Versatile Biodegradable Poly(acrylic acid)-Based Hydrogels Infiltrated in Porous Titanium Implants to Improve the Biofunctional Performance.

This research work proposes a synergistic approach to improve implants' performance through the use of porous Ti substrates to reduce the mismatch between Young's modulus of Ti (around 110 GPa) and the cortical bone (20-25 GPa), and the application of a biodegradable, acrylic acid-based polymeric coating to reduce bacterial adhesion and proliferation, and to enhance osseointegration. First, porous commercially pure Ti substrates with different porosities and pore size distributions were fabricated by using space-holder techniques to obtain substrates with improved tribomechanical behavior. On the other hand, a new diacrylate cross-linker containing a reduction-sensitive disulfide bond was synthesized to prepare biodegradable poly(acrylic acid)-based hydrogels with 1, 2, and 4% cross-linker. Finally, after the required characterization, both strategies were implemented, and the combination of 4% cross-linked poly(acrylic acid)-based hydrogel infiltrated in 30 vol % porosity, 100-200 µm average pore size, was revealed as an outstanding choice for enhancing implant performance.

A Culturomics-Based Bacterial Synthetic Community for Improving Resilience towards Arsenic and Heavy Metals in the Nutraceutical Plant Mesembryanthemum crystallinum.

Plant-growth-promoting bacteria (PGPB) help plants thrive in polluted environments and increase crops yield using fewer inputs. Therefore, the design of tailored biofertilizers is of the utmost importance. The purpose of this work was to test two different bacterial synthetic communities (SynComs) from the microbiome of Mesembryanthemum crystallinum, a moderate halophyte with cosmetic, pharmaceutical, and nutraceutical applications. The SynComs were composed of specific metal-resistant plant-growth-promoting rhizobacteria and endophytes. In addition, the possibility of modulating the accumulation of nutraceutical substances by the synergetic effect of metal stress and inoculation with selected bacteria was tested. One of the SynComs was isolated on standard tryptone soy agar (TSA), whereas the other was isolated following a culturomics approach. For that, a culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard TSA and MA media, stablishing two independent collections. All bacteria were tested for PGP properties, secreted enzymatic activities, and resistance towards As, Cd, Cu, and Zn. The three best bacteria from each collection were selected in order to produce two different consortiums (denominated TSA- and MA-SynComs, respectively), whose effect on plant growth and physiology, metal accumulation, and metabolomics was evaluated. Both SynComs, particularly MA, improved plant growth and physiological parameters under stress by a mixture of As, Cd, Cu, and Zn. Regarding metal accumulation, the concentrations of all metals/metalloids in plant tissues were below the threshold for plant metal toxicity, indicating that this plant is able to thrive in polluted soils when assisted by metal/metalloid-resistant SynComs and could be safely used for pharmaceutical purposes. Initial metabolomics analyses depict changes in plant metabolome upon exposure to metal stress and inoculation, suggesting the possibility of modulating the concentration of high-value metabolites. In addition, the usefulness of both SynComs was tested in a crop plant, namely Medicago sativa (alfalfa). The results demonstrate the effectiveness of these biofertilizers in alfalfa, improving plant growth, physiology, and metal accumulation.

Use of native plants and their associated bacteria rhizobiomes to remediate-restore Draa Sfar and Kettara mining sites, Morocco.

