Effect of Plant Growth-Promoting Rhizobacteria on Salicornia ramosissima Seed Germination under Salinity, CO2 and Temperature Stress

In a scenario of climate change and growing population, halophyte root microbiota interactions may be a sustainable solution to improve alternative crop production while combating abiotic stress. In this work, seeds of the cash crop halophyte Salicornia ramosissima were inoculated with five different plant growth-promoting rhizobacteria consortia, isolated from the rhizosphere of five halophytes in southwestern Spain salt marshes. For the first time, we recorded seed germination response to three interactive abiotic stressors, CO2 (400 and 700 ppm), temperature (25 and 29 ℃) and salinity (171, 510 and 1030 mM NaCl), all of them related to climate change. Salinity played a decisive role, as no significant differences were registered between treatments at 171 mM NaCl and no germination took place at 1030 mM NaCl. At 510 mM NaCl, one rhizobacterial consortium improved seed parameters notably, increasing up to 114% germination percentage and 65% seedlings biomass. These first findings encourage us to think that cash crop halophytes like S. ramosissima and halophyte root microbiota may be valuable resources for human or animal feeding in a future climate reality.Ministerio de Economía y Competitividad (CGL2016-75550-R)Ministerio de Educación, Cultura y Deporte (FPU014/03987

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-

Endophytic cultivable bacteria of the metal bioaccumulator Spartina maritima improve plant growth but not metal uptake in polluted marshes soils

Endophytic bacterial population was isolated from Spartina maritima tissues, a heavy metal bioaccumulator cordgrass growing in the estuaries of Tinto, Odiel, and Piedras River (south west Spain), one of the most polluted areas in the world. Strains were identified and ability to tolerate salt and heavy metals along with plant growth promoting and enzymatic properties were analyzed. A high proportion of these bacteria were resistant toward one or several heavy metals and metalloids including As, Cu, and Zn, the most abundant in plant tissues and soil. These strains also exhibited multiple enzymatic properties as amylase, cellulase, chitinase, protease and lipase, as well as plant growth promoting properties, including nitrogen fixation, phosphates solubilization, and production of indole-3-acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The best performing strains (Micrococcus yunnanensis SMJ12, Vibrio sagamiensis SMJ18, and Salinicola peritrichatus SMJ30) were selected and tested as a consortium by inoculating S. maritima wild plantlets in greenhouse conditions along with wild polluted soil. After 30 days, bacterial inoculation improved plant photosynthetic traits and favored intrinsic water use efficiency. However, far from stimulating plant metal uptake, endophytic inoculation lessened metal accumulation in above and belowground tissues. These results suggest that inoculation of S. maritima with indigenous metal-resistant endophytes could mean a useful approach in order to accelerate both adaption and growth of this indigenous cordgrass in polluted estuaries in restorative operations, but may not be suitable for rhizoaccumulation purposes

Impact of Plant Growth Promoting Bacteria on Salicornia ramosissima Ecophysiology and Heavy Metal Phytoremediation Capacity in Estuarine Soils

