Investigating the synergistic effects of biochar, trans-zeatin riboside, and Azospirillum brasilense on soil improvement and enzymatic activity in water-stressed wheat.

BACKGROUND: Water stress is a major danger to crop yield, hence new approaches to strengthen plant resilience must be developed. To lessen the negative effects of water stress on wheat plants, present study was arranged to investigate the role of synergistic effects of biochar, trans-zeatin riboside (t-ZR), and Azospirillum brasilense on soil improvement and enzymatic activity in water-stressed wheat. RESULTS: In a three-replication experiment comprising of four treatments (T0: Control, T1: Drought stress (DS), T2: DS + t-ZR with biochar, T3: DS + A. brasilense with biochar), we observed notable improvements in soil quality and enzymatic activities in water-stressed wheat plants with the application of t-ZR and A. brasilense with biochar. In drought stress, Treatment having the application of A. brasilense with biochar performs best as compared to the other and significant increased the enzymatic activities such as peroxidase (7.36%), catalase (8.53%), superoxide dismutase (6.01%), polyphenol oxidase (14.14%), and amylase (16.36%) in wheat plants. Different enzymatic activities showed different trends of results. Soil organic C, dissolved organic C, dissolved organic N also enhanced 29.46%, 8.59%, 22.70% respectively with the application of A. brasilense with biochar under drought stress condition. CONCLUSIONS: The synergistic action of A. brasilense and biochar creates an effective microbiological environment that supports essential plant physiological processes during drought stress. This enhancement is attributed to improved soil fertility and increased organic matter content, highlighting the potential of these novel strategies in mitigating water stress effects and enhancing crop resilience.

An Azospirillum brasilense chemoreceptor that mediates nitrate chemotaxis has conditional roles in the colonization of plant roots.

Motile plant-associated bacteria use chemotaxis and dedicated chemoreceptors to navigate gradients in their surroundings and to colonize host plant surfaces. Here, we characterize a chemoreceptor that we named Tlp2 in the soil alphaproteobacterium Azospirillum brasilense. We show that the Tlp2 ligand-binding domain is related to the 4-helix bundle family and is conserved in chemoreceptors found in the genomes of many soil- and sediment-dwelling alphaproteobacteria. The promoter of tlp2 is regulated in an NtrC- and RpoN-dependent manner and is most upregulated under conditions of nitrogen fixation or in the presence of nitrate. Using fluorescently tagged Tlp2 (Tlp2-YFP), we show that this chemoreceptor is present in low abundance in chemotaxis-signaling clusters and is prone to degradation. We also obtained evidence that the presence of ammonium rapidly disrupts Tlp2-YFP localization. Behavioral experiments using a strain lacking Tlp2 and variants of Tlp2 lacking conserved arginine residues suggest that Tlp2 mediates chemotaxis in gradients of nitrate and nitrite, with the R159 residue being essential for Tlp2 function. We also provide evidence that Tlp2 is essential for root surface colonization of some plants (teff, red clover, and cowpea) but not others (wheat, sorghum, alfalfa, and pea). These results highlight the selective role of nitrate sensing and chemotaxis in plant root surface colonization and illustrate the relative contribution of chemoreceptors to chemotaxis and root surface colonization.IMPORTANCEBacterial chemotaxis mediates host-microbe associations, including the association of beneficial bacteria with the roots of host plants. Dedicated chemoreceptors specify sensory preferences during chemotaxis. Here, we show that a chemoreceptor mediating chemotaxis to nitrate is important in the beneficial soil bacterium colonization of some but not all plant hosts tested. Nitrate is the preferred nitrogen source for plant nutrition, and plants sense and tightly control nitrate transport, resulting in varying nitrate uptake rates depending on the plant and its physiological state. Nitrate is thus a limiting nutrient in the rhizosphere. Chemotaxis and dedicated chemoreceptors for nitrate likely provide motile bacteria with a competitive advantage to access this nutrient in the rhizosphere.

Thermal and salt stress effects on the survival of plant growth-promoting bacteria Azospirillum brasilense in inoculants for maize cultivation.

