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

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

Exploring through the use of physiological and isotopic techniques the potential of a PGPR-based biofertilizer to improve nitrogen fertilization practices efficiency in strawberry cultivation

The use of microorganisms as a biofertilizer in strawberry has focused mainly on pathogen biocontrol, which has led to the underestimation of the potential of microorganisms for the improvement of nutritional efficiency in this crop. A study was established to investigate the impact of a plant growth-promoting rhizobacteria (PGPR) based biofertilizer integrated by self-compatible stress tolerant strains with multiple PGP properties, including atmospheric nitrogen fixation, on strawberry (Fragaria × ananassa cv. Rociera) tolerance to N deficiency in terms of growth and physiological performance. After 40 days of nitrogen fertilization shortage, inoculated plants were able to maintain root development and fertility structures (i.e. fruits and flowers) at a level similar to plants properly fertilized. In addition, inoculation lessened the negative impact of nitrogen deficiency on leaves’ dry weight and relative water content. This effect was mediated by a higher root/shoot ratio, which would have allowed them to explore larger volumes of soil for the acquisition of water. Moreover, inoculation was able to buffer up to 50% of the reduction in carbon assimilation capacity, due to its positive effect on the diffusion efficiency of CO2 and the biochemical capacity of photosynthesis, as well as on the activity of photosystem II light harvesting. Furthermore, the higher leaf C/N ratio and the maintained δ15N values close to control plants were related to positive bacterial effects at the level of the plant nutritional balance. Despite these positive effects, the application of the bacterial inoculum was unable to completely counteract the restriction of fertilization, being necessary to apply a certain amount of synthetic fertilizer for the strawberry nutrition. However, according to our results, the complementary effect of this PGPR-based biofertilizer could provide a higher efficiency in environmental and economic yields on this crop

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

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

Diseño de consorcios bacterianos potenciadores de la nodulación para la recuperación de estuarios degradados por estreses abióticos utilizando Medicago sativa

El deterioro de los suelos es uno de los problemas más acuciantes en la actualidad. Distintos tipos de estrés abiótico están presentes en suelos agrícolas y estuarinos que antes eran idóneos para cultivos y presentaban una gran biodiversidad. El manejo inadecuado de fertilizantes químicos, la salinidad, los metales pesados y otros contaminantes han contribuido a la pérdida de nutrientes y microorganismos que aportan beneficios a los suelos. En Huelva (Andalucía, España) se encuentran estuarios con gran importancia ecológica y económica, como el conjunto de estuarios de los ríos Tinto y Odiel, muy estudiados por ser una de las regiones con mayor contaminación por metales pesados del mundo, y el estuario del río Piedras, con zonas descritas como suelos pobres en nutrientes. Para poder contrarrestar el daño causado por estos estreses abióticos se han propuesto alternativas biotecnológicas de biorremediación, más sostenibles con el medio ambiente y más económicas que los tratamientos de remediación físico-químicos convencionales. En este sentido, las leguminosas son una excelente opción para recuperar suelos degradados que se encuentran expuestos a estrés abiótico, a través de la fijación biológica de nitrógeno. Estas plantas son idóneas para la fitoestabilización de metales pesados, y las bacterias promotoras del crecimiento vegetal (PGPB, Plant Growth Promoting Bacteria) se pueden utilizar como inoculantes para mejorar el crecimiento, la nodulación y, en definitiva, la adaptación de las leguminosas a los suelos contaminados. Las medidas de minerales y macronutrientes realizadas en las marismas del río Piedras mostraron una deficiencia nutricional y un bajo contenido de materia orgánica, convirtiéndolo en un suelo degradado medioambientalmente. Por otro lado, la concentración de metales/loides presentes en los suelos del estuario del río Odiel sobrepasaron los valores permitidos por la legislación autonómica y estatal en parques naturales, suelos agrícolas e industriales. En esta Tesis Doctoral se propone la utilización de microorganismos autóctonos, incluyendo bacterias rizosféricas, endófitas no rizobios asociadas al nódulo y rizobios, con propiedades que promueven el crecimiento de la planta (PGP) para mejorar el crecimiento y la adaptación de plantas de Medicago sativa, así como incrementar su capacidad de fitoestabilizar metales, para la recuperación de los suelos estuarinos degradados y/o contaminados. Se aislaron rizobacterias y bacterias endófitas cultivables de nódulos de plantas de Medicago spp. que crecen en las marismas de los ríos Piedras y Odiel, respectivamente. La selección de las bacterias para realizar experimentos in vitro y en condiciones de invernadero se realizó en base a sus propiedades PGP como las auxinas, ACC desaminasa y la formación de biofilm entre otras, la presencia de enzimas líticas, entre ellas la quitinasa, pectinasa y amilasa entre otras, y la resistencia a metales pesados. Las bacterias endófitas seleccionadas fueron dos Pseudomonas (Pseudomonas sp. N4 y Pseudomonas sp. N8) y dos rizobios (Ensifer sp. N10 y Ensifer sp. N12) y tres rizobacterias (Pseudomonas sp. L1, Chryseobacterium soli L2 y Priestia megaterium L3). También se seleccionaron tres bacterias de colección por sus propiedades PGP y la resistencia a metales. Estas últimas incluyen Ensifer medicae MA11, un rizobio que nodula Medicago spp., y dos endófitos del género Variovorax (V. paradoxus S110T y V. gossypii JM-310T). Se emplearon inoculantes individuales, combinaciones rizobacterias-rizobios o rizobios-endófitos, y se diseñaron tres consorcios bacterianos compuestos por tres rizobacterias (Pseudomonas sp. L1, Chryseobacterium soli L2 y Priestia megaterium L3) y un rizobio (Ensifer medicae MA11; CSL), el mismo rizobio (Ensifer medicae MA11) y los dos endófitos del género Variovorax (V. paradoxus S110T y V. gossypii JM-310T; CSV) y, por último, un consorcio compuesto por dos endófitos no rizobios (Pseudomonas sp. N4 y Pseudomonas sp. N8) y dos rizobios autóctonos (Ensifer sp. N10 y Ensifer sp. N12; CSN). Estas inoculaciones mejoraron la germinación, la nodulación y el crecimiento de las plantas en experimentos in vitro. En condiciones de invernadero con suelos de los estuarios mejoraron la nodulación, el estado fisiológico de la planta, los parámetros fotosintéticos, el contenido de clorofila y nitrógeno, la respuesta al estrés de la planta, además de mejorar la acumulación de metales en las raíces M. sativa en suelos degradados con niveles de moderados a altos de contaminación. Partiendo de la mencionada propuesta, en esta Tesis Doctoral se ha demostrado que las inoculaciones de M. sativa con los consorcios bacterianos compuestos por rizobios y endófitos resistentes a metales o rizobacterias con propiedades PGP son herramientas útiles para la fitoestabilización de metales/oides y para promover el crecimiento de leguminosas en suelos con distintos tipos de estrés abiótico, contribuyendo a la recuperación de estos ecosistemas. Con esta estrategia se garantiza, además, una mínima translocación de metales a la parte aérea de las plantas, por lo que podrían utilizarse, además de en estrategias de fitoestabilización de metales, como plantas forrajeras

Role of Nodulation-Enhancing Rhizobacteria in the Promotion of Medicago sativa Development in Nutrient-Poor Soils

Legumes are usually used as cover crops to improve soil quality due to the biological nitrogen fixation that occurs due to the interaction of legumes and rhizobia. This symbiosis can be used to recover degraded soils using legumes as pioneer plants. In this work, we screened for bacteria that improve the legume–rhizobia interaction in nutrient-poor soils. Fourteen phosphate solubilizer-strains were isolated, showing at least three out of the five tested plant growth promoting properties. Furthermore, cellulase, protease, pectinase, and chitinase activities were detected in three of the isolated strains. Pseudomonas sp. L1, Chryseobacterium soli L2, and Priestia megaterium L3 were selected to inoculate seeds and plants of Medicago sativa using a nutrient-poor soil as substrate under greenhouse conditions. The effects of the three bacteria individually and in consortium showed more vigorous plants with increased numbers of nodules and a higher nitrogen content than non-inoculated plants. Moreover, bacterial inoculation increased plants’ antioxidant activities and improved their development in nutrient-poor soils, suggesting an important role in the stress mechanisms of plants. In conclusion, the selected strains are nodulation-enhancing rhizobacteria that improve leguminous plants growth and nodulation in nutrient-poor soils and could be used by sustainable agriculture to promote plants’ development in degraded soils

Nodule Synthetic Bacterial Community as Legume Biofertilizer under Abiotic Stress in Estuarine Soils.

Estuaries are ecologically important ecosystems particularly affected by climate change and human activities. Our interest is focused on the use of legumes to fight against the degradation of estuarine soils and loss of fertility under adverse conditions. This work was aimed to determine the potential of a nodule synthetic bacterial community (SynCom), including two Ensifer sp. and two Pseudomonas sp. strains isolated from Medicago spp. nodules, to promote M. sativa growth and nodulation in degraded estuarine soils under several abiotic stresses, including high metal contamination, salinity, drought and high temperature. These plant growth promoting (PGP) endophytes were able to maintain and even increase their PGP properties in the presence of metals. Inoculation with the SynCom in pots containing soil enhanced plant growth parameters (from 3- to 12-fold increase in dry weight), nodulation (from 1.5- to 3-fold increase in nodules number), photosynthesis and nitrogen content (up to 4-fold under metal stress) under all the controlled conditions tested. The increase in plant antioxidant enzymatic activities seems to be a common and important mechanism of plant protection induced by the SynCom under abiotic stress conditions. The SynCom increased M. sativa metals accumulation in roots, with low levels of metals translocation to shoots. Results indicated that the SynCom used in this work is an appropriate ecological and safe tool to improve Medicago growth and adaptation to degraded estuarine soils under climate change conditions.MCIN/AEI/UE NextGenerationEU/PRTR PDC2021-120951-I00Junta de Andalucía, I+D+I FEDER - US-1262036Plan Andaluz de Investigación, Desarrollo e Innovación de la Junta de Andalucía - PAIDI2020 - P20_0068

Improved <i>Medicago sativa</i> Nodulation under Stress Assisted by <i>Variovorax</i> 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

Enhanced legume growth and adaptation to degraded estuarine soils using Pseudomonas sp. nodule endophytes

The joint estuary of Tinto and Odiel rivers (SW Spain) is one of the most degraded and polluted areas in the world and its recovery is mandatory. Legumes and their associated bacteria are recommended sustainable tools to fight against soils degradation and loss of fertility due to their known positive impacts on soils. The aim of this work was to isolate and characterize plant growth promoting nodule endophytes (PGPNE) from inside nodules of Medicago spp. naturally growing in the estuary of the Tinto and Odiel Rivers and evaluate their ability to promote legume adaptation in degraded soils. The best rhizobia and non-rhizobia among 33 endophytes were selected based on their plant growth promoting properties and bacterial enzymatic activities. These strains, identified as Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10 and Ensifer sp. N12, were used for in vitro studies using Medicago sativa plants. The effects of individual or combined inoculation on seed germination, plant growth and nodulation were studied, both on plates and pots containing nutrient-poor soils and moderately contaminated with metals/loids from the estuary. In general, inoculation with combinations of rhizobia and Pseudomonas increased plant biomass (up to 1.5-fold) and nodules number (up to 2-fold) compared to single inoculation with rhizobia, ameliorating the physiological state of the plants and helping to regulate plant stress mechanisms. The greatest benefits were observed in plants inoculated with the consortium containing the four strains. In addition, combined inoculation with Ensifer and Pseudomonas increased As and metals accumulation in plant roots, without significant differences in shoot metal accumulation. These results suggest that PGPNE are useful biotools to promote legume growth and phytostabilization potential in nutrient-poor and/or metals contaminated estuarine soils.Ministerio de Ciencia e Innovación PDC2021-120951-I00Junta de Andalucía US-1262036, PAIDI2020, P20_0068