Soil and mine tailings are unreceptive to plant growth representing an imminent threat to the environment and resource sustainability. Using indigenous plants and their associated rhizobacteria to restore mining sites would be an eco-friendly solution to mitigate soil-metal toxicity. Soil prospection from Draa Sfar and Kettara mining sites in Morocco was carried out during different seasons for native plant sampling and rhizobacteria screening. The sites have been colonized by fifteen tolerant plant species having different capacities to accumulate Cu, Zn, and P in their shoots/root systems. In Draa Sfar mine, Suaeda vera J.F. Gmel., Sarcocornia fruticosa (L.) A.J. Scott., and Frankenia corymbosa Desf. accumulated mainly Cu (more than 90 mg kg-1), Atriplex halimus L. accumulated Zn (mg kg-1), and Frankenia corymbosa Desf. accumulated Pb (14 mg kg-1). As for Kettara mine, Aizoon canariense L. mainly accumulated Zn (270 mg kg-1), whereas Forsskalea tenacissima L. was the best shoot Cu accumulator with up to 50 mg kg-1, whereas Cu accumulation in roots was 21 mg kg-1. The bacterial screening revealed the strains' abilities to tolerate heavy metals up to 50 mg kg-1 Cu, 250 mg kg-1 Pb, and 150 mg kg-1 Zn. Isolated strains belonged mainly to Bacillaceae (73.33%) and Pseudomonadaceae (10%) and expressed different plant growth-promoting traits, alongside their antifungal activity. Results from this study will provide an insight into the ability of native plants and their associated rhizobacteria to serve as a basis for remediation-restoration strategies.

Restoring the plant productivity of heavy metal-contaminated soil using phosphate sludge, marble waste, and beneficial microorganisms.

Assisted natural remediation (ANR) has been highlighted as a promising, less expensive, and environmentally friendly solution to remediate soil contaminated with heavy metals. We tested the effects of three amendments (10% compost, C; 5 or 15% phosphate sludge, PS5 and PS15; and 5 or 15% marble waste, MW5 and MW15) in combination with microorganism inoculation (rhizobacteria consortium alone, mycorrhizae alone, and the two in-combination) on alfalfa in contaminated soil. Plant concentrations of Zn, Cu, and Pb were measured, along with proline and malondialdehyde production. The microbiological and physicochemical properties of the mining soil were evaluated. Application of the amendments allowed germination and promoted growth. Inoculation with the rhizobacteria consortium and/or mycorrhizae stimulated plant growth. PS and MW stimulated the production of proline. Inoculation of alfalfa with the rhizobacteria-mycorrhizae mixture and the application of MW allowed the safe cultivation of the legume, as shown by the low concentrations of metals in plant shoots. Zn and Pb concentrations were below the limits recommended for animal grazing and accumulated essentially in roots. Soil analyses showed the positive effect of the amendments on the soil physicochemical properties. All treatments increased soil pH (around 7), total organic carbon, and assimilable phosphorus content. Notably, an important decrease in soluble heavy metals concentrations was observed. Overall, our findings revealed that the applied treatments reduced the risk of metal-polluted soils limiting plant growth. The ANR has great potential for success in the restoration of polymetallic and acidic mining soils using the interaction between alfalfa, microorganisms, and organo-mineral amendments.

Pseudoalteromonas rhizosphaerae sp. nov., a novel plant growth-promoting bacterium with potential use in phytoremediation.

Strain RA15T was isolated from the rhizosphere of the halophyte plant Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). RA15T cells were Gram stain-negative, non-spore-forming, aerobic rods and formed cream-coloured, opaque, mucoid, viscous, convex, irregular colonies with an undulate margin. Optimal growth conditions were observed on tryptic soy agar (TSA) plates supplemented with 2.5 % NaCl (w/v) at pH 7.0 and 28 °C, although it was able to grow at 4-32 °C and at pH values of 5.0-9.0. The NaCl tolerance range was from 0 to 15 %. The major respiratory quinone was Q8 but Q9 was also present. The most abundant fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 1 ω8c and C16 : 0. The polar lipids profile comprised phosphatidylglycerol and phosphatidylethanolamine as the most abundant representatives. Phylogenetic analyses confirmed the well-supported affiliation of strain RA15T within the genus Pseudoalteromonas, close to the type strains of Pseudoalteromonas neustonica, Pseudoalteromonas prydzensis and Pseudoalteromonas mariniglutinosa. Results of comparative phylogenetic and phenotypic studies between strain RA15T and its closest related species suggest that RA15T could be a new representative of the genus Pseudoalteromonas, for which the name Pseudoalteromonas rhizosphaerae sp. nov. is proposed. The type strain is RA15T (=CECT 9079T=LMG 29860T). The whole genome has 5.3 Mb and the G+C content is 40.4 mol%.

