Arthrocnemum macrostachyum y su microbioma como herramienta para la recuperación de suelos degradados

La contaminación por metales y pesados y la salinidad afectan a la calidad de los suelos agrícolas, disminuyendo el número de tierras utilizadas para la agricultura. Estos problemas afectan directamente a todos los seres vivos, incluidos los humanos. En esta Tesis Doctoral, se usó a la planta Arthrocnemum macrostachyum y a su microbioma como herramienta para la fitorremediación de los suelos degradados por estos contaminantes. A. macrostachyum es una planta halófita hiperacumuladora e hipertolerante al cadmio y podría ser usada como herramienta de fitorremediación. Esta planta crece de manera natural en las marismas del Odiel, un ecosistema contaminado con altos niveles de metales pesados. Durante este trabajo se aislaron un total de 48 bacterias (18 de la rizosfera de A. macrostachyum y 30 endófitos de esta misma planta) y todas ellas pudieron crecer en presencia de metales pesados y sal. Al menos una de las principales propiedades PGP (promotoras del crecimiento de la planta) estuvo presente en todas las bacterias aisladas y se observaron altos valores de estas propiedades. La presencia de metales pesados en el medio afectó a la producción de las propiedades PGP por las bacterias. Este efecto puede ser negativo o positivo, dependiendo del metal, la cepa y la propiedad PGP. Por otro lado, un alto número de estas bacterias aisladas podrían ser nuevas especies no descritas y estas cepas también fueron estudiadas. Los resultados obtenidos sugieren que estas bacterias podrían promover el crecimiento de las plantas incluso en presencia de metales pesados y que el conjunto de planta y bacterias podría ser una interesante herramienta de fitorremediación. Por ello, dos consorcios bacterianos formados por las mejores bacterias rizosféricas (CR) y los mejores endófitos (CE) fueron seleccionados en base a su resistencia a metales pesados y a sus propiedades PGP. La cinética de germinación de semillas de A. macrostachyum fue observada en presencia y ausencia de metales pesados usando estos dos consorcios. Por otro lado, un experimento de invernadero fue diseñado a partir de las semillas para investigar el papel de los consorcios en la capacidad de A. macrostachyum de captar y tolerar metales pesados cuando crece en sedimentos contaminados con metales. A las plantas se les asignaron tres tratamientos (inoculadas con el consorcio CR, inoculadas con el consorcio CE y sin inocular). Para empezar, la inoculación bacteriana aceleró la germinación de las semillas tanto en presencia como en ausencia de metales pesados. Ambos consorcios mejoraron la capacidad de A. macrostachyum para acumular iones en sus raíces y, además, también se amortiguaron los niveles de toxicidad. Las bacterias tuvieron un efecto beneficioso en el aparato fotosintético de las plantas, así como en la modulación de la maquinaria de estrés oxidativo. Estos resultados sugieren que la inoculación de A. macrostachyum con las bacterias seleccionadas podrían favorecer el establecimiento y el crecimiento de esta planta en marismas contaminadas y mejorar la eficiencia de la remediación. Con respecto al problema de la salinización de suelos agrícolas, hubo con creciente interés en la revegetación de estos suelos usando planta halófitas. En esta Tesis Doctoral se aislaron 8 endófitos de la parte aérea de plantas de A. macrostachyum crecidas en suelos agrícolas de la zona de Lebrija. Estas bacterias aisladas pertenecieron al género Bacillus y otros géneros relacionados. Se determinaron la presencia de propiedades PGP, la presencia de actividades enzimáticas y la tolerancia a sal. Las mejores bacterias fueron seleccionadas para la formación de un consorcio, con el cuál se estudió los efectos en la inoculación en la germinación de las semillas y en el desarrollo de las plantas en presencia de distintos tratamientos con sal (0, 510 y 1030 mM). La inoculación mejoró considerablemente la germinación de las semillas y el porcentaje de germinación. Además, la inoculación con