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The new strategies that are trying to be developed to protect microorganisms for a successful application have generated various types of granulated, powdered, or liquid formulations. In this work, we have developed a rhizobial encapsulation system for legumes accompanied by metabolites to enhance microorganism-plant communication. This novel way of producing a biofertilizer for legumes was developed based on alginate, a degradable compound that allows environmentally friendly use. This way of generating an inoculant allows it designing by making different molecular combinations for different purposes, being a double inoculant, biological and molecular.

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Here, we estimate fast changes in the fluidity of Sinorhizobium meliloti membranes submitted to cyclic temperature changes (10°C-40°C-10°C) by monitoring the fluorescence polarization (P) of DPH and TMA-DPH of the whole cell (WC) as well as in its outer (OM) and inner (IM) membranes. Additionally, the long-term response to thermal changes is demonstrated through the dynamics of the phospholipid and fatty acid composition in each membrane. This allowed membrane homeoviscous adaptation by the return to optimal fluidity levels as measured by the PDPH/TMA-DPH in WC, OM, IM, and multilamellar vesicles of lipids extracted from OM and IM. Due to probe-partitioning preferences and membranes' compositional characteristics, DPH and TMA-DPH exhibit different behaviors in IM and OM. The rapid effect of cyclic temperature changes on the P was the opposite in both membranes with the IM being the one that exhibited the thermal behavior expected for lipid bilayers. Interestingly, only after the incubation at 40°C, cells were unable to recover the membrane preheating P levels when cooled up to 10°C. Solely in this condition, the formation of threads and nodular structures in Medicago sativa infected with S. meliloti were delayed, indicating that the symbiotic interaction was partially altered but not halted.

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Immobilizarion of PGPR for agricultural applications aims to provide temporary physical protection from stressful environmental conditions and the gradual release of cells for successful root colonization, release the cells gradually. In this work, we immobilized Bradyrhizobium sp. SEMIA6144 or Azospirillum brasilense Az39 cells in 2% alginate beads prepared by ionic gelation process, and then stored up to 12 months at 4 °C. Alginate matrix showed interaction with the immobilized bacteria (FTIR), allowed a constant release of cells, and improved their viability and capability to interact with Arachis hypogaea. Cell number into beads reached 107 CFU.bead-1; however, viability decreased from 4 months of storage for Az39, while it was maintained up to 12 months for SEMIA6144, showing a low metabolic activity measured by the MTT assay. Adhesion of SEMIA6144 and Az39 from new beads to peanut root was 11.5% and 16%, respectively, higher than non-immobilized bacteria. Peanut inoculation with 12 months storage SEMIA6144 beads significantly increased root length and biomass at 30 days of growth, and under restrictive water condition (RWC), nodulation and total plant N content increased compared with liquid inoculation. Our results demonstrate that immobilization of SEMIA6144 and Az39 in alginate matrix is a potential alternative to enhance peanut growth even under RWC. KEY POINTS: • Alginate encapsulation enhances viability of SEMIA6144 or Az39 under storage at 4 °C for 1 year. • Alginate beads 2% ensure the gradual release of the microorganisms. • Cells from beads stored for long periods present chemotaxis and adhesion to peanut root. • Peanut inoculation with 1-year-old SEMIA6144 beads improves nodulation and growth in RWC.

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Plant growth promoting rhizobacteria (PGPR) is an alternative to chemical fertilizers for sustainable, environment friendly agriculture. There is a need to develop strategies to potentiate the interaction between rhizobacteria and plants. Flavonoids and organic acids (components of root exudates) play specific beneficial roles as carbon sources and signal molecules in the plant - rhizobacteria interactions. The goal of this work is to encapsulate signal molecules, namely citric acid and naringin, an organic acid and a flavonoid, respectively, by a biodegradable polymer, polycaprolactone (PCL), in order to maintain the stability and activity of those signal molecules and enable their slow or controlled release over a selected period of time, according to the needs of the plants. This approach is expected to potentiate food crops, namely peanut crop, in adverse environmental conditions (water deficit), by promoting the beneficial interaction between the peanut plant (A. hypogaea) and rhizobacteria. The microcapsules (MCs) are obtained by an emulsion process combined with solvent evaporation technique and are characterized by scanning electron microscopy, thermogravimetry and Fourier transformed infrared spectroscopy. The kinetics of in vitro release of encapsulated molecules, in a period where the uptake of the compound in plants can occur, is studied. The encapsulation synthesis parameters that lead to the best encapsulation process yield and efficiency, as well as to the best final performance in terms of release, are identified. The effect of pH and molecular weight of PCL is found to mediate the release properties of the molecules for different types of soil. PCL 45000 Mw dissolved at 16% in dichloromethane leads to an encapsulation efficiency of 75% and the resulting MCs containing naringin exhibit a slow release profile for 30 days, unmodified by pH, enabling their use in soils of different characteristics. This research makes possible the manufacturing of smart materials for sustainable agriculture practices.

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The aim of this work was to clarify the mechanism related to plant growth promoting of a bacterial strain (L115) isolated from Arachis hypogaea rhizospheres and the effects of high growth temperature and salinity on phospholipids and fatty acids composition. L115 was isolated from peanut rhizospheres and identified according to the sequence analysis of the 16S rRNA gene. Phenotypic, metabolic and plant growth promoting rhizobacteria (PGPR) characteristics of L115 were tested. Inoculation test in plant growth chamber was performed. In addition, L115 was exposed to a 37 °C and 300 mM NaCl and phospholipids and fatty acid composition were evaluated. L115 strain was identified as Ochrobactrum intermedium and was able to increase the peanut shoot and root length as well as dry weight, indicating a PGPR role by being able to produce indole acetic acid and siderophores and present ACC deaminase activity. In addition, L115 showed tolerance to both high growth temperature and 300 mM NaCl. The most striking change was a decreased percentage of 18:1 fatty acid and an increase in 16:0 and 18:0 fatty acids, under high growth temperature or a combination of increased temperature and salinity. The most important change in phospholipid levels was an increase in phosphatidylcholine biosynthesis in all growth conditions. L115 can promote the growth of peanut and can tolerate high growth temperature and salinity modifying the fatty acid unsaturation degree and increasing phosphatidylcholine levels. This work is the first to report the importance of the genus Ochrobactrum as PGPR on peanut growth as well as on the metabolic behaviour against abiotic stresses that occur in soil. This knowledge will be useful for developing strategies to improve the growth of this bacterium under stress and to enhance its bioprocess for the production of inoculants.

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