Halomonas radicis sp. nov., isolated from Arthrocnemum macrostachyum growing in the Odiel marshes(Spain) and emended descriptions of Halomonas xinjiangensis and Halomonas zincidurans.

Strain EAR18T was isolated as an endophyte from the roots of a halophyte plant, Arthrocnemum macrostachyum, growing in the Odiel marshes (Huelva, Spain). Cells of strain EAR18T were Gram- stain-negative, motile, non-spore-forming aerobic rods. It grew optimally on tryptic soy agar supplemented with 2.5 % NaCl (w/v), at pH 7 and 30 °C for 48 h. It tolerated NaCl from 0 to 25 % (w/v). It presented Q9 as the major quinone and C19 : 0 cyclo ω8c, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0 as the predominant fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and four unidentified phospholipids. The results of phylogenetic analysis based on 16S rRNA gene sequences revealed that strain EAR18T formed a well-supported clade with species Halomonas zincidurans B6T and Halomonas xinjiangensis TRM 0175T (similarities of 98.3 and 96.1 % respectively). Furthermore, digital DNA-DNA hybridization analysis resulted in values of 20.4 % with H. xinjiangensis TRM 0175T and 35.50 % with H. zincidurans B6T, and ANIb/ANIm results in values of 73.8 %/84.2 % with H. xinjiangensis TRM 0175T and 86.8 %/89.4 % with H. zincidurans B6T. Based on phylogeny and differential phenotypic properties in comparison with its closest related species, strain EAR18T is suggested to represent a new species in the genus Halomonas, for which the name Halomonas radicis sp. nov. is proposed. The type strain is EAR18T (=CECT 9077T=LMG 29859T). The whole genome was sequenced, and it had a total length of 4.6 Mbp and a G+C content of 64.9 mol%.

Soil phenanthrene phytoremediation capacity in bacteria-assisted Spartina densiflora.

Polycyclic aromatic hydrocarbons (PAH) have become a threat for the conservation of wetlands worldwide. The halophyte Spartina densiflora has shown to be potentially useful for soil phenanthrene phytoremediation, but no studies on bacteria-assisted hydrocarbon phytoremediation have been carried out with this halophyte. In this work, three phenanthrene-degrading endophytic bacteria were isolated from S. densiflora tissues and used for plant inoculation. Bacterial bioaugmentation treatments slightly improved S. densiflora growth, photosynthetic and fluorescence parameters. But endophyte-inoculated S. densiflora showed lower soil phenanthrene dissipation rates than non-inoculated S. densiflora (30% below) or even bulk soil (23% less). Our work demonstrates that endophytic inoculation on S. densiflora under greenhouse conditions with the selected PAH-degrading strains did not significantly increase inherent phenanthrene soil dissipation capacity of the halophyte. It would therefore be advisable to provide effective follow-up of bacterial colonization, survival and metabolic activity during phenanthrene soil phytoremediation.

Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., plant growth promoting endophytes isolated from the halophyte plant Arthrocnemum macrostachyum.

Two endophytic bacteria (EAod3T and EAod7T) were isolated from the aerial part of plants of Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). Phylogenetic analysis based on 16S rRNA gene sequences indicated their affiliation to the genus Kushneria. 16S rRNA gene sequences of strains EAod3T and EAod7T showed the highest similarity to Kushneria marisflavi DSM 15357T (99.0 and 97.6 %, respectively). Digital DNA-DNA hybridization studies between the draft genomes of strain EAod3T and K. marisflavi DSM 15357T corresponded to 28.5 % confirming the novel lineage of strain EAod3T in the genus Kushneria. Cells of both strains were Gram-staining-negative, aerobic and motile rods able to grow at 4-37 °C, at pH 5.0-8.0 and tolerate 0.5-25 % NaCl (w/v). They presented ubiquinone Q9 and C16 : 0, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c as the major fatty acids. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Based on the phenotypic and phylogenetic results, strains EAod3T (=CECT 9073T=LMG 29856T) and EAod7T (=CECT 9075T=LMG 29858T) are proposed as new representatives of the genus Kushneria, and the proposed names are Kushneria phyllosphaerae sp. nov. and Kushneria endophytica sp. nov., respectively. The whole genome sequence of strain EAod3T has a total length of 3.8 Mbp and a G+C content of 59.3 mol%.