el consorcio disminuyó los efectos de la salinidad, mejorando el crecimiento de las plantas e incrementando el potencial de A. macrostachyum de acumular Na+ en los tallos. Estos resultados sugieren que las bacterias aisladas de la filosfera de las plantas de A. macrostachyum parecen jugar un papel importante en la tolerancia de la planta a la sal bajo concentraciones de sal estresantes, mejorando la germinación de las semillas y el desarrollo de la planta.In the world, there are problems with contamination of heavy metals and problems about salinity. Both problems affect the quality of soils, the agriculture and, finally, to humans. In this work, Arthrocnemum macrostachyum and its microbiome were used as tool for the phytoremediation of degraded soils. Arthrocnemum macrostachyum is a halophyte plant Cd- hyperaccumulator and hypertolerant and it could be used as phytoremediation tool. This plant grows naturally in Odiel marshes, a polluted ecosystem with high levels of heavy metals. A total of 48 bacteria were isolated (18 from the rhizosphere and 30 endophytes) and all of them could grow in presence of heavy metals and salt. At least one of the PGP properties was present in the isolated bacteria and there were several strains that showed high values of these properties. The heavy metals presence affected to the PGP production by the bacteria both negatively and positively. On the other hand, a high number of isolated bacteria could be new species and they were studied. The results suggest that the isolated bacteria could promote the plant growth even in presence of heavy metals and the set of plant and bacteria could be an interesting tool for the phytoremediation. Two bacterial consortia with the best-performing endophytic (CE) or rhizospheric strains (CR) were selected on basis on the heavy metals resistance and PGP properties. A germination kinetics was observed in presence and absence of heavy metals using both bacterial consortia. On the other hand, a glasshouse experiment was designed to investigate the role of bacterial consortia on its metal uptake capacity and tolerance in plants grown in metal polluted sediments. Plants were assigned to three treatments (with CR, CE and without inoculation). Bacterial inoculation accelerated germination of A. macrostachyum seeds in both the absence and presence of heavy metals. Bioaugmentation with both bacterial consortiums enhanced A. macrostachyum capacity to accumulate ions in its roots. Furthermore, bioaugmentation ameliorated the phytotoxicity levels. Bacteria had a beneficial effect on photochemical apparatus and on the modulation of its oxidative stress machinery. These results suggest that inoculation of A. macrostachyum with the selected bacteria could ameliorate plant establishment and growth in contaminated marshes and improve the metal remediation efficiency. Concernig to the salinity, there is an increasing interest to use halophytes for revegetation of salt affected ecosystems, as well as in understanding their mechanisms of salt tolerance. 8 endophytic bacteria belonging to Bacillus and closely related genera were isolated from phyllosphere of A. macrostachyum growing in salty agricultural soils. The presence of PGP properties and enzymatic activities and tolerance towards NaCl was determined. Effects of inoculation on seeds germination and adult plant growth under experimental NaCl treatments (0, 510 and 1030 mM NaCl) were studied. Inoculation with a consortium including the best performing bacteria improved considerably the kinetics of germination and the final germination percentage of A. macrostachyum seeds. Inoculation of plants mitigated the effects of high salinity on plant growth and physiological performance and, in addition, this consortium appears to have increased the potential of A. macrostachyum to accumulate Na+ in its shoots. Results suggest that bacteria isolated from A. macrostachyum phyllosphere seem to play an important role in plant salt tolerance under stressing salt concentrations, improving seed germination and plant development