Salicornia ramosissima is a C3 halophyte that grows naturally in South Western Spain salt marshes, under soil salinity and heavy metal pollution (mostly Cu, Zn, As, and Pb) caused by both natural and anthropogenic pressure. However, very few works have reported the phytoremediation potential of S. ramosissima. In this work, we studied a microbe-assisted phytoremediation strategy under greenhouse conditions. We inoculated plant growth promoting (PGP) and heavy metal resistant bacteria in pots with S. ramosissima and natural non-polluted and polluted sediments collected from Spanish estuaries. Then, we analyzed plant ecophysiological and metal phytoaccumulation response. Our data suggested that inoculation in polluted sediments improved S. ramosissima plant growth in terms of relative growth rate (RGR) (32%) and number of new branches (61%). S. ramosissima photosynthetic fitness was affected by heavy metal presence in soil, but bacteria inoculation improved the photochemical apparatus integrity and functionality, as reflected by increments in net photosynthetic rate (21%), functionality of PSII (Fm and Fv/Fm) and electron transport rate, according to OJIP derived parameters. Beneficial effect of bacteria in polluted sediments was also observed by augmentation of intrinsic water use efficiency (28%) and slightly water content (2%) in inoculated S. ramosissima. Finally, our results demonstrated that S. ramosissima was able to accumulate great concentrations of heavy metals, mostly at root level, up to 200 mg Kg–1 arsenic, 0.50 mg Kg–1 cadmium, 400 mg Kg–1 copper, 25 mg Kg–1 nickel, 300 mg Kg–1 lead, and 300 mg Kg–1 zinc. Bioaugmentation incremented S. ramosissima heavy metal phytoremediation potential due to plant biomass increment, which enabled a greater accumulation capacity. Thus, our results suggest the potential use of heavy metal resistant PGPB to ameliorate the capacity of S. ramosissima as candidate for phytoremediation of salty polluted ecosystems.Ministerio de Economía y Competitividad (CGL2016-75550-R, AEI/FEDER, UE

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

Persistent Overactive Cytotoxic Immune Response in a Spanish Cohort of Individuals With Long-COVID: Identification of Diagnostic Biomarkers

Long-COVID is a new emerging syndrome worldwide that is characterized by the persistence of unresolved signs and symptoms of COVID-19 more than 4 weeks after the infection and even after more than 12 weeks. The underlying mechanisms for Long-COVID are still undefined, but a sustained inflammatory response caused by the persistence of SARS-CoV-2 in organ and tissue sanctuaries or resemblance with an autoimmune disease are within the most considered hypotheses. In this study, we analyzed the usefulness of several demographic, clinical, and immunological parameters as diagnostic biomarkers of Long-COVID in one cohort of Spanish individuals who presented signs and symptoms of this syndrome after 49 weeks post-infection, in comparison with individuals who recovered completely in the first 12 weeks after the infection. We determined that individuals with Long-COVID showed significantly increased levels of functional memory cells with high antiviral cytotoxic activity such as CD8+ TEMRA cells, CD8±TCRγδ+ cells, and NK cells with CD56+CD57+NKG2C+ phenotype. The persistence of these long-lasting cytotoxic populations was supported by enhanced levels of CD4+ Tregs and the expression of the exhaustion marker PD-1 on the surface of CD3+ T lymphocytes. With the use of these immune parameters and significant clinical features such as lethargy, pleuritic chest pain, and dermatological injuries, as well as demographic factors such as female gender and O+ blood type, a Random Forest algorithm predicted the assignment of the participants in the Long-COVID group with 100% accuracy. The definition of the most accurate diagnostic biomarkers could be helpful to detect the development of Long-COVID and to improve the clinical management of these patients.This work was supported by the Coordinated Research Activities at the National Center of Microbiology (CNM, Instituto de Salud Carlos III) (COV20_00679) to promote an integrated response against SARS-CoV-2 in Spain (Spanish Ministry of Science and Innovation), which is coordinated by Dr Inmaculada Casas (WHO National Influenza Center of the CNM); a generous donation provided by Chiesi España, S.A.U. (Barcelona, Spain); the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00); and the Spanish AIDS Research Network RD16CIII/0002/0001 that is included in Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2016-2020, Instituto de Salud Carlos III, European Region Development Fund (ERDF). The work of ML-H and SR-M is financed by NIH grant R01AI143567. The work of MT is supported by Instituto de Salud Carlos III (COV20_00679). The work of LV is supported by a pre-doctoral grant from Instituto de Salud Carlos III (FIS PI16CIII/00034-ISCIII-FEDER). The work of FR-M is financed by the Spanish Ministry of Science and Innovation (PID2019-110275RB-I00).S

Anti-trbc1 antibody-based flow cytometric detection of t-cell clonality: Standardization of sample preparation and diagnostic implementation