Vibrio palustris sp. nov. and Vibrio spartinae sp. nov., two novel members of the Gazogenes clade, isolated from salt-marsh plants (Arthrocnemum macrostachyum and Spartina maritima).

Two bacterial strains, EAod9T and SMJ21T, isolated from salt-marsh plants, were determined to be related to species of the genus Vibriofrom from 16S rRNA sequence comparisons. Their closest phylogenetic relatives are members of the Gazogenes clade, Vibrio mangrovi and Vibrio rhizosphaerae , which show the greatest similarity to the SMJ21TrRNA sequence (97.3 and 97.1 %, respectively), while EAod9T had less than 97.0 % similarity to any other species of the genus Vibrio. Both strains share the basic characteristics of the genus Vibrio, as they are Gram-stain negative, motile, slightly halophilic, facultatively anaerobic bacteria. In addition, they are oxidase-negative and unable to grow on TCBS Agar; they grow between 15 to 26 °C, pH 6 to 8 and in up to 10 % (w/v) total salinity. They produce indol, are positive in the Voges-Proskauer test and are negative for arginine dihydrolase, lysine and ornithine decarboxylases. Strain SMJ21T is aerogenic and red-pigmented, due to prodigiosin production, while strain EAod9T ferments glucose without gas and is not pigmented. The major cellular fatty acids of both novel strains were C16 : 1ω7c/C16 : 1ω6c and C16 : 0. WGSobtained for both strains, along with the other five members of the clade, allowed the determination of ANI indexes and in silico estimations of DDH values, which confirmed that the two strains represent two novel species of the genus Vibrio: Vibriopalustris sp. nov. (with EAod9T=CECT 9027T=LMG 29724T as the proposed type strain) and Vibrio spartinae sp. nov. (with SMJ21T=CECT 9026T=LMG 29723T as the proposed type strain).

Marinomonas spartinae sp. nov., a novel species with plant-beneficial properties.

Two strains of Gram-stain-negative, chemo-organotrophic, aerobic and halophilic gammaproteobacteria, isolated from within the stem and roots of Spartina maritima in salt marshes from the south Atlantic Spanish coast, were found to represent a novel species in the genus Marinomonas through phylogenetic analysis of their 16S rRNA genes and phenotypic characterization. 16S rRNA gene sequences of the two strains shared < 96.2% similarity with other Marinomonas species, with Marimonas alcarazii being the most similar in sequence. They required sodium ions for growth, were able to thrive at low (4 °C) temperatures and at salinities of 12-15%, were unable to hydrolyse any tested macromolecule except casein, and grew with different monosaccharides, disaccharides, sugar alcohols, organic acids and amino acids. The novel species differed from other Marinomonas species in the use of several sole carbon sources, its temperature and salinity ranges for growth, ion requirements and cellular fatty acid composition, which included C16:0, C16:1 and C18:1 as major components and C10:0 3-OH, C12:0 and C12:0 3-OH as minor components. The name Marinomonas spartinae sp. nov. is proposed, with SMJ19T (=CECT 8886T=KCTC 42958T) as the type strain.

Unraveling the effect of arsenic on the model Medicago-Ensifer interaction: a transcriptomic meta-analysis.