Biostimulant Capacity of an Enzymatic Extract From Rice Bran Against Ozone-Induced Damage in Capsicum annum

Ozone is a destructive pollutant, damaging crops, and decreasing crop yield. Therefore, there is great interest in finding strategies to alleviate ozone-induced crop losses. In plants, ozone enters leaves through the stomata and is immediately degraded into reactive oxygen species (ROS), producing ROS stress in plants. ROS stress can be controlled by ROS-scavenging systems that include enzymatic or non-enzymatic mechanisms. Our research group has developed a product from rice bran, a byproduct of rice milling which has bioactive molecules that act as an antioxidant compound. This product is a water-soluble rice bran enzymatic extract (RBEE) which preserves all the properties and improves the solubility of proteins and the antioxidant components of rice bran. In previous works, the beneficial properties of RBEE have been demonstrated in animals. However, to date, RBEE has not been used as a protective agent against oxidative damage in agricultural fields. The main goal of this study was to investigate the ability of RBEE to be used as a biostimulant by preventing oxidative damage in plants, after ozone exposure. To perform this investigation, pepper plants (Capsicum annuum) exposed to ozone were treated with RBEE. RBEE protected the ozone-induced damage, as revealed by net photosynthetic rate and the content of photosynthetic pigments. RBEE also decreased the induction of antioxidant enzyme activities in leaves (catalase, superoxide dismutase, and ascorbate peroxidase) due to ozone exposure. ROS generation is a common consequence of diverse cellular traumas that also activate the mitogen-activated protein kinase (MAPK) cascade. Thus, it is known that the ozone damages are triggered by the MAPK cascade. To examine the involvement of the MAPK cascade in the ozone damage CaMPK6-1, CaMPK6-2, and CaMKK5 genes were analyzed by qRT-PCR. The results showed the involvement of the MAPK pathway in both, not only in ozone damage but especially in its protection by RBEE. Taken together, these results support that RBEE protects plants against ozone exposure and its use as a new biostimulant could be proposed

Safe Cultivation of Medicago sativa in Metal-Polluted Soils from Semi-Arid Regions Assisted by Heatand Metallo-Resistant PGPR

Soil contamination with heavy metals is a constraint for plant establishment and development for which phytoremediation may be a solution, since rhizobacteria may alleviate plant stress under these conditions. A greenhouse experiment was conducted to elucidate the effect of toxic metals on growth, the activities of ROS (reactive oxygen species)-scavenging enzymes, and gene expression of Medicago sativa grown under different metal and/or inoculation treatments. The results showed that, besides reducing biomass, heavy metals negatively affected physiological parameters such as chlorophyll fluorescence and gas exchange, while increasing ROS-scavenging enzyme activities. Inoculation of M. sativa with a bacterial consortium of heat- and metallo-resistant bacteria alleviated metal stress, as deduced from the improvement of growth, lower levels of antioxidant enzymes, and increased physiological parameters. The bacteria were able to effectively colonize and form biofilms onto the roots of plants cultivated in the presence of metals, as observed by scanning electron microscopy. Results also evidenced the important role of glutathione reductase (GR), phytochelatin synthase (PCS), and metal transporter NRAMP1 genes as pathways for metal stress management, whereas the gene coding for cytochrome P450 (CP450) seemed to be regulated by the presence of the bacteria. These outcomes showed that the interaction of metal-resistant rhizobacteria/legumes can be used as an instrument to remediate metal-contaminated soils, while cultivation of inoculated legumes on these soils is still safe for animal grazing, since inoculation with bacteria diminished the concentrations of heavy metals accumulated in the aboveground parts of the plants to below toxic levelsMarruecos. Centre National pour la Recherche Scientifique et Technique (CNRST)-España, Ministerio de Economía y Competitividad (MINECO)-PPR2 /2016/42Unión Europea (FEDER)-CGL2016-75550-

Sustainable agricultural management of saline soils in arid and semi-arid Mediterranean regions through halophytes, microbial and soil-based technologies

Soil salinization is an important global issue since marginal salt-affected soils have harmful consequences in agriculture and ecosystems. This article reviews different sustainable strategies adopted for marginal soil reclamation in Mediterranean climates. An innovative approach to soil salinity management includes a wide range of technologies, such as: phytoremediation, phytodesalination, vegetative bioremediation, amendments application and Technosols as well as inoculation with beneficial microorganisms like plant growth promoting bacteria and arbuscular mycorrhizal fungi. Besides that, the role of Mediterranean halophyte crops in acceler- ating salt-affected area’s recovery while providing food and feed, and beneficial halophilic microorganisms for new bioinoculant production, are discussed. We conclude that the combined use of plant, soil- and microbial- based technologies is a valuable option to relieve saline stress exposure and improve crops growth and yield in saline conditions.Federation of European Microbiological Societies (FEMS) (FEMS-GO-2020- 203),) (FEMS-GO-2020–203 University), University of Sevilla (Spain; Plan Propio de Investigación y Tranferencia 2021 Ayuda A1-I.3A1)info:eu-repo/semantics/publishedVersio