BACKGROUND: The plant growth-promoting bacteria (PGPB) Azospirillum brasilense is widely used as an inoculant for important grass crops, providing numerous benefits to the plants. However, one limitation to develop viable commercial inoculants is the control of PGPB survival, requiring strategies that guarantee their survival during handling and field application. The application of sublethal stress appears to be a promising strategy to increase bacterial cells tolerance to adverse environmental conditions since previous stress induces the activation of physiological protection in bacterial cell. In this work, we evaluated the effects of thermal and salt stresses on the survival of inoculant containing A. brasilense Ab-V5 and Ab-V6 strains and we monitored A. brasilense viability in inoculated maize roots after stress treatment of inoculant. RESULTS: Thermal stress application (> 35 °C) in isolated cultures for both strains, as well as salt stress [sodium chloride (NaCl) concentrations > 0.3 mol L-1], resulted in growth rate decline. The A. brasilense enumeration in maize roots obtained by propidium monoazide quantitative polymerase chain reaction (PMA-qPCR), for inoculated maize seedlings grown in vitro for 7 days, showed that there is an increased number of viable cells after the salt stress treatment, indicating that A. brasilense Ab-V5 and Ab-V6 strains are able to adapt to salt stress (0.3 mol L-1 NaCl) growth conditions. CONCLUSION: Azospirillum brasilense Ab-V5 and Ab-V6 strains had potential for osmoadaptation and salt stress, resulting in increased cell survival after inoculation in maize plants. © 2024 Society of Chemical Industry.

Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes.

BACKGROUND: PROTEIN PHOSPHATASE 2A (PP2A) expression is crucial for the symbiotic association between plants and various microbes, and knowledge on these symbiotic processes is important for sustainable agriculture. Here we tested the hypothesis that PP2A regulatory subunits, especially B'φ and B'θ, are involved in signalling between plants and mycorrhizal fungi or plant-growth promoting bacteria. RESULTS: Treatment of tomato plants (Solanum lycopersicum) with the plant growth-promoting rhizobacteria (PGPR) Azospirillum brasilense and Pseudomonas simiae indicated a role for the PP2A B'θ subunit in responses to PGPR. Arbuscular mycorrhizal fungi influenced B'θ transcript levels in soil-grown plants with canonical arbuscular mycorrhizae. In plant roots, transcripts of B'φ were scarce under all conditions tested and at a lower level than all other PP2A subunit transcripts. In transformed tomato plants with 10-fold enhanced B'φ expression, mycorrhization frequency was decreased in vermiculite-grown plants. Furthermore, the high B'φ expression was related to abscisic acid and gibberellic acid responses known to be involved in plant growth and mycorrhization. B'φ overexpressor plants showed less vigorous growth, and although fruits were normal size, the number of seeds per fruit was reduced by 60% compared to the original cultivar. CONCLUSIONS: Expression of the B'θ gene in tomato roots is strongly influenced by beneficial microbes. Analysis of B'φ overexpressor tomato plants and established tomato cultivars substantiated a function of B'φ in growth and development in addition to a role in mycorrhization.

Application of Azospirillum on seeds and leaves, associated with Rhizobium inoculation, increases growth and yield of common bean.

Association of rhizobia with other plant growth-promoting bacteria (PGPB), such as Azospirillum, have the potential to increase crop yields. This work aimed to assess how Rhizobium tropici and Azospirillum brasilense alone or in combination, affect the growth and yields of common bean grains (Phaseolus vulgaris L.). In a field experiment, R. tropici and A. brasilense were inoculated on seeds, alone or in combination, associated or not with foliar spraying of A. brasilense. Shoot biomass, nitrogen accumulation, thousand-grain weight, and grain yield were evaluated. Application of A. brasilense, on seed or by foliar spraying, and seed inoculation of R. tropici, had an additive effect, increasing biomass and accumulated nitrogen, thousand-grain weight, and grain yield.

Day and blue light modify growth, cell physiology and indole-3-acetic acid production of Azospirillum brasilense Az39 under planktonic growth conditions.

AIM: In this work, we evaluated the effects of light on growth, cell physiology and stress response of Azospirillum brasilense Az39, a non-photosynthetic rhizobacteria, under planktonic growth conditions. METHODS AND RESULTS: Exponential cultures of Az39 were exposed to blue (BL), red (RL) and daylight (DL) or maintained in darkness for 24, 48 and 72 h. The biomass production and indole 3-acetic acid (IAA) biosynthesis increased by exposition to DL. Conversely, BL decreased IAA concentration through a direct effect on the molecule. The DL increased superoxide dismutase activity, hydrogen peroxide and thiobarbituric acid reactive substances levels, but the last one was also increased by BL. Both DL and BL increased cell aggregation but only BL increased biofilm formation. CONCLUSIONS: We demonstrated that both BL and DL are stress effectors for A. brasilense Az39 under planktonic growth conditions. The DL increased biomass production, IAA biosynthesis and bacterial response to stress, whereas BL induced cell aggregation and biofilms formation, but decreased the IAA concentration by photooxidation. SIGNIFICANCE AND IMPACT OF THE STUDY: Blue light and DL changes growth capacity, cell physiology and plant growth promotion ability of A. brasilense Az39 and these changes could be considered to improve the production and functionality of biofertilizers.

Natural occurrence of Azospirillum brasilense in petunia with capacity to improve plant growth and flowering.

