS samples than CT samples. On the other hand, unamended soils exhibited significantly higher values of relative abundance for genes related with stress induced by glucose limitation and metal resistance to cadmium and other metals. The category of plant interaction covers a wide range of different functional genes involved in microbial interactions with plants, including genes related to fungal function, soil benefit, soilborne pathogens and virulence. The analyses performed showed significant differences (p<0.1) in some subcategories, as shown in Figure 13GJ. There were not any significant differences in the genes in the categories of soil benefit or fungi function. Nevertheless, CT samples exhibited a higher significant relative abundance of detected genes from the oomycetes subcategory (soilborne pathogen), which included different genes from this pathogenic group. Genes related to virulence processes such as iron oxidation or secretion had a higher significant relative abundance for amended soils; whereas unamended soils exhibited significantly higher values for genes involved in virulence actions such as iron uptake (aerobactin genes) and pilin formation CHAPTER II: Microbiome of a suppressive soil 101 CHAPTER II: Microbiome of a suppressive soil 108 values of glucose, fructose or sucrose, The characteristics and composition of AS makes this susbstrate an acceptable growing media for soilless culture (Valverde et al., 2013). Moreover, it must be taken into account that the avocado is a shallow rooted tree, with needs good aeration. Roots are helped by the presence of rich surface of organic mulch, as shown by the tendency of healthy feeder roots to grow into any decomposing litter layer (Chanderbali et al., 2013). In this work, a metagenomics approach to the community composition of amended and unamended avocado soils have been performed for the first time. The use of metabarcoding and GeoChip techniques allowed a better knowledge on the community composition and their potential activities. In first place, an attempt to identify key factors involved in this enhanced suppressivity after the addition of organic amendments revealed the crucial role of the microbiota present in the orgaic amended soil. The microbiota evolved in the composted almond shells was crucial for suppressiveness because the reduction of the bacterial population after a heat treatment in the organic amendment resulted in a more conducive phenotype (heat-treated soil samples harbour 105 cfu/g, most likely composed mainly by sporulated bacterial and fungal microorganisms). Moreover, total or partial suppressiveness was recovered when these heat-treated soil samples were complemented with a portion of soil influenced by AS, but it remained conducive when complemented with a portion of conventional soil (CT). This effect has been previously described for different suppressive soils, where sterilization by autoclaving, steam pasteurization, and irradiation rendered soils conducive to the pathogen studied (Malajczuk, 1983; Weller et al., 2002; Mendes et al., 2011). Suppressiveness experiments performed do not excluded the possibility that the disinfected avocado root used could harbour endophytic microorganisms, but our results significantly pointed out the role of the composted almond shells in the plant protection against R. necatrix. Thus, our results CHAPTER II: Microbiome of a suppressive soil 109 support the crucial role of microbes present in AS for turning the conducive CT soil into a more suppressive soil against R. necatrix. To gain insights into the microbial diversity present in the soil samples, we used several different approaches. Phylogenetic marker analysis based on the sequencing of 16S rDNA and ITSs revealed a relatively similar array of prokaryotic and eukaryotic populations present in the AS and CT soil samples; however, a different response has been described in the literature for other types of organic matter from different sources, such as composted municipal waste (Zaccardelli et al., 2013). It is remarkable that in our model system, the group of fast-growing, easily cultivable Proteobacteria is the dominant group of prokaryotes in both soil samples. These data are similar to those previously observed for other soil and rhizosphere samples with a high presence of organic matter (Lynch and Whipps, 1990; Paul and Clark, 1996; Hawkes et al., 2007; Mendes et al. 2011). Moreover, the representation of the other phyla different than Proteobacteria were quite similar among AS-amended and unamended soils, thus contradicting the idea that a highly specific community is stimulated by the addition of AS. Diversity analysis confirmed the previously obtained results (Bonilla et al., 2015), highlighting the enhancement of specific microbial populations in AS-amended samples, such as Betaproteobacteria (Burkholderiales) and the class of Gammaproteobacteria, which have been reported to protect plants from fungal infections in other suppressive soils (Mendes et al., 2011). It is important to note the clear enhancement in AS-amended soil of the order Steroidobacter, previously reported to play an essential role in the positive interactions with plants; for example, controlling seed germination, stem and root elongation or stress protection in plants (Zarraonaindia et al., 2015). In contrast, analysis of eukaryotic ITS revealed a different abundance distribution of microbes among the two types of soil samples. Fungal clones were the most common and dominant microbial eukaryotes in the soil. AS-amended soil samples had an CHAPTER II: Microbiome of a suppressive soil 110 increased relative abundance of Ascomycota. This fact is not surprising considering that Ascomycetes are the largest group on true fungi (Larena et al., 1999). Moreover, the dominance of Ascomycota has been observed during different composting processes (De Gannes et al., 2013; Neher et al., 2013), where most of them are saprophytic and live on dead organic material that they help decompose (Agrios, 1997; Viebahn et al., 2005). This behaviour easily explains their higher abundance when composted almond shells are added to the soil as a mulch. Within Ascomycota, the group that exhibited the most apparent and highest increase of abundance in ASamended soil samples was the fungal class of Dothideomycetes. A high abundance of Dothideomycetes in soils with at high hydrocarbon concentrations has been previously reported (Ferrari et al., 2011), suggesting its preference for those habitats with a high concentration of organic matter where it participates in biomass conversion (Shrestha et al., 2011). Moreover, the large increase of the phylum Pleosporales (Dothideomycetes) is also not surprising because this group is very well known to contain species that chlorinate lignin as a first step of biomass conversion during plant litter degradation (Ortíz-Bermúdez et al., 2007). Interestingly, it has been shown that several genera of Dothideomycetes exhibit an increased presence in suppressive soils because they harbour endohyphal bacteria from groups that are capable of hydrocarbon biodegradation, such as the Xanthomonadales, Pseudomonadales, Burkholderiales and Sphingomonadales (Hoffman and Arnold, 2010). Dothideomycetes have also been shown to increase slightly in AS-amended soils. However, the group that shows an apparent decrease in