Coastal Ecosystems as Sources of Biofertilizers in Agriculture: From Genomics to Application in an Urban Orchard

Pantoea agglomerans RSO7, a rhizobacterium previously isolated from Spartina maritima grown on metal polluted saltmarshes, had demonstrated good plant growth promoting activity for its host halophyte, but was never tested in crops. The aims of this study were: (1) testing PGP activity on a model plant (alfalfa) in vitro; (2) testing a bacterial consortium including RSO7 as biofertilizer in a pilot experiment in urban orchard; and (3) identifying the traits related to PGP activities. RSO7 was able to enhance alfalfa growth in vitro, particularly the root system, besides improving plant survival and protecting plants against fungal contamination. In addition, in a pilot experiment in urban orchard, a consortium of three bacteria including RSO7 was able to foster the growth and yield of several winter crops between 1.5 and 10 fold, depending on species. Moreover, the analysis of chlorophyll fluorescence revealed that photosynthesis was highly ameliorated. Genome analysis of RSO7 depicted the robustness of this bacterial strain which showed resilience to multiple stresses (heat, cold, UV radiation, several xenobiotics). Together with wide metabolic versatility, genes conferring resistance to oxidative stress were identified. Many genes involved in metal resistance (As, Cu, Ni, Co, Zn, Se, Te) and in tolerance toward high osmolality (production of a battery of osmoprotectans) were also found. Regarding plant growth promoting properties, traits for phosphate solubilization, synthesis of a battery of siderophores and production of IAA were detected. In addition, the bacterium has genes related to key processes in the rhizosphere including flagellar motility, chemotaxis, quorum sensing, biofilm formation, plant-bacteria dialog, and high competitiveness in the rhizosphere. Our results suggest the high potential of this bacterium as bioinoculant for an array of crops. However, the classification in biosecurity group 2 prevents its use according to current European regulation. Alternative formulations for the application of the bioinoculant are discussed.Junta de Andalucía P11-RNM-7274-MOFIUS, University of Seville FIUS19/0065FEDER, Junta de Andalucía US-1262036University of Seville AE2-1

Improved Medicago sativa Nodulation under Stress Assisted by Variovorax sp. Endophytes

Legumes are the recommended crops to fight against soil degradation and loss of fertility because of their known positive impacts on soils. Our interest is focused on the identification of plant-growth-promoting endophytes inhabiting nodules able to enhance legume growth in poor and/or degraded soils. The ability of Variovorax paradoxus S110T and Variovorax gossypii JM-310T to promote alfalfa growth in nutrient-poor and metal-contaminated estuarine soils was studied. Both strains behaved as nodule endophytes and improved in vitro seed germination and plant growth, as well as nodulation in co-inoculation with Ensifer medicae MA11. Variovorax ameliorated the physiological status of the plant, increased nodulation, chlorophyll and nitrogen content, and the response to stress and metal accumulation in the roots of alfalfa growing in degraded soils with moderate to high levels of contamination. The presence of plant-growth-promoting traits in Variovorax, particularly ACC deaminase activity, could be under the observed in planta effects. Although the couple V. gossypii-MA11 reported a great benefit to plant growth and nodulation, the best result was observed in plants inoculated with the combination of the three bacteria. These results suggest that Variovorax strains could be used as biofertilizers to improve the adaptation of legumes to degraded soils in soil-recovery programs.España MCIN/AEI/10.13039/501100011033España Ministry of Science and Innovation UE “NextGenerationEU/PRTR (PDC2021-120951-I00

Unravelling the combined use of soil and microbial technologies to optimize cultivation of Halophyte Limonium algarvense (Plumbaginaceae) using saline soils and water

Salt-affected soils have detrimental effects on agriculture and ecosystems. However, these soils can still be used for halophyte (salt-tolerant plants) cultivation using brackish and/or saline water. In this study, we employed soil technologies and mutualistic microorganisms as a sustainable strategy to improve the growth and reproduction of the halophyte Limonium algarvense Erben’s growth and reproduction under saline conditions. A microcosm assay was conducted under controlled greenhouse conditions to cultivate L. algarvense using a saline Fluvisol (FLU) amended—or not—with a Technosol (TEC). Plants were inoculated with the arbuscular mycorrhizal fungus (AMF) Rhizoglomus irregulare and/or a consortium of plant growth-promoting bacteria (PGPB), and they were irrigated with estuarine water. Soil enzyme analysis and physicochemical characterisation of the soils, collected at the beginning and at the end of the assay, were carried out. The physiological status of non-inoculated and inoculated plants was monitored during the assay for 4 months, and AMF root colonisation was evaluated. In FLU, only plants inoculated with the AMF survived. These plants had lower number of leaves, and shoot and root dry biomass than the ones grown in the TEC by the end of the assay. In the TEC, PGPB inoculation led to higher NDVI and PRI values, and AMF inoculation promoted higher reproductive development but not pollen fertility. The findings show that the combined use of soil and microbial technologies can be successfully applied to cultivate L. algarvense, suggesting their generalized use for other Limonium species with economic interest, while contributing to the sustainable use of marginal lands.Federation of European Microbiological Societies (FEMS) (FEMSGO- 2020-203), University of Sevilla (Spain; Plan Propio de Investigación y Tranferencia 2021 Ayuda A1-I.3A1) for grants to support the stays at ISA-ULisboa (Portugal).info:eu-repo/semantics/publishedVersio

Helping Legumes under Stress Situations: Inoculation with Beneficial Microorganisms

In the upcoming years, legume crops will be subjected to multiple, diverse, and overlapping environmental stressors (raise in global temperatures and CO2, drought, salinity, and soil pollution). These factors will menace legume productivity and food quality and security. In this context, tolerant plant growth promoting rhizobacteria (PGPR) are useful biotechnological tools to assist legume establishment and growth. In this chapter, tolerant PGPR able to promote legume growth will be revised. Besides, in the era of -omics, the mechanisms underlying this interaction are being deciphered, particularly transcriptomic, proteomic, and metabolomic changes modulated by PGPR, as well as the molecular dialog legume-rhizobacteria

Innovative compounds to battle multirresistance to antibiotics: use of pva-tannic acid nano- particles to inhibit staphylococcus pseudintermedius growth

Antibiotic resistance is an increasing public health problem that affects to numerous pathogens, including Staphylococcus pseudintermedius, which has a high prevalence of methicillin resistance and can be transmitted to humans. The development of new compounds to prevent the appearance of antibiotic resistances and find alternatives to classic therapies is essential to health protection. The main objective of the present study is to determine whether the nanoparticles of polyvinyl alcohol and tannic acid “PVA-TA NPs” are effective to inhibit Staphylococcus pseudintermedius and can be considered as an alternative therapy. The study includes increasing concentration essays of the nanoparticles and establishes, using the determination of turbidity by refractometry that could be a useful tool to inhibit bacterial growth, resulting successful. Nonetheless, further studies to identify the underlying action mechanisms of these nano-particles are going on in our group

Looking for new compounds to battle antibiotic resistance: optimization of organic solvents

A high number of pathogens have shown the capacity of resist to the action of antibiotics. This fact presents a very important problem to public health and highlights the necessity of looking for new substances as alternative to antibiotics. Since some of these substances have a hydrophobic nature they need to be solved in organic solvents. Some of these solvents are also toxic to bacteria, mainly affecting membranes. For these reasons it is necessary to perform a study of the toxicity of organic solvents to bacteria, which has been the aim of this work. Five organic solvents, DMF (dimethylformamide), TBME (tert-butyl methyl ether), THF (tetrahydrofuran), DMSO (dimethyl sulfoxide) and CH2Cl2 (dichloromethane), have been tested, using two of the most prevalent pathogens (Staphylococcus pseudintermedius and Pseudomonas aeruginosa,) both in animals and humans. The results show that, when possible, DMSO followed by TBME are the best options for testing new compounds in Staphylococcus pseudintermedius, whether DMF followed by TBME are the preferred solvents for testing hydrophobic compounds in Pseudomonas aeruginosa