New Inoculation Strategy for Legume Based on Rhizobium-Metabolite Co-encapsulation.

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.

Membrane Homeoviscous Adaptation in Sinorhizobium Submitted to a Stressful Thermal Cycle Contributes to the Maintenance of the Symbiotic Plant-Bacteria Interaction.

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.

Immobilization of Bradyrhizobium and Azospirillum in alginate matrix for long time of storage maintains cell viability and interaction with peanut.

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.

Structure of the unusual Sinorhizobium fredii HH103 lipopolysaccharide and its role in symbiosis.

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-l-glycero-l-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing ß-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its α-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum.

Polycaprolactone microcapsules containing citric acid and naringin for plant growth and sustainable agriculture: physico-chemical properties and release behavior.

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.

Restrictive water condition modifies the root exudates composition during peanut-PGPR interaction and conditions early events, reversing the negative effects on plant growth.

Water deficit is one of the most serious environmental factors that affect the productivity of crops in the world. Arachis hypogaea is a legume with a high nutritional value and 70% is cultivated in semi-arid regions. This research aimed to study the effect of water deficit on peanut root exudates composition, analyzing the importance of exudates on peanut-PGPR interaction under restrictive water condition. Peanut seedlings were subjected to six treatments: 0 and 15 mM PEG, in combination with non-inoculated, Bradyrhizobium sp. and Bradyrhizobium-Azospirillum brasilense inoculated treatments. We analyzed the 7-day peanut root exudate in response to a water restrictive condition and the presence of bacterial inocula. Molecular analysis was performed by HPLC, UPLC and GC. Bacteria motility, chemotaxis, bacterial adhesion to peanut roots and peanut growth parameters were analyzed. Restrictive water condition modified the pattern of molecules exuded by roots, increasing the exudation of Naringenin, oleic FA, citric and lactic acid, and stimulation the release of terpenes of known antioxidant and antimicrobial activity. The presence of microorganisms modified the composition of root exudates. Water deficit affected the first events of peanut-PGPR interaction and the root exudates favored bacterial mobility, the chemotaxis and attachment of bacteria to peanut roots. Changes in the profile of molecules exuded by roots allowed A. hypogaea-Bradyrhizobium and A.hypogaea-Bradyrhizobium-Azospirillum interaction thus reversing the negative effects of restrictive water condition on peanut growth. These findings have a future potential application to improve plant-PGPR interactions under water deficit by formulating inoculants containing key molecules exuded during stress.

Changes in the lipid composition of Bradyrhizobium cell envelope reveal a rapid response to water deficit involving lysophosphatidylethanolamine synthesis from phosphatidylethanolamine in outer membrane.

We evaluate the behavior of the membrane of Bradyrhizobium sp. SEMIA6144 during adaptation to polyethylene glycol (PEG). A dehydrating effect on the morphology of the cell surface, as well as a fluidizing effect on the membrane was observed 10 min after PEG shock; however, the bacteria were able to restore optimal membrane fluidity. Shock for 1 h caused an increase of lysophosphatidylethanolamine in the outer membrane at the expense of phosphatidylcholine and phosphatidylethanolamine (PE), through an increase in phospholipase activity. The amount of lysophosphatidylethanolamine did not remain constant during PEG shock, but after 24 h the outer membrane was composed of large amounts of phosphatidylcholine and less amount of lysophosphatidylethanolamine similar to the control. The inner membrane composition was also modified after 1 h of shock, observing an increase of phosphatidylcholine at the expense of PE, the proportions of these phospholipids were then modified to reach 24 h of shock values similar to the control. Vesicles prepared with the lipids of cells exposed to 1 h shock presented higher rigidity compared to the control, indicating that changes in the composition of phospholipids after 1 h of shock restoring fluidity after the PEG effect and would allow cells to maintain surface morphology.

Arachis hypogaea PGPR isolated from Argentine soil modifies its lipids components in response to temperature and salinity.

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.

Swimming and swarming motility properties of peanut-nodulating rhizobia.

Motility allows populations of bacteria to rapidly reach and colonize new microniches or microhabitats. The motility of rhizobia (symbiotic nitrogen-fixing bacteria that nodulate legume roots) is an important factor determining their competitive success. We evaluated the effects of temperature, incubation time, and seed exudates on swimming and swarming motility of five strains of Bradyrhizobium sp. (peanut-nodulating rhizobia). Swimming motility was increased by exudate exposure for all strains except native Pc34. In contrast, swarming motility was increased by exudate exposure for native 15A but unchanged for the other four strains. All five strains displayed the ability to differentiate into swarm cells. Morphological examination by scanning electron microscopy showed that the length of the swarm cells was variable, but generally greater than that of vegetative cells. Our findings suggest the importance of differential motility properties of peanut-nodulating rhizobial strains during agricultural inoculation and early steps of symbiotic interaction with the host.