AS-amended soils is Mortierellales. This group has a complex phylogeny (Wagner et al., 2013) and is considered to be ubiquitous in the bulk and rhizospheric soil, implying that it could play a role in maintenance of the microecological balance (Miao et al., 2015). Interestingly, the group of Glomeromycota, which contains different groups of symbiotic fungi previously detected in avocado (Hass and Menge, 1990; González-Cortés et al., 2012), it is clearly CHAPTER II: Microbiome of a suppressive soil 111 detected in unamended soils, but decreased in the amended ones (below 1%). A possible explanation could be that in the AS amended soils, take place a strong competition with other decomposing fungi, such as the Dothideomycetes, more adapted to an environment with high amount of decomposing organic matter. Finally, it should be noted that members of Xylariaceae, to which R. necatrix belongs (Pliego et al., 2012), are less abundant in AS-amended soils, thus revealing a negative effect on this fungal group. These results indicate that the soil fungal community was affected by the soil amendment with AS. Phylogenetic markers such as the prokaryotic 16S and eukaryotic ITS region do not carry explicit functional information. For this, the use of GeoChip-based analysis allowed for the analysis of microbial functional genes encoding key enzymes involved in major biogeochemical processes that facilitate linking microbial community structure to potential ecological functions (Torsvik and Ovreas, 2002). Using this technique, we screened potential functional gene diversity among unamended and ASamended soil samples. Probe signals and DCA analysis indicated that the microbial community functional structures differed between CT and AS soil samples. The sample sites are very close together, so the differences observed in the microbial communities are thought to be the result of amendment with organic matter. Generally, similar abundance patterns of functional genes involved in nutrient cycling processes such a nitrogen, phosphorous or sulphur cycling, were found in both types of samples. However, AS-amended samples had higher signal intensities for C degradation (carbon cycle) genes than CT, with some differences being statistically significant. Substrates for this group of genes ranged from labile C to more recalcitrant C (e.g., starch, hemicelluloses, cellulose, chitin and lignin). These results suggest that AS-amended microbial have a greater capacity for C degradation than CT communities. This suggests, as expected, an important role of carbon cycling in CHAPTER II: Microbiome of a suppressive soil 112 response to the addition of organic matter to the soil. However, no differences in gene abundance for N, P or S cycling was observed. This can be explained because almond shells are a lignin-rich waste resulting from the almond industry, mostly composed of approximately 27% lignin and 73% holocellulose (Caballero et al., 1996), and those cycles were not compromised. However, statistical differences in the abundance of genes related to organic remediation and metal resistance were observed in ASamended soil displaying lower levels than CT. This observation may be due to a decrease in the available compounds due to the high sorption ability of the composted almond shells and derivate compounds from its degradation, which have been previously reported to be able to remove such substances from the soil (Pehlivan et al., 2009). Interestingly, both soil samples shared a core of probes corresponding to approximately 90% of the assayed sequences (27364 probes). However, approximately 10% of the total probes analysed were unique for AS-amended samples (2766 probes). When the sequence of these probes were analysed, they resulted in a very similar distribution to that previously shown for the whole GeoChip analysis, with above 34.5% corresponding to C cycling, followed by probes related to organic remediation (14.5%), stress (13.4%), metal resistance (11.9%) or the N cycle (8.6%). These results support the following previously described results: systems associated with organic matter-mediated general suppression; suppression typically occurs as a result of the activation of the indigenous microbial community (Lockwood, 1990); and suppressive activities can be generated by one to few populations of organisms (Gerlagh, 1968; Cook and Baker, 1983; Hoitink and Boehm, 1999; Weller et al., 2002). Postma et al. (2000) found that qualitative rather than quantitative shifts in the bacterial community correlate with disease suppressiveness, and several studies indicated that mechanisms within the microbial activity of the soil are responsible for CHAPTER II: Microbiome of a suppressive soil 113 the suppression of pathogens (Rovira and Wildermuth, 1981; Nitta, 1991; Workneh and van Bruggen, 1994; van Os and van Ginkel, 2001). Among the specific taxa stimulated, Pseudomonadaceae, Burkholderiaceae, Xanthomonadales and Actinobacteria, harbour genera and species with activity against plant pathogenic fungi (Postma et al., 2010). Additionally, it is important to note that Pseudomonas, Rhizobium, Bacillus, Variovorax, Phyllobacterium, and Azospirillum, are considered the most efficient plant growth-promoting bacteria (Bertrand et al., 2001). Sequencing of specific probes present in AS-amended soils revealed the presence in such soil samples of genes for bacterial and fungal catalases, phenazine biosynthetic genes (from Proteobacteria) or the presence of potential antibiotics produced by Actinobacteria (data not shown). Nearly all of these probes corresponded to the GeoChip category “soil benefit”, where the antimicrobials from different groups were analysed. To the best of our knowledge, no probes from Bacilli were used, so the role of antimicrobials such as iturin or fengicins, produced by Bacillus spp., cannot be discussed based on our results. It is important to note that the genus Pseudomonas (class Gammaproteobacteria) and Bacillus (class Bacilli) are two of the most prominent bacteria that can be isolated from avocado soil and rhizosphere displaying antifungal activity and plant protection against soilborne pathogens (Cazorla et al., 2006; 2007; González-Sánchez et al., 2010). Our results reinforce the importance of such microorganisms in the soil and root ecology of the avocado crop. These groups of microorganisms can produce metabolites, such as siderophores and antibiotics, with specific suppressive activity against soilborne pathogens. Antagonistic pseudomonads, including Pseudomonas chlororaphis, play a role in white root rot suppressiveness (Cazorla et al., 2006; Calderón et al., 2014). However, other types of rhizobacterial taxa may differ in prevalence between suppressive and conducive soils, suggesting that the microbial CHAPTER II: Microbiome of a suppressive soil 114 basis of white root rot could be far more complex than solely a Pseudomonas property; it has also been observed for other pathosystems such as Thielaviospsis basicolamediated black root rot of tobacco (Almario et al., 2014). 