To evaluate the natural occurrence of the plant growth-promoting bacterium Azospirillum brasilense and petunia plants, local strains were isolated and characterized by biochemical and molecular methods. Three strains were assessed in greenhouse conditions using Petunia × hybrida Ultra™. Treatments: Plants without bacterial inoculation or chemical fertilization; fertilized with NPK and KNO3 ; and independently inoculated with the strains 2A1, 2A2, and 2E1 by submerging their roots in a bacterial suspension (~106 CFU·ml-1 ). Root length, dry weight of roots and shoots, leaf area, leaf greenness, and nutrient content were evaluated. The number of days from transplanting to the opening of the first flower and the number of flowers per plant were also determined. As a result, five isolates were characterized as A. brasilense, showing the capacity to produce indoles and siderophores, to solubilize phosphate, nitrogenase activity, and nifH-PCR amplification. In general, all the parameters of the plant assay were improved in plants inoculated with A. brasilense, with variations among the strains, as well as the onset of flowering and the number of flowers per plant, compared with uninoculated or fertilized plants. This is the first report on the natural occurrence of A. brasilense in petunia with the capacity to improve plant growth and flowering.

Specific Root Exudate Compounds Sensed by Dedicated Chemoreceptors Shape Azospirillum brasilense Chemotaxis in the Rhizosphere.

Plant roots shape the rhizosphere community by secreting compounds that recruit diverse bacteria. Colonization of various plant roots by the motile alphaproteobacterium Azospirillum brasilense causes increased plant growth, root volume, and crop yield. Bacterial chemotaxis in this and other motile soil bacteria is critical for competitive colonization of the root surfaces. The role of chemotaxis in root surface colonization has previously been established by endpoint analyses of bacterial colonization levels detected a few hours to days after inoculation. More recently, microfluidic devices have been used to study plant-microbe interactions, but these devices are size limited. Here, we use a novel slide-in chamber that allows real-time monitoring of plant-microbe interactions using agriculturally relevant seedlings to characterize how bacterial chemotaxis mediates plant root surface colonization during the association of A. brasilense with Triticum aestivum (wheat) and Medicago sativa (alfalfa) seedlings. We track A. brasilense accumulation in the rhizosphere and on the root surfaces of wheat and alfalfa. A. brasilense motile cells display distinct chemotaxis behaviors in different regions of the roots, including attractant and repellent responses that ultimately drive surface colonization patterns. We also combine these observations with real-time analyses of behaviors of wild-type and mutant strains to link chemotaxis responses to distinct chemicals identified in root exudates to specific chemoreceptors that together explain the chemotactic response of motile cells in different regions of the roots. Furthermore, the bacterial second messenger c-di-GMP modulates these chemotaxis responses. Together, these findings illustrate dynamic bacterial chemotaxis responses to rhizosphere gradients that guide root surface colonization.IMPORTANCE Plant root exudates play critical roles in shaping rhizosphere microbial communities, and the ability of motile bacteria to respond to these gradients mediates competitive colonization of root surfaces. Root exudates are complex chemical mixtures that are spatially and temporally dynamic. Identifying the exact chemical(s) that mediates the recruitment of soil bacteria to specific regions of the roots is thus challenging. Here, we connect patterns of bacterial chemotaxis responses and sensing by chemoreceptors to chemicals found in root exudate gradients and identify key chemical signals that shape root surface colonization in different plants and regions of the roots.

Azospirillum brasilense viable cells enumeration using propidium monoazide-quantitative PCR.

Azospirillum brasilense is a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations or in planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability of A. brasilense in inoculant and in maize roots. A. brasilense was grown in culture medium and was exposed to 50 â„ƒ. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 104 CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.

Bradyrhizobium sp. enhance ureide metabolism increasing peanuts yield.

This study aimed to evaluate the effects of seed inoculation with Bradyrhizobium sp. and co-inoculation with Azospirillum brasilense. The seed treatments were as follows: control (without inoculation); A. brasilense (2 mL per kg-1 of seed); A. brasilense (4 mL per kg-1 of seed); Bradyrhizobium sp. (2 mL per kg-1 of seed); Bradyrhizobium sp. (4 mL per kg-1 of seed); A. brasilense + Bradyrhizobium sp. (2 mL of each strain per kg-1 of seed); and A. brasilense + Bradyrhizobium sp. (4 mL of each strain per kg-1 of seed). Peanut plants from seeds inoculated with Bradyrhizobium sp. and A. brasilense exhibited highest leaf concentration of photosynthetic pigments, carotenoids, nitrate, ammonia and amino acids. The inoculation of seeds with Bradyrhizobium sp. resulted in plants with increased concentrations of total soluble sugars, and ureides compared to the untreated plants. In contrast, seeds treated with A. brasilense alone resulted in plants exhibiting highest concentration of amino acids, which represent the highest concentration of nitrogen compounds in peanut plants. Seed inoculation with Bradyrhizobium sp. at a rate of 2 mL kg-1 was identified as the best treatment to promote increased biological nitrogen fixation and generate higher peanut yields.