Biochemical and molecular evidence of a Δ9 fatty acid desaturase from Ensifer meliloti 1021.

It has been reported that Ensifer meliloti presents a high proportion of monounsaturated fatty acids and has a putative desaturase gene designated as PhFAD12 (National Centre for Biotechnology Information), encoding a putative Δ12 desaturase-like protein. In this work, we report the desaturation capacity and characterisation of this gene encoding the putative fatty acid desaturase of E. meliloti 1021. This gene was also isolated from the rhizobial strain and overexpressed in Escherichia coli. Compared to a control, the expression of this gene in the transformed strain decreased the levels of palmitic and stearic acids, enhanced palmitoleic and cis-vaccenic levels, and allowed for the detection of oleic acid. E. coli overexpressing the putative desaturase gene was capable of desaturating palmitic and stearic acids to monounsaturated fatty acids, similarly to the rhizobial strain. Our studies show that AAK64726 encodes a Δ9 desaturase instead of a Δ12 desaturase as previously indicated. This work describes evidence for the presence of a desaturase-mediated mechanism in monounsaturated fatty acid synthesis in E. meliloti 1021, which is modified by high growth temperature. This mechanism supplements the anaerobic mechanism for unsaturated fatty acid synthesis.

Growth temperature and salinity impact fatty acid composition and degree of unsaturation in peanut-nodulating rhizobia.

Growth and survival of bacteria depend on homeostasis of membrane lipids, and the capacity to adjust lipid composition to adapt to various environmental stresses. Membrane fluidity is regulated in part by the ratio of unsaturated to saturated fatty acids present in membrane lipids. Here, we studied the effects of high growth temperature and salinity (NaCl) stress, separately or in combination, on fatty acids composition and de novo synthesis in two peanut-nodulating Bradyrhizobium strains (fast-growing TAL1000 and slow-growing SEMIA6144). Both strains contained the fatty acids palmitic, stearic, and cis-vaccenic + oleic. TAL1000 also contained eicosatrienoic acid and cyclopropane fatty acid. The most striking change, in both strains, was a decreased percentage of cis-vaccenic + oleic (≥ 80% for TAL1000), and an associated increase in saturated fatty acids, under high growth temperature or combined conditions. Cyclopropane fatty acid was significantly increased in TAL1000 under the above conditions. De novo synthesis of fatty acids was shifted to the synthesis of a higher proportion of saturated fatty acids under all tested conditions, but to a lesser degree for SEMIA6144 compared to TAL1000. The major adaptive response of these rhizobial strains to increased temperature and salinity was an altered degree of fatty acid unsaturation, to maintain the normal physical state of membrane lipids.

Phosphatidylcholine levels of peanut-nodulating Bradyrhizobium sp. SEMIA 6144 affect cell size and motility.

Phosphatidylcholine, the major phospholipid in eukaryotes, is found in rhizobia and in many other bacteria interacting with eukaryotic hosts. Phosphatidylcholine has been shown to be required for a successful interaction of Bradyrhizobium japonicum USDA 110 with soybean roots. Our aim was to study the role of bacterial phosphatidylcholine in the Bradyrhizobium-peanut (Arachis hypogaea) symbiosis. Phospholipid N-methyltransferase (Pmt) and minor phosphatidylcholine synthase (Pcs) activities were detected in crude extracts of the peanut-nodulating strain Bradyrhizobium sp. SEMIA 6144. Our results suggest that phosphatidylcholine formation in Bradyrhizobium sp. SEMIA 6144 is mainly due to the phospholipid methylation pathway. Southern blot analysis using pmt- and pcs-probes of B. japonicum USDA 110 revealed a pcs and multiple pmt homologues in Bradyrhizobium sp. SEMIA 6144. A pmtA knockout mutant was constructed in Bradyrhizobium sp. SEMIA 6144 that showed a 50% decrease in the phosphatidylcholine content in comparison with the wild-type strain. The mutant was severely affected in motility and cell size, but formed wild-type-like nodules on its host plant. However, in coinoculation experiments, the pmtA-deficient mutant was less competitive than the wild type, suggesting that wild-type levels of phosphatidylcholine are required for full competitivity of Bradyrhizobium in symbiosis with peanut plants.

Different and new Nod factors produced by Rhizobium tropici CIAT899 following Na+ stress.

The root nodule bacterium Rhizobium tropici strain CIAT899 is highly stress resistant. It grows under acid conditions, in large amounts of salt, and at high osmotic pressure. An earlier study reported a substantial qualitative and quantitative effect of acid stress on the biosynthesis of Nod factors. The aim of the present work was to investigate the effect of high salt (NaCl) concentrations, another common stress factor, on Nod factor production. For this purpose, thin-layer chromatography, HPLC and MS analyses were carried out. The expression of nodulation genes was also studied using a nodP:lacZ fusion. High concentrations of sodium enhanced nod gene expression and Nod factor biosynthesis. The effect is sodium specific because high potassium or chloride concentrations did not have this effect. Under salt stress conditions, 46 different Nod factors were identified in a CIAT899 culture, compared with 29 different Nod factors under control conditions. Only 15 Nod factor structures were common to both conditions. Under salt stress conditions, 14 different new Nod factor structures were identified that were not observed as being produced under neutral or acid conditions. The implications of our results are that stress has a great influence on Nod factor biosynthesis and that new, very interesting regulatory mechanisms, worth investigating, are involved in controlling Nod factor biosynthesis.

Attachment of bacteria to the roots of higher plants.

Attachment of soil bacteria to plant cells is supposedly the very early step required in plant-microbe interactions. Attachment also is an initial step for the formation of microbial biofilms on plant roots. For the rhizobia-legume symbiosis, various mechanisms and diverse surface molecules of both partners have been proposed to mediate in this process. The first phase of attachment is a weak, reversible, and unspecific binding in which plant lectins, a Ca(+2)-binding bacterial protein (rhicadhesin), and bacterial surface polysaccharide appear to be involved. The second attachment step requires the synthesis of bacterial cellulose fibrils that cause a tight and irreversible binding of the bacteria to the roots. Cyclic glucans, capsular polysaccharide, and cellulose fibrils also appear to be involved in the attachment of Agrobacterium to plant cells. Attachment of Azospirillum brasilense to cereals roots also can be divided in two different steps. Bacterial surface proteins, capsular polysaccharide and flagella appear to govern the first binding step while extracellular polysaccharide is involved in the second step. Outer cell surface proteins and pili are implicated in the adherence of Pseudomonas species to plant roots.

Adaptational changes in lipids of Bradyrhizobium SEMIA 6144 nodulating peanut as a response to growth temperature and salinity.

Phospholipids provide the membrane with its barrier function and play a role in a variety of processes in the bacterial cell, as responding to environmental changes. The aim of the present study was to characterize the physiological and metabolic response of Bradyrhizobium SEMIA 6144 to saline and temperature stress. This study provides metabolic and compositional evidence that nodulating peanut Bradyrhizobium SEMIA 6144 is able to synthesize fatty acids, to incorporate them into its phospholipids (PL), and then modify them in response to stress conditions such as temperature and salinity. The fatty acids were formed from [1-(14)C]acetate and mostly incorporated in PL (95%). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) were found to be the major phospholipids in the bacteria analyzed. The amount and the labeling of each individual PL was increased by NaCl, while they were decreased by temperature stress. The amount of PC, PE, and PG under the combined stresses decreased, as in the temperature effect. The results indicate that synthesized PL of Bradyrhizobium SEMIA 6144 are modified under the tested conditions. Because in all conditions tested the PC amount was always modified and PC was the major PL, we suggest that this PL may be involved in the bacteria response to environmental conditions.

Factors affecting the attachment of rhizospheric bacteria to bean and soybean roots.

The plant rhizosphere is an important soil ecological environment for plant-microorganism interactions, which include colonization by a variety of microorganisms in and around the roots that may result in symbiotic, endophytic, associative, or parasitic relationships within the plant, depending on the type of microorganisms, soil nutrient status, and soil environment. Rhizosphere competence may be attributable to the differences in the extent of bacterial attachment to the root surface. We present results of the effect of various factors on the attachment to bean (Phaseolus vulgaris) and soybean (Glycine max) roots of some bacterial species of agronomic importance, such as Rhizobium tropici, Rhizobium etli, Ensifer fredii (homotypic synonym Sinorhizobium fredii), and Azospirillum brasilense; as well as the attachment capability of the plant growth promoting rhizobacteria Pseudomonas fluorescens and Chryseobacterium balustinum. Additionally, we have studied various bacterial traits, such as autoaggregation and flagella movements, which have been postulated to be important properties for bacterial adhesion to surfaces. The lack of mutual incompatibility between rhizobial strains and C. balustinum has been demonstrated in coinoculation assays.

A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots.

As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp. strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L. (peanut). Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria. Optimal attachment occurred when bacteria were at the late log or early stationary phase. Cell surface proteins from the Bradyrhizobium sp. strains inhibited the attachment when supplied prior to the attachment assay. Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment. The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots. Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.