5. Conclusion In conclusion, and taking together the results obtained in this work and in previous works related, a theoretical model about the role of the microorganisms in enhancing suppressiveness after amendment with composted almond shells can be proposed (Figure 15). Soil amendments with composted almond shells resulted in an extra input of organic matter rich in lignin that could be initially degraded by fungal members of the community (such as Dothideomycetes) and Actinobacterias. Lignin degradation from composting almond shells would produce a progressive release to the soil of more simple compounds.Those compounds, together with others also present in the almond shells, could lead to an increase in carbon sources available, such as cellulose, hemicellulose, and aromatic compounds. At this point, some Proteobacteria already present in the soil (such as Gammaproteobacteria and Betaproteobacteria) could take advantage metabolizing that available organic matter, thus slightly enhancing their population. These groups of microorganisms could harbor, among other, genes involved in antifungal enzymatic activities and production of antimicrobial compounds that could have an effect on the interaction with other microbes. The resulting modified microbiota after addition of composted almond shells could be more active against some groups of phytopathogenic fungi (as Xylariales, where R. necatrix is included) finally showing a phenotype of induced suppressiveness effect. CHAPTER II: Microbiome of a suppressive soil 115 Figure 15: Hipothetical mode of action of almond shells amendment. Theoretical model proposed about the role of microorganisms in increasing suppressiveness after adition of composted almond shells to agricultural soil. The extra input of organic matter rich in lignin could be degraded by Ascomycetes and and Actinobacterias. An increase in carbon sources available occur and other polysacharides, as cellulose and hemicellulose, and aromatic compounds could be metabolize by Proteobacteria. These groups of microorganisms produce enzymes with antifungal activities (such as chitinases) and antimicrobial compounds (such as phenazine). Modified microbiota could have specific activity against fungi pathogens as Xilariales, where Rosellinia necatrix is included. The effect of microbial community changes induced a suppressiveness in the agricultural soil. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 117 CHAPTER III Characterization of biocontrol strains after an amendment with composted almond shells from a suppressiveness-induced soil SUBMITTED IN: Vida, C., Cazorla, F.M. and de Vicente, A. (2017) Characterization of biocontrol strains after an amendment with composted almond shells from a suppressivenessinduced soil. Res. Microbiol. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 118 Abstract The improvement of soil quality in avocado crops through organic amendments with composted almond shells has a positive effect on crop yield and plant health and serves as an enhancement of soil suppressiveness against the phytopathogenic fungus Rosellinia necatrix. In previous studies, induced soil suppressiveness against this pathogen was related to the stimulation of Gammaproteobacteria, especially some members of Pseudomonas spp. with biocontrol-related activities. In this work, we isolated bacteria from this suppressiveness-induced amended soil using a selective medium for Pseudomonas-like microorganisms. We characterized the obtained bacterial collection to aid in identification, including metabolic profiles, antagonistic responses, hybridization to biosynthetic genes of antifungal compounds, production of lytic exoenzymatic activities, and plant growth promotion-related traits, and sequenced and compared amplified 16S rDNA genes from some representative bacteria. The final selection of representative strains mainly belonged to the genus Pseudomonas but also included the genera Serratia and Stenotrophomonas. Their biocontrol-related activities were assayed using the experimental avocado model, and the results showed that all the selected strains protected the avocado roots against R. necatrix. This work confirmed the biocontrol activity of these Gammaproteobacteria-related members against R. necatrix following specific stimulation in a suppressiveness-induced soil after a composted almond shell application. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 125 Bacterial colonies from the culture plates were spotted onto a nylon membrane (Nytran®N, GE Healthcare Life Science, USA) and processed by colony blotting as previously described (Matas et al., 2014). The spotted membrane was dried, and the DNA was crosslinked in a UV chamber. Prior to DNA fixation, 0.5 µl of DNA was extracted from the control reference strains using the UltraClean® Microbial DNA Isolation Kit (MOBIO Laboratories, Inc., Carlsbad, CA, USA) following the manufacturer’s instructions and was then added to the test membrane. For membrane hybridization, we used the DIG Easy Hyb kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. Immunological detection was performed with an anti-digoxigenin antibody conjugated to alkaline phosphatase. The DIG-labelled nucleic acids were detected by chemiluminescence using a Molecular Imager ChemiDoc system (Bio-Rad). Table 6: Oligonucleotide sequences used in this study to obtain probes for detection of genes responsible of antifungal production. *Published in Calderon et al., 2013 * CHAPTER III: Isolation of bacterial strains with biocontrol abilities 126 2.5 Enzymatic activities Plate-based assays were used for the direct characterization of bacterial hydrolytic activities. We tested for lipase, protease, amylase, β-glucanase, cellulase, and chitinase enzymatic activity. Luria Bertani (LB) agar (0.8%) plates were supplemented with different test substrates to evaluate each enzymatic activity after incubation at 25°C for 2-5 days. LB plates with Tween 80 (2%, Sigma-Aldrich, Madrid, Spain) were used for lipase detection (Howe and Ward, 1976); the assay was considered positive when the formation of a calcium oleate precipitate was observed. LB plates with powdered milk (3%) were used for protease detection (Gerhardt, 1994) and plates with colloidal chitin (0.2%, Sigma-Aldrich, Madrid, Spain) were used for chitinase detection (Murthy and Bleakley, 2012); in the protease and chitinase assays, the presence of clearing zones (halos) surrounding bacterial growth was considered a positive result. LB plates with starch (0.75%, Panreac, Barcelona, Spain) were used to evaluate amylase activity, whereas plates with lichenan (0.1%, Megazyme, Barcelona, Spain) were used to detect β-glucanase activity (Walsh et al., 1995) and plates with 1carboxymethylcellulose (0.75%, Panreac, Barcelona, Spain) were used to detect cellulase activity (Hankin and Anagnostakis, 1995); for the amylase, β-glucanase and cellulase assays, the plates were stained with Congo red (0.3%) for 30 min, and clearing halos around the colonies represented a positive enzymatic response. 2.6 PGP-related activities The collection of 246 bacterial isolates was tested for phosphate solubilisation and siderophore production as activities related to plant growth promotion (PGP). To identify phosphate-solubilizing activity, bacterial isolates were grown in glucose-yeast (GY) broth containing 8 g of agar, 50 ml of K2HPO4 (10%) and 100 ml of CaCl2 (10%) per litre. The plates were incubated for 5 days at 25°C, and the formation of visible CHAPTER III: Isolation of bacterial strains with biocontrol abilities 127 clearing halos around the tested colonies indicated phosphate solubilisation (SylvesterBradley et al., 1982). Siderophore-producing bacteria were detected using a mixture of M9 medium salt (1x) without Na2HPO4, 30.24 g of PIPES buffer, 9 g of agar, 30 ml of casamino acid solution (10%), 10 ml of glucose solution (20%) (Cordero et al., 2012) and 100 ml of chromazurol S complex [CAS/iron(III)/hexadecyl-trimethyl ammonium bromide] per litre. The plates were incubated at 25°C for 48 h. Siderophore-producing bacteria formed a yellow-orange halo in the blue-green medium background. From the isolate collection, 24 representatives were selected based on their differential responses in the previous tests (Figure 18). In vitro seedling growth promotion assays were performed following the procedure of Ryu et al., 2005 with modifications. The 24 representative selected isolates were assayed for the ability to promote tomato (Solanum lycopersicum L.) seed germination and plant growth. Tomato seeds (c.v. “Moneymaker”) were disinfected by immersion in 0.1% NaOCl for 20 min and then washed (20 min) with sterile distilled water. Ten tomato seedlings were dipped in 1 ml of a 1×109 cfu/ml suspension of each selected test isolate. Bacillus amyloliquefaciens CECT8237 was used as a positive control for PGPR activity (Magno et al., 2015) and P. chlororaphis PCL1606 was used as a negative control. After 20 min of inoculation, the excess suspension was poured off, and the inoculated seedlings were transferred to test tubes with 3 ml of Murashige and Skoog agar (0.8%) and incubated at 25°C in the dark for 3 days. Then, the seedlings were grown at 25°C under a diurnal cycle of white light/dark (16/8 h). The average fresh weight (mg) was calculated after 10 days of growth. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 128 2.7 Identification using 16S rRNA gene partial sequencing To help characterize the 24 selected isolates, sequencing of the variable region of the bacterial 16S rRNA gene was performed. PCR amplification was performed using the primers 8F (5′-AGR GTT YGA TYM TGG CTC AG -3′) and 1391R (5′-GAC GGG CGG TGT GTR CA-3′) (Klindworth et al., 2013). The PCR products were checked by electrophoresis in an agarose gel (0.8%) and sequenced by Macrogen Europe (Amsterdam, the Netherlands). Identity studies comparing the partial sequences to previously deposited sequences were performed using the National Center for Biotechnology Information (NCBI) GenBank Blast software (Bethesda, MD, USA). Figure 18: Workflow used in this study to characterize soil bacterial isolates with biocontrol ability against avocado fungal pathogen Rosellinia necatrix. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 129 The corresponding sequences displaying a similarity higher than 85% were deposited in the GenBank database (Table 7). 2.8 Biocontrol activities Based on the previous results, 8 representative bacterial isolates were selected for the biocontrol experiments (Figure 18). Biocontrol assays against white root rot caused by * ** *, pair of bases, **, percentage of identity with others bacterial uploaded sequences in GenBank database obtained by Blast tool. Table 7: Information of partial sequences of 16S rRNA gene obtained by PCR methology from 24 selected bacterial isolates submitted to GenBank database. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 130 the virulent strain R. necatrix CH53 were conducted using the susceptible pathosystem on avocado plants (Cazorla et al., 2006). Briefly, the biocontrol assays were performed with six-month-old commercial avocado plants supplied by Brokaw nurseries (Brokaw España, S.L., Vélez-Málaga, Spain). The roots from the avocado plants were disinfected by immersion in 0.1% NaOCl for 20 min and then washed (20 min) with sterile distilled water. The roots were bacterized by immersion in a suspension of the bacterial isolates or mixtures (108 cfu/ml) or in sterile LB medium as a negative control for 20 min. The avocado plants were placed into square plastic pots containing potting soil. Fungal infection with R. necatrix was performed using infected wheat grains as previously described (Freeman et al., 1986). Non-bacterized plants were used as the controls. Fifteen avocado plants were tested per treatment. The plants were grown in a chamber at 25°C with 70% relative humidity and 16 h of daylight and were watered twice per week. Aerial symptoms of the disease in the avocadoes were recorded on a scale of 0–3, and the disease index (DI) was calculated at the end of the assay (Cazorla et al., 2006). 2.9 Statistical methods The biocontrol and seed growth promotion test data were statistically analysed using analysis of variance (Sokal and Rohl, 1986), followed by Fisher’s least significant difference test (p = 0.05) using the SPSS 22 software (SPSS Inc., Chicago). CHAPTER III: Isolation of bacterial strains with biocontrol abilities 131 3. Results 3.1 Isolation and characterization A collection of culturable bacteria from composted almond shells-amended soil was constructed using a selective medium for Pseudomonads (Larkin and Honeycutt, 2006) and related groups (Sands and Rovira, 1970). A total of 246 bacterial isolates were initially selected based on the abundance and differences in colony morphology displayed on the plates. Physiological and metabolic tests were performed to group the isolates. All 246 isolates responded as Gram-negative and catalase-positive bacteria. The diversity observed in the additional tests allowed the bacterial isolates to be clustered into 5 groups. One hundred and forty-eight isolates (60.2%) that were glucose fermentative and oxidase negative with non-fluorescent and non-pigmented colonies were grouped as Enterobacteriaceae-like (group A). Twenty-six isolates (10.5%) were glucose oxidative, oxidase positive and producers of fluorescent pigments under UV light; these isolates were considered fluorescent Pseudomonadaceae-like (group B). Eleven isolates (4.5%) were glucose oxidative, oxidase positive and non-producers of fluorescent pigment; thus, these isolates were grouped as non-fluorescent Pseudomonadaceae-like (group B’). Twelve isolates (4.9%) were glucose oxidative, oxidase negative and yellow pigmented colonies and were clustered as Xanthomonadaceae-like (group C). Finally, 49 bacterial isolates (19.9%) could not metabolize glucose and were oxidase negative and were grouped as unclassified isolates (group D). 