The genetic regulation underlying the effect of arsenic (As(III)) on the model symbiosis Medicago-Ensifer was investigated using a combination of physiological (split-roots), microscopy and genetic (microarrays, qRT-PCR and composite plants) tools. Nodulation was very sensitive to As(III) (median inhibitory dose (ID50) = 20 µM). The effect on root elongation and on nodulation was local (nonsystemic). A battery of stress (salt, drought, heat shock, metals, etc.)-related genes were induced. Glutathione played a pivotal role in tolerance/detoxification, together with secondary metabolites ((iso)flavonoids and phenylpropanoids). However, antioxidant enzymes were not activated. Concerning the symbiotic interaction, molecular evidence suggesting that rhizobia alleviate As stress is for the first time provided. Chalcone synthase (which is involved in the first step of the legume-rhizobia cross-talk) was strongly enhanced, suggesting that the plants are biased to establish symbiotic interactions under As(III) stress. In contrast, 13 subsequent nodulation genes (involved in nodulation factors (Nod factors) perception, infection, thread initiation and progression, and nodule morphogenesis) were repressed. Overexpression of the ethylene responsive factor ERN in composite plants reduced root stress and partially restored nodulation, whereas overexpression of the early nodulin ENOD12 enhanced nodulation both in the presence and, particularly, in the absence of As, without affecting root elongation. Several transcription factors were identified, which could be additional targets for genetic engineering aiming to improve nodulation and/or alleviate root stress induced by this toxic.

Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia.

The rhizobia-legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain Ensifer medicae MA11 in Cu-contaminated substrates. The copAB genes from a Cu-resistant Pseudomonas fluorescens strain were expressed in E. medicae MA11 under the control of the nifH promoter. The resulting strain E. medicae MA11-copAB was able to alleviate the toxic effect of Cu in Medicago truncatula. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate twofold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered E. medicae strain increased Cu accumulation in roots and decreased the content in shoots. Thus, E. medicae MA11-copAB increased the capacity of M. truncatula to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the nifH promoter-copAB construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia microsymbionts of authoctonous legumes growing in Cu-contaminated soils.

Uncovering the Role of Selenite and Selenium Nanoparticles (SeNPs) in Adolescent Rat Adipose Tissue beyond Oxidative Balance: Transcriptomic Analysis.

Studies on adolescent rats, when body composition is changing deeply, reveal that the administration of sodium selenite and selenium nanoparticles (SeNPs), at the same dose, have opposite effects on adipogenesis in white adipose tissue (WAT). To investigate the mechanisms involved in these contrasting effects by means of transcriptomic analysis, three groups of male adolescent rats (n = 18) were used: control (C), selenite supplemented (S), and SeNPs supplemented (NS). Both treated groups received a twofold increase in Se dose compared to the control group through water intake for three weeks. Following treatment, WAT was removed and frozen at -80 °C until subsequent use for RNA extraction, endogenous antioxidant enzymatic activities determination, and quantification of H2O2 and malondialdehyde. NS rats displayed a larger number of differentially expressed genes and cellular processes impacted than S rats. Remarkably, these changes involved upregulation of gene expression associated with the immune system, catabolism, mitochondrial function, and oxidative balance. NS rats presented an increase in antioxidant enzymes activity, alongside an accumulation of H2O2 and malondialdehyde levels. The expression level of 81 genes related to oxidative stress was significantly affected in NS rats. Analyzing the KEGG pathway enrichment revealed that NS rats exhibited increased activity in key catabolic pathways and decreased activity in crucial growth signaling processes. These changes contribute to the mass decrease in WAT found in NS rats. These results suggest a possible application of SeNPs in WAT reduction and induction of the immune response during adolescence.

Nitrate modulates the physiological tolerance responses of the halophytic species Sarcocornia fruticosa to copper excess.

Coexistence impact of pollutants of different nature on halophytes tolerance to metal excess has not been thoroughly examined, and plant functional responses described so far do not follow a clear pattern. Using the Cu-tolerant halophyte Sarcocornia fruticosa as a model species, we conducted a greenhouse experiment to evaluate the impact of two concentration of copper (0 and 12 mM CuSO4) in combination with three nitrate levels (2, 14 and 50 mM KNO3) on plant growth, photosynthetic apparatus performance and ROS-scavenging enzymes system. The results revealed that S. fruticosa was able to grow adequately even when exposed to high concentrations of copper and nitrate. This response was linked to the plant capacity to uptake and retain a large amount of copper in its roots (up to 1500 mg kg-1 Cu), preventing its transport to aerial parts. This control of translocation was further magnified with nitrate concentration increment. Likewise, although Cu excess impaired S. fruticosa carbon assimilation capacity, the plant was able to downregulate its light-harvesting complexes function, as indicated its lowers ETR values, especially at 12 mM Cu + 50 mM NO3. This downregulation would contribute to avoid excess energy absorption and transformation. In addition, this strategy of avoiding excess energy was accompanied by the upregulation of all ROS-scavenging enzymes, a response that was further enhanced by the increase in nitrate concentration. Therefore, we conclude that the coexistence of nitrate would favor S. fruticosa tolerance to copper excess, and this effect is mediated by the combined activation of several tolerance mechanisms.

Microbiota-Liver-Bile Salts Axis, a Novel Mechanism Involved in the Contrasting Effects of Sodium Selenite and Selenium-Nanoparticle Supplementation on Adipose Tissue Development in Adolescent Rats.

Adolescence is a period during which body composition changes deeply. Selenium (Se) is an excellent antioxidant trace element related to cell growth and endocrine function. In adolescent rats, low Se supplementation affects adipocyte development differently depending on its form of administration (selenite or Se nanoparticles (SeNPs). Despite this effect being related to oxidative, insulin-signaling and autophagy processes, the whole mechanism is not elucidated. The microbiota-liver-bile salts secretion axis is related to lipid homeostasis and adipose tissue development. Therefore, the colonic microbiota and total bile salts homeostasis were explored in four experimental groups of male adolescent rats: control, low-sodium selenite supplementation, low SeNP supplementation and moderate SeNPs supplementation. SeNPs were obtained by reducing Se tetrachloride in the presence of ascorbic acid. Supplementation was received orally through water intake; low-Se rats received twice more Se than control animals and moderate-Se rats tenfold more. Supplementation with low doses of Se clearly affected anaerobic colonic microbiota profile and bile salts homeostasis. However, these effects were different depending on the Se administration form. Selenite supplementation primarily affected liver by decreasing farnesoid X receptor hepatic function, leading to the accumulation of hepatic bile salts together to increase in the ratio Firmicutes/Bacteroidetes and glucagon-like peptide-1 (GLP-1) secretion. In contrast, low SeNP levels mainly affected microbiota, moving them towards a more prominent Gram-negative profile in which the relative abundance of Akkermansia and Muribaculaceae was clearly enhanced and the Firmicutes/Bacteroidetes ratio decreased. This bacterial profile is directly related to lower adipose tissue mass. Moreover, low SeNP administration did not modify bile salts pool in serum circulation. In addition, specific gut microbiota was regulated upon administration of low levels of Se in the forms of selenite or SeNPs, which are properly discussed. On its side, moderate-SeNPs administration led to great dysbiosis and enhanced the abundance of pathogenic bacteria, being considered toxic. These results strongly correlate with the deep change in adipose mass previously found in these animals, indicating that the microbiota-liver-bile salts axis is also mechanistically involved in these changes.

Culturomics and Circular Agronomy: Two Sides of the Same Coin for the Design of a Tailored Biofertilizer for the Semi-Halophyte Mesembryanthemum crystallinum.

According to the EU, the global consumption of biomass, fossil fuels, metals, and minerals is expected to double by 2050, while waste will increase by 70%. In this context, the Circular Economy Action Plan (CEAP) intends to integrate development and sustainability. In this regard, tailored biofertilizers based on plant growth-promoting bacteria (PGPB) can improve plant yield with fewer inputs. In our project, an autochthonous halophyte of the Andalusian marshes, namely Mesembryanthemum crystallinum, was selected for its interest as a source of pharmaceuticals and nutraceuticals. The aim of this work was to use a culturomics approach for the isolation of specific PGPB and endophytes able to promote plant growth and, eventually, modulate the metabolome of the plant. For this purpose, a specific culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard tryptone soy agar (TSA) and MA in order to obtain two independent collections. A higher number of bacteria were isolated on TSA than in MA (47 vs. 37). All the bacteria were identified, and although some of them were isolated in both media (Pseudomonas, Bacillus, Priestia, Rosellomorea, etc.), either medium allowed the isolation of specific members of the M. crystallinum microbiome such as Leclercia, Curtobacterium, Pantoea, Lysinibacillus, Mesobacillus, Glutamicibacter, etc. Plant growth-promoting properties and extracellular degrading activities of all the strains were determined, and distinct patterns were found in both media. The three best bacteria of each collection were selected in order to produce two different consortia, whose effects on seed germination, root colonization, plant growth and physiology, and metabolomics were analyzed. Additionally, the results of the plant metabolome revealed a differential accumulation of several primary and secondary metabolites with pharmaceutical properties. Overall, the results demonstrated the feasibility of using "low cost media" based on plant biomass to carry out a culturomics approach in order to isolate the most suitable bacteria for biofertilizers. In this way, a circular model is established in which bacteria help plants to grow, and, in turn, a medium based on plant wastes supports bacterial growth at low prices, which is the reason why this approach can be considered within the model of "circular agronomy".

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.

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.

Plant Growth-Promoting Rhizobacteria Improve Rice Response to Climate Change Conditions.

Rice is one of the most important crops in the world and is considered a strategic crop for food security. Furthermore, the excessive use of chemical fertilizers to obtain high yields causes environmental problems. A sustainable alternative includes taking advantage of beneficial bacteria that promote plant growth. Here, we investigate the effect of five bacterial biofertilizers from halophytes on growth, and we investigate photosynthetic efficiency in rice plants grown under saline conditions (0 and 85 mmol L-1 NaCl) and future climate change scenarios, including increased CO2 concentrations and temperature (400/700 ppm and 25/+4 °C, respectively). Biofertilizers 1-4 increased growth by 9-64% in plants grown with and without salt in both CO2- temperature combinations, although there was no significant positive effect on the net photosynthetic rate of rice plants. In general, biofertilizer 1 was the most effective at 400 ppm CO2 and at 700 ppm CO2 +4 °C in the absence of salt. Inocula 1-5 also stimulated plant length at high CO2 levels without salt. Finally, the positive effect of biofertilization was attenuated in the plants grown under the interaction between salt and high CO2. This highlights the significance of studying biofertilization under stress interaction to establish the real potential of biofertilizers in the context of climate change conditions.

Improving Grapevine Heat Stress Resilience with Marine Plant Growth-Promoting Rhizobacteria Consortia.

Amid climate change, heatwave events are expected to increase in frequency and severity. As a result, yield losses in viticulture due to heatwave stress have increased over the years. As one of the most important crops in the world, an eco-friendly stress mitigation strategy is greatly needed. The present work aims to evaluate the physiological fitness improvement by two marine plant growth-promoting rhizobacteria consortia in Vitis vinifera cv. Antão Vaz under heatwave conditions. To assess the potential biophysical and biochemical thermal stress feedback amelioration, photochemical traits, pigment and fatty acid profiles, and osmotic and oxidative stress biomarkers were analysed. Bioaugmented grapevines exposed to heatwave stress presented a significantly enhanced photoprotection capability and higher thermo-stability, exhibiting a significantly lower dissipation energy flux than the non-inoculated plants. Additionally, one of the rhizobacterial consortia tested improved light-harvesting capabilities by increasing reaction centre availability and preserving photosynthetic efficiency. Rhizobacteria inoculation expressed an osmoprotectant promotion, revealed by the lower osmolyte concentration while maintaining leaf turgidity. Improved antioxidant mechanisms and membrane stability resulted in lowered lipid peroxidation product formation when compared to non-inoculated plants. Although the consortia were found to differ significantly in their effectiveness, these findings demonstrate that bioaugmentation induced significant heatwave stress tolerance and mitigation. This study revealed the promising usage of marine PGPR consortia to promote plant fitness and minimize heatwave impacts in grapevines.