Kinetics and structural features of dimeric glutamine-dependent bacterial NAD+ synthetases suggest evolutionary adaptation to available metabolites.

NADH (NAD+) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD+ also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD+ biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD+ synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadENH3 and octameric glutamine-dependent NadEGln, and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadEGln in bacteria. Substrate preferences and structural analyses suggested that dimeric NadEGln enzymes may constitute evolutionary intermediates between dimeric NadENH3 and octameric NadEGln The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.

Modulation of nitrogen metabolism of maize plants inoculated with Azospirillum brasilense and Herbaspirillum seropedicae.

Maize is highly responsive to the application of nitrogen to achieve high productivity. Inoculation with diazotrophic bacteria can improve plant growth with low N fertilization. The objective was to evaluate the inoculation of two species of diazotrophs on N metabolism in maize plants, in the presence of two concentrations of nitrogen in a hydroponic system. A factorial arrangement composed of two N levels (3.0 and 0.3 mM), with the presence of Hs-Herbaspirillum seropedicae, and Ab-Azospirillum brasilense or not. The parameters used were dry mass; N, P, and K accumulation; nitrate reductase activity; soluble fractions in roots and leaves. The inoculation altered the N metabolism and promoted greater development of maize plants, as well as a higher accumulation of P and K in the shoots. A more intensive process of N assimilation was evidenced when the plants were inoculated with H. seropedicae, leading to increased levels of NO3- and reduced N-amino, sugars, and NH4+ in leaves associated with high N level, opposite of A. brasilense.

Can co-inoculation of Bradyrhizobium and Azospirillum alleviate adverse effects of drought stress on soybean (Glycine max L. Merrill.)?

Harnessing the beneficial potential of plant growth-promoting rhizobacteria may be an alternative strategy to improve plant tolerance to drought stress. The effect of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense either alone or in combination on the plant growth and drought tolerance of soybean [Glycine max (L.) Merrill.] was investigated in this study in greenhouse conditions. Treatments were arranged in a randomized block design in a 3 × 4 factorial: three irrigation regimes [100% of pot capacity-PC (well-watered control), 50% of PC (moderate stress) and 25% of PC (severe stress)] and four inoculation treatments [control (non-inoculated), inoculation with B. japonicum, inoculation with A. brasilense, and co-inoculation with B. japonicum and A. brasilense]. Leaf relative water content, cell membrane stability, root nodulation, plant growth, and morphophysiological indexes were recorded. The inoculation of soybean plants with B. japonicum and A.brasilense either alone or in combination improved leaf membrane stability under drought stress conditions when compared to non-inoculated plants; however, this lower damage to cell membranes was not sufficient to maintain the leaf water content of the plant under drought stress. Plants co-inoculated with B. japonicum and A.brasilense improved the root nodulation under severe drought conditions. Inoculation of B. japonicum and A. brasilense either alone or in combination reduced the pod abortion rate under moderate drought stress, but had no effect under severe drought stress. In summary, the co-inoculation of A. brasilense and B. japonicum alleviate adverse effects limited by drought stress to the growth of soybeans.Author: Please check and confirm that the authors [Elijanara Raissa Silva, Carlos Eduardo Silva Oliveira, Alan Mario Zuffo, Eduardo Pradi Vendruscolo] and their initials have been correctly identified and amend if necessary.The authors were correctly identified.

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense.

The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth-promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

Azospirillum brasilense Az39, a model rhizobacterium with AHL quorum-quenching capacity.

AIMS: The aim of this research was to analyse the quorum-sensing (QS) and quorum-quenching (QQ) mechanisms based on N-acyl-l-homoserine lactones (AHLs) in Azospirillum brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions. METHODS AND RESULTS: We performed an in silico and in vitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained in vitro using the reporter strains Chromobacterium violaceum and Agrobacterium tumefaciens and liquid chromatography coupled with mass-mass spectrometry analysis showed that although Az39 does not produce AHL molecules, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated with the bacterial cell. In Az39 cultures supplemented with 500 nmol l-1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than in noninoculated LB media controls. Similar results were observed upon the addition of AHLs with hydroxy (OH-) and keto (oxo-) substitutions in C3. These results not only demonstrate the ability of Az39 to degrade AHLs. They also show the wide spectrum of molecules that can be degraded by this bacterium. CONCLUSIONS: Although A. brasilense Az39 is a silent bacterium unable to produce AHL signals, it is able to interrupt the communications between other bacteria and/or plants by