3.2 Antagonistic activity of bacterial isolates from soil Further characterization of biocontrol-related traits was performed for the collection of 246 isolates. The antagonistic abilities against the phytopathogenic soilborne fungi R. necatrix CH53 (Rn) and F. oxysporum f. sp. radicis-lycopersici ZUM2407 (Fo) and CHAPTER III: Isolation of bacterial strains with biocontrol abilities 132 the phytopathogenic oomycete P. cinnamomi 344 (Pc) were assessed using dual plate assays. A total of 22% of the isolates exhibited antagonistic behaviour (n=55) (Figure 19A). The antagonistic isolates were mainly present in groups A (n=31) and B+B’ (n=17+4), with many representative isolates displaying antagonism to all three soilborne pathogens assayed (A=8 and B=8). The number of strains antagonistic to the avocado phytopathogens R. necatrix and P. cinnamomi were very similar (n=27 and n=29 isolates, respectively). However, more strains were antagonistic to F. oxysporum (n=37). To gain insights into the nature of the antagonistic activity, colony blotting was performed using digoxigenin-labelled probes to assess antifungal compounds (Castric, 1975; Howell and Stipanovic, 1979; Chin-A-Woeng et al., 1998; Cazorla et al., 2006). A total of 11% of the bacterial isolates exhibited hybridization to biosynthetic antimicrobial product genes (n=27). Group B+B’ showed the highest number (n=12+4) of representatives with hybridization to the different probes (Figure 19B). Some of the bacterial isolates showed multiple hybridization signals to 2 or 3 antimicrobial biosynthetic genes. Specifically, 13 isolates showed the presence of both HPR biosynthetic genes and PCA and HCN biosynthetic genes; this group was the most abundant. In this specific group, we primarily found 8 fluorescent Pseudomonadaceae-like isolates as the more represented group of strains (group B) as well as 3 hypothetical Enterobacteriaceae-like (group A) bacteria. No hybridization signals were observed for the PRN and DAPG biosynthetic genes with the exception of the control strains PCL1606, BL915 and Pf5 (Table 8). 3.3 Enzyme production The total collection of 246 bacterial isolates were analysed for the production of exoenzymatic activities (lipases, proteases, amylases, β-glucanases, cellulases and chitinases). Seventy-eight percent of the isolates (Figure 19C) were positive for at least CHAPTER III: Isolation of bacterial strains with biocontrol abilities 133 Figure 19: Characterization of biocontroland PGP-related traits for the collection of bacterial isolates (n= 246) and the number of isolates displaying each response (n). A) Antagonism against soilborne phytopathogens. (B) Detection of biosynthetic genes for antifungal production. (C) Enzymatic activities. (D) PGP-related activities. Rn: Rosellinia necatrix; Fo: Fusarium oxysporum f. sp. radicis-lycopersici; Pc: Phytophthora cinnamomi; Lip: lipase; Pro: protease; Chi: chitinase; Pho: phosphate solubilisation; Sid: siderophores; HPR: 2-hexyl 5-propyl resorcinol; PCA: phenazine-1-carboxylic acid; HCN: hydrogen cyanide; PLT: pyoluteorin; PRN: pyrrolnitrin; DAPG: 2,4-diacetylphloroglucinol. CHAPTER III: Isolation of bacterial strains with biocontrol abilities 134 one of the enzymatic activities tested (n=191). Of the bacterial isolates with enzymatic degradation abilities, 172 isolates exhibited lipase activity, 148 strains produced proteases and 43 isolates had positive results for chitinase. Some of the isolates shared the production of 2 or 3 exoenzymes. Forty-three percent (n=106) of the bacteria exhibited lipase and protease activity (A=48, B+B’=8+3, C=7, and D=40). Furthermore, 28 strains produced lipase, protease and chitinase enzymes (A=20, B’=2, C=1, and D=5). Eight strains shared lipase and chitinase production, all of which were from the Enterobacteriaceae-like group, and only 2 of the isolates produced the protease and chitinase enzymes. No amylase, β-glucanase or cellulase activity was detected in any of the isolates analysed in this study. 3.4 Plant growth promotion-related activities Plant growth promotion-related activities were analysed for the bacterial isolate collection (n=246). Seventy-four percent of the isolates only had the capacity to solubilize a non-soluble phosphate source (A=44, B+B’=4+1, and D=5), and only 61 isolates produced siderophores (A=14, B+B’=7+1, C=2, and D=17) (Figure 19D). Twenty isolates produced both activities, 18 of which were Enterobacteriaceae-like. To gain insights into the effective contribution of representative selected isolates (n=24) to plant growth, a tomato seedling growth promotion assay was performed. The bacterial isolates ‘17’ (Enterobacteriaceae-like) and ‘20’ (fluorescent Pseudomonadaceae-like, Figure 20) showed a significant increase in the average fresh weight (mg) at the end of the experiment similar to the positive control plant growthpromoting bacterium Bacillus amyloliquefaciens CECT8237 (Magno et al., 2015). CHAPTER III: Isolation of bacterial strains with biocontrol abilities 141 observed in the obtained isolates agreed with previous observations reporting specific isolation of Gram-negative bacteria with different metabolic profiles (Sands and Rovira, 1970). In this study, we used a workflow of experiments chosen to characterize the collection of suppressiveness-inducing soil isolates (Lugtenberg and Kamilova, 2009). In this case, dual plate antagonism assays were performed against different fungal (R. necatrix CH53 and F. oxysporum f. sp. radicis-lycopersici ZUM2407) and oomycete (P. cinnamomi 344) phytopathogens. The results showed a slightly higher number of soil isolates with antagonistic activity to F. oxysporum (n= 37) than to the avocado pathogens R. necatrix (n= 27) and P. cinnamomi (n=29). Interestingly, many of the isolates with antagonism belonged to the Enterobacteriaceae-like group (A; n=31), followed by the fluorescent Pseudomonadaceae-like group (B; n=17) and the nonfluorescent Pseudomonadaceae-like group (B’; n=4). Several Pseudomonas spp. have been described previously as biocontrol agents for avocado crops (Cazorla et al., 2006; Pliego et al., 2008; González-Sánchez et al., 2013). Previous studies showed that antagonism to different phytopathogens was a prevalent trait in the selected strains, suggesting that antagonism could be a useful strategy to select biocontrol strains for this plant-pathogen system (González-Sánchez et al., 2013). Fungal antagonism is usually mediated by different compounds, of which the antifungal antimicrobial compounds are of major importance (Raaijmakers et al., 2002). A fast and easy-to-perform method to predict the putative production of antifungal compounds is genetic analysis of the presence of biosynthetic genes (Zhang et al., 2006). The antifungal compounds produced by Pseudomonas spp. are well known and have been used in the colony blotting assays because these biosynthetic genes are available. Thus, the colony blotting detection assays for biosynthetic genes of antimicrobial compounds with antifungal activity showed a higher number of isolates with a triple combination of antibiotics (hydrogen cyanide (HCN), phenazine- CHAPTER III: Isolation of bacterial strains with biocontrol abilities 142 1-carboxylic acid (PCA) and 2-hexyl 5-propyl resorcinol (HPR)), mostly from the fluorescent Pseudomonadaceae-like group (B; n=8). Different examples of biocontrol fluorescent Pseudomonads have been described based on their ability to control a wide range of soilborne pathogens producing these mentioned antibiotics (Haas and Defago, 2005; Cazorla et al., 2006). Nevertheless, we did not detect any isolates displaying hybridization signals to the biosynthetic genes for 2,4-diacetylphloroglucinol (DAPG) and pyrrolnitrin (PRN) production, probably because the antagonistic bacteria with these antibiotics are more closely related to herbaceous plants in the literature (Hammer et al., 1997; De Souza et al., 2003; Barahona et al., 2010; Bankhead et al., 2016). However, other antifungal compounds different from the Pseudomonas spp. compounds produced by the Enterobacteriaceae-like group were not taken into account in this study. Because the production of lytic exoenzymes has been described with implications for biocontrol activity (Haran et al., 1996), we characterized the exoenzymatic production profiles of the bacterial collection. In this characterization, we only detected the production of lipases, proteases and chitinases by the different isolates and not amylases, β-glucanases or cellulases; these exoenzymatic activities are probably related to other groups of soil microorganisms, such as fungi (Haran et al., 1996). A high number of isolates was able to produce lipases and proteases, suggesting the broad distribution of these activities in the soil bacterial community, probably due to their involvement in general metabolism, such as the degradation of lipids (Jaeger et al., 1994) and proteins (Frees et al., 2013). However, a large number of isolates exhibited triple lytic activity, including the production of chitinases, which was in agreement with previous observations of this activity in suppressive soils (Cretoiu et al., 2013) and more specifically in suppressiveness soils induced by the amendment of composted almond shells (Vida et al., 2016). CHAPTER III: Isolation of bacterial strains with biocontrol abilities 143 The PGP-related activities assessed with the plate assays confirmed their presence (ability to solubilize an insoluble phosphate source and synthetize siderophores) in a higher number of Enterobacteriaceae-like group isolates that were described as PGPR bacteria in previous studies (Vacheron et al., 2013). The maintenance of an adequate level of mineral nutrients (especially P and the available form of Fe3+) can have a beneficial effect on crop production (Ghosh et al., 2015) and a high concentration of high-affinity siderophores in the rhizosphere can inhibit the growth of fungal pathogens when the Fe3+ concentration is low (Lugtenberg and Kamilova, 2009). Interestingly, most isolates from the Enterobacteriaceae-like group were producers of lytic exoenzymes and exhibited PGP-related activities, whereas the putative antibiotic producers were mainly allocated into the Pseudomonadaceae-like groups, suggesting the specialization of these groups of bacteria and the presence of different modes of action for biological control. After the first screening, we selected a group of representative isolates with a wide range of responses to different previous assays. Partial sequencing of the 16S rRNA gene was performed to aid in the preliminary identification of the putative genera to which the strains belonged. The limitation of 16S rDNA partial sequencing for the identification of a bacterial strain at the species level has been demonstrated (Loong et al., 2016). Thus, we could only assign a genus name to nine of the isolates with a correlation between the identity analysis (<95%) and the metabolic pattern. Additionally, in vitro seed growth promotion experiments revealed very low activity (only 2 strains) probably due to the source of the bacterial strains, suggesting that the PGP activity was more related to bacteria directly inhabiting the root environment (Kloepper and Schroth, 1980; Hartmann et al., 2009), and supporting the hypothesis of the presence of different groups of microorganisms in soil related to plant growth promotion and biocontrol traits (Bashan and Holguin, 1998). CHAPTER III: Isolation of bacterial strains with biocontrol abilities 144 The biological control activity of eight selected bacteria on avocado plants showed that the isolation of Gammaproteobacteria strains from a composted almond shells suppressiveness-induced soil could represent a strategy for selecting microorganisms with biocontrol ability against R. necatrix, potentially Serratia spp., Pseudomonas spp. and Stenotrophomonas spp. The strains belonging to the genera Serratia spp. and Pseudomonas spp. (isolates ‘3’, ‘5’, ‘9’, ‘15’ and ‘20’) are widely described in the literature due to their diverse plant growth-promoting activities and antagonistic interactions with a broad range of soilborne pathogen (Gkarmiri et al., 2015; Kamou et al., 2016). 5. Conclusion In this work, we demonstrated that specific representatives from the Gammaproteobacteria class isolated from a suppressiveness-induced soil after amendment with composted almond shells displayed biocontrol abilities and associated traits that could be involved in the suppressiveness of the avocado phytopathogenic fungus R. necatrix. CHAPTER IV: Bacterial interactions in biocontrol process 147 CHAPTER IV Development of a synthetic bacterial consortium to study microbial interactions in the rhizosphere during the biocontrol activity against Rosellinia necatrix CHAPTER IV: Bacterial interactions in biocontrol process 148 Abstract Induced microbial community from an avocado crop soil after organic amendment, have a key role in suppressiveness against avocado white root rot (WRR) caused by the fungus Rosellinia necatrix. This suppressive phenotype is the result of the soil microorganisms activities that do not act as individuals but as a dynamic community where different kind of interactions could take place. However, due to the difficulties of working about the interactions inside natural communities, a promising way to afford such objective is to create artificial microbial communities that could retain the traits of their natural microbiome. In this study, we start the modelling of a bacterial consortium with three biological control agents against WRR, Pseudomonas chlororaphis PCL1601, Pseudomonas chlororaphis PCL1606 and Pseudomonas pseudoalcaligenes AVO110. Stability and compatibility among the members of this artificial consortium have been confirmed. Furthermore, roots visualization assays revealed the colonization pattern of the putative synthetic community and the stability of the bacterial consortium along the avocado and wheat root. Additionally, biological control experiments against R. necatrix were performed and confirmed that the bacterial consortium retained the biocontrol activity. In order to further understand microbial interactions that could happen during biocontrol process, the genome of PCL1601 was sequenced, allowing genome comparisons and predictions of secondary metabolites production of the three bacterial partners. In this work, we included the study about production of volatile organic compounds (VOCs) due to the importance in microbial communication and antifungal activity. The results showed that VOCs can constitute a source of antifungal compounds, helping in the biocontrol phenotype, with some of these VOCs produced only when the bacterial assayed were organised as a synthetic community, but not as individual organism. CHAPTER IV: Bacterial interactions in biocontrol process 149 1. Introduction Soil microbial suppressiveness can be considered as a direct result of the activities from soil microorganisms, who contribute to all biogeochemical cycles. Most of the soil processes are the result of the microbial activities in their natural environment, where microbes do not act as single individuals, but do it as a dynamically microbial community, where all cells could interact and communicate each with another (Mitri and Foster, 2013). However, it is difficult to stablish what is the role of the different microbes into a natural community. A promising way to overcome the difficulties of studying complex communities is to create artificial microbial consortia that can retain some characters of their natural microbiome to be further studied. These communities could act as a model system to evaluate the role of ecological, structural and functional features of communities in a controlled way (Großkopf and Soyer, 2014). Recently, special interest have being gained for the microbial community enhanced into an agricultural suppressiveness-induced soil of avocado (Persea americana Mill.) against the white root rot (WRR), caused by the soilborne fungus Rosellinia necatrix. Studies have demonstrated that the use of composted almond shells as organic amendments or mulches increases soil suppressiveness (Bonilla et al., 2015). In fact, this suppressive activity were directly related with the microbial composition and activities, specifically with the increase of representatives from the bacterial class Gammaproteobacteria (including the genera Pseudomonas, Serratia and Stenotrophomonas) and the fungal class Dothydeomycetes (Vida et al., 2016). Each host provides a microhabitat with different abiotic conditions, which directly influence the structure of the root microbial community (Berg et al., 2015). Prevoius studies have described many bacterial strains isolated from soil and rhizosphere of avocado as biocontrol agents against R. necatrix, many of them included into Pseudomonas spp. genus (Pliego et al., 2012; Vida et al., 2017a under review). CHAPTER IV: Bacterial interactions in biocontrol process 150 Pseudomonads is a large genus placed into the Gammaproteobacteria class, well known for its frequent isolation from soil and rhizosphere environments (Haas and Défago, 2005), utilization of a wide range of organic compounds (Wu et al., 2011) and production of secondary metabolites (Gross and Loper, 2009). Members of the genus Pseudomonas (sensu stricto) show remarkable metabolic and physiologic versatility, enabling colonization of diverse habitats, and showing potential in biotechnological applications (Silby et al., 2011). Many Pseudomonads from P. fluorescens, P. chlororaphis and P. aeruginosa groups interact with plants in order to contribute in plant health by antagonizing plant-pathogenic microorganisms (biocontrol strains) and directly influencing plant disease resistance and growth (plant growth-promoting bacteria) (Haas and Defago, 2005). Using the avocado-R. necatrix test system, three biocontrol agents have been isolated and well-described in previous works. Two of them, P. chlororaphis PCL1601 and PCL1606 were isolated from the rhizosphere of healthy avocado trees and screened for their antagonistic and biocontrol activity against R. necatrix (Cazorla et al., 2006). This ability were related with the production of different antimicrobial compounds. Pseudomonas chlororaphis PCL1601 produced proteases, lipases, hydrogen cyanide (HCN), phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) whereas P. chlororaphis PCL1606 produced proteases, lipases, siderophores, HCN, pyrrolnitin (PRN) and 2-hexyl 5-propyl resorcinol (HPR) antifungal compounds, crucial for biological control against R. necatrix and for avocado root colonization (Cazorla et al., 2006; Calderón et al., 2014). A third bacterial strain is P. pseudoalcaligenes AVO110, also isolated from roots of healthy avocado trees, but selected because its ability to efficiently colonize avocado roots, and also displaying biocontrol towards R. necatrix. Previous studies showed that the mode of action of this strain could be related with the competition for niches and nutrients because it cannot CHAPTER IV: Bacterial interactions in biocontrol process 151 produce any well-known antifungal metabolites from Pseudomonads (Pliego et al., 2007; 2008). The construction of an artificial bacterial consortium with these characterized strains could represent an approach to understand how cells live in close proximity, and to unravel their interaction during the biocontrol activity. In this sense, colonization of root system have been described in literature as an essential trait for further applications (de Weert et al., 2002), including biocontrol of soilborne diseases (ChinA-Woeng et al., 1998; Lugtenberg and Kamilova, 2009; Barahona et al., 2010). In this environment, the efficient root colonisers can compete for niches and nutrients in the rhizosphere and promote the production of antifungal compounds, so the knolewdge of the colonization patterns of an artificial consortium is crucial in order to evaluate them from an ecological and biotechnological point of view (De Roy et al., 2014). Because these different biocontrol Pseudomonas spp. displayed different phenotypes, it is not surprising that its diversity could extends to the genomic sequence level. Bacterial genome sequences can now be generated faster and cheaper enough to be considered part of the toolbox for investigating bacteria (Edwards and Holt, 2013). Furthermore, this technology can provide, for example, insight into mode of life and essential processes of bacterial strains. The analysis of core sequences among bacteria or regions that are unique to a specific strain could help us to unravel the social interactions established between microorganisms. The genome comparative analysis could provide information about the hypothetical role of the different strains during biocontrol process, analysing the putative secondary metabolites production and activities implicated in biogeochemical cycling of nutrients and interand intraspecific interactions (Silby et al., 2011). Additionally, important molecules in communication between bacteria are the volatile organic compounds (VOCs), commonly produced by bacteria and emitted to environment. Many VOCs play a significant role in the communication between CHAPTER IV: Bacterial interactions in biocontrol process 152 organisms, affecting to growth, antibiotic production and gene expression of soil bacteria (Garbeva et al., 2014). Volatile molecules can diffuse through liquid and gaseous phases of the soil (Effmert et al., 2012), playing key roles in interspecific bacterial interaction physically separated in the porous soil matrix. Concretely, Pseudomonas spp. have been described to produce different VOCs that inhibit the growth of different soilborne pathogens (Kai et al., 2009). Historically, most studied is the compound hydrogen cyanide (Knowles, 1976), volatile compound that inhibits several metal-containing enzymes such as cytochrome c oxidase of the respiratory chain (Effmert et al., 2012). For this reason, HCN could act as a toxic for most aerobic organisms living in the same habitat as Pseudomonads. Pseudomonas chlororaphis PCL1601 and PCL1606 have been described to produce HCN (Cazorla et al., 2006), but P. pseudoalcaligenes AVO110 is a non-producer of this volatile (Pliego et al., 2007). However, nothing is known about the production of other VOCs from these strains. In this study, in order to inititate a broader study on the interaction that take place on the rhizosphere during the biocontrol against R. necatrix, we initiated the construction and study of a 3-bacterial consortium biocontrol strains previously isolated from an avocado agricultural soil. First, absence of negative interactions among them were confirmed by compatibility plate assays, and visualization of spatial distribution in root surface of the microbial consortium in presence or absence of the pathogen R. necatrix. The results confirmed the stability of the artificial consortium on avocado roots. 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Microbiology | www.frontiersin.org 14 January 2016 | Volume 7 | Article 4 Draft Genome Sequence of the Rhizobacterium Pseudomonas chlororaphis PCL1601, Displaying Biocontrol against Soilborne Phytopathogens Carmen Vida,Antonio de Vicente,Francisco M. Cazorla Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora, Universidad de Málaga, Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC); Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain ABSTRACT In this study, we present the draft genome sequence of the bacterial strain Pseudomonas chlororaphis PCL1601. This bacterium was isolated from the rhizosphere of healthy avocado trees and displayed antagonistic and biological control activities against different soilborne phytopathogenic fungi and oomycete. Pseudomonas chlororaphis PCL1601 is a Gram-negative aerobic bacterium isolated from the rhizosphere of a healthy avocado tree allocated in an area affected by avocado white root rot (1), a fungal disease caused by the soilborne phytopathogen Rosellinia necatrix (2). The bacterial isolation was carried out from avocado root samples, with further isolation of different nutrient media with cycloheximide (100 ␮ g/ml) to avoid fungal growth interference. Pseudomonas chlororaphis PCL1601 formed opaque and light-yellow colonies when grown on solid nutrient medium, and the colonies were fluorescent when grown in King’s B (KB) medium. Furthermore, PCL1601 presented antagonistic activity against several soilborne pathogens, such as Fusarium oxysporum and Rhizoctonia solani, but especially to the avocado soilborne pathogens R. necatrix and Phytophthora cinnamomi (1). Additionally, P. chlororaphis PCL1601 showed biological control activity against R. necatrix on avocado and to F. oxysporum f. sp. radicis-lycopersici on tomato (1). This strain is able to produce some antimicrobial compounds, such as hydrogen cyanide (HCN), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN) (1). Here, we report the draft genome sequence of P. chlororaphis PCL1601. Genomic DNA of P. chlororaphis PCL1601 was extracted with the PowerSoil DNA isolation kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) after overnight growth in liquid King’s B medium at 25°C. Genome sequencing was performed at ChunLab, Inc. (Seoul, South Korea) using the Pacific Biosciences 20 K method. Sequencing depth was 223.26⫻ coverage of the genome, which was assembled de novo into 25 contigs with the PacBio SMRT Analysis pipeline version 2.3.0 (ChunLab, Inc.). The resulting draft genome sequence was ordered using the genome sequence of Pseudomonas chororaphis PA23 as the template (3). The resulting draft genome sequence was annotated with the NCBI Prokaryotic Genome Annotation Pipeline. Additionally, the secondary metaboliteand antibiotic-encoding gene clusters were predicted with antiSMASH (4). The draft genome of PCL1601 is 6,755,444 bp in length, containing a G⫹C content of 64% and 5,897 predicted coding sequences, 17 rRNAs, and 68 tRNAs, features similar to those previously described for the biocontrol strain P. chlororaphis PCL1606, also isolated from avocado rhizosphere (5). However, genome annotation displayed a higher range of putative genes involved in general metabolism (carbohydrates, amino Received 6 February 2017 Accepted 7 February 2017 Published XXX Citation Vida C, de Vicente A, Cazorla FM. 2017. Draft genome sequence of the rhizobacterium Pseudomonas chlororaphis PCL1601, displaying biocontrol against soilborne phytopathogens. Genome Announc 0(999):e00130-17. https://doi.org/10.1128/ genomeA.00130-17. Copyright © 2017 Vida et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Francisco M. Cazorla, [email protected]. PROKARYOTES Volume 5 Issue 999 e00130-17 genomea.asm.org 1 AQ: au AQ: aff AQ: A AQ: B Editor: Section: Designation: Prokaryotes jga-genome/jga99917/jga5104d17z xppws S⫽3 2/13/17 10:48 ArtID: DOI:10.1128/genomeA.00130-17 CE: MT acids, lipids, etc.) and transport (such as inorganic ion transport and metabolism, intracellular trafficking, secretion, and vesicular transport). Using antiSMASH, we found 13 potential biosynthetic gene clusters potentially involved in secondary metabolite production, highlighting the phenazine biosynthetic gene cluster, but also bacteriocins (n⫽4), siderophores (n⫽2), and nonribosomal peptide synthetases (NRPS; n⫽2), most of them displaying architecture (higher than 90%) similar to other biosynthetic operons also described in other P. chlororaphis strains. The remaining clusters have lower homologies and need further characterization. Accession number(s). This whole-genome shotgun project has been deposited in GenBank under the accession no. MSCT00000000 (from MSCT01000001 to MSCT01000025). The version described in this paper is the first version, MSCT01000000. ACKNOWLEDGMENTS This work was supported by Plan Nacional I ⫹D⫹I from Ministerio de Economía (MINECO) (grants AGL11-30354-C02-01 and AGL14-52518-C2-IR), cofinanced by FEDER funds (EU). C. Vida was supported by a Ph.D. fellowship from the FPI program of MINECO. REFERENCES 1. Cazorla FM, Duckett SB, Bergström ET, Noreen S, Odijk R, Lugtenberg BJJ, Thomas-Oates JE, Bloemberg GV. 2006. Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol. Mol Plant Microbe Interact 19:418–428. https://doi.org/10.1094/MPMI-19-0418. 2. Pliego C, López-Herrera C, Ramos C, Cazorla FM. 2012. Developing tools to unravel the biological secrets of Rosellinia necatrix, and emergent threat to woody crops. Mol Plant Pathol 13:226–239. https://doi.org/ 10.1111/J.1364-3703.2011.00753.X. 3. Loewen PC, Villenueva J, Fernando WG, de Kievit T. 2014. Genome sequence of Pseudomonas chlororaphis strain PA23. Genome Announc 2(4):e00689-14. https://doi.org/10.1128/genomeA.00689-14. 4. Weber T, Blin K, Duddela S, Krug D, Kim HU, Bruccoleri R, Lee SY, Fischbach MA, Müller R, Wohlleben W, Breitling R, Takano E, Medema MH. 2015. antiSMASH 3.0–a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res 43:W237–W243. https:// doi.org/10.1093/nar/gkv437. 5. Calderón CE, Ramos C, de Vicente A, Cazorla FM. 2015. Comparative genomic analysis of Pseudomonas chlororaphis PCL1606 reveals new insight into antifungal compounds involved in biocontrol. Mol Plant Microbe Interact 28:249–260. https://doi.org/10.1094/MPMI-10-14-0326 -FI. Vida et al. Volume 5 Issue 999 e00130-17 genomea.asm.org 2 jga-genome/jga99917/jga5104d17z xppws S⫽3 2/13/17 10:48 ArtID: DOI:10.1128/genomeA.00130-17 CE: MT Taking together our results, we showed the positive effect of application of composted almond shells as organic amendment in biological control of avocado soil-borne pathogen Rosellinia necatrix.In this work, we assigned this biocontrol activity to soil microbial community, where different groups of Gammaproteobacterias, including Pseudomonas spp.,were naturally selected. Isolation of culturable members from the suppressive soil of Pseudomonas spp., Serratia spp. and Stenotrophomonas spp., showed the ability of these microorganisms to control the disease index cause by the pathogen, both in avocado roots as in wheat root, using different biological control methods. Due to the importance of genus Pseudomonas sp. in this suppressive soil, and using Pseudomonas spp. previously described by their biocontrol activity against R. necatrix,we design a bacterial consortium in order to improve the knowledge of the putative community interactions that occur during biological control process.