© 2021 by the authors.A single antibody (anti-TRBC1; JOVI-1 antibody clone) against one of the two mutually exclusive T-cell receptor β-chain constant domains was identified as a potentially useful flow-cytometry (FCM) marker to assess Tαβ-cell clonality. We optimized the TRBC1-FCM approach for detecting clonal Tαβ-cells and validated the method in 211 normal, reactive and pathological samples. TRBC1 labeling significantly improved in the presence of CD3. Purified TRBC1+ and TRBC1− monoclonal and polyclonal Tαβ-cells rearranged TRBJ1 in 44/47 (94%) and TRBJ1+TRBJ2 in 48 of 48 (100%) populations, respectively, which confirmed the high specificity of this assay. Additionally, TRBC1+/TRBC1− ratios within different Tαβ-cell subsets are provided as reference for polyclonal cells, among which a bimodal pattern of TRBC1-expression profile was found for all TCRVβ families, whereas highly-variable TRBC1+/TRBC1− ratios were observed in more mature vs. naïve Tαβ-cell subsets (vs. total T-cells). In 112/117 (96%) samples containing clonal Tαβ-cells in which the approach was validated, monotypic expression of TRBC1 was confirmed. Dilutional experiments showed a level of detection for detecting clonal Tαβ-cells of ≤10−4 in seven out of eight pathological samples. These results support implementation of the optimized TRBC1-FCM approach as a fast, specific and accurate method for assessing T-cell clonality in diagnostic-FCM panels, and for minimal (residual) disease detection in mature Tαβ+ leukemia/lymphoma patients.This work was supported by the CB16/12/00400 (CIBERONC) and PI20-01346 grants, from the Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (Madrid, Spain) and FONDOS FEDER, and by the EuroFlow Foundation (Leiden, The Netherlands). N. Muñoz-García is supported by a pre-doctoral grant (Ref. IBPredoc17/00012) from IBSAL (Salamanca, Spain). M. Lima, N. Villamor, A.W. Langerak, J.J.M. van Dongen, A. Orfao, and J. Almeida are members of the EuroFlow Consortiu

Assessing the Biofortification of Wheat Plants by Combining a Plant Growth-Promoting Rhizobacterium (PGPR) and Polymeric Fe-Nanoparticles: Allies or Enemies?

Biofortification has been widely used to increase mineral nutrients in staple foods, such as wheat (Triticum aestivum). In this study, a new approach has been used by analyzing the effect of inoculation with a plant growth-promoting rhizobacterium (PGPR), namely, Bacillus aryabhattai RSO25 and the addition of 1% (v/v) of organometallic Fe-containing polymeric nanoparticles (FeNPs) alone and in combination. Previously, the minimal inhibitory concentration of FeNPs for the bacterium was determined in order not to inhibit bacterial growth. All treatments had minor effects on seed germination and plant survival. Considering the physiology of plants, several photosynthetic parameters were significantly improved in individual treatments with FeNPs or the bacterium, particularly the efficiency of the photosystem II and the electron transport rate, which is indicative of a better photosynthetic performance. However, at the end of the experiment, a significant effect on final plant growth was not observed in shoots or in roots. When using FeNPs alone, earlier spike outgrow was observed and the final number of spikes increased by 20%. Concerning biofortification, FeNPs increased the concentration of Fe in spikes by 35%. In fact, the total amount of Fe per plant base rose to 215% with regard to the control. Besides, several side effects, such as increased Ca and decreased Na and Zn in spikes, were observed. Furthermore, the treatment with only bacteria decreased Na and Fe accumulation in grains, indicating its inconvenience. On its side, the combined treatment led to intermediate Fe accumulation in spikes, since an antagonist effect between RSO25 and FeNPs was observed. For this reason, the combined treatment was discouraged. In conclusion, of the three treatments tested, FeNPs alone is recommended for achieving efficient Fe biofortification in wheat.Ministry of Science and Innovation of Spain grant PID2019-109371GB-I00Junta de Andalucía (Spain) Project FQM-135University of Seville, Spain PPIT-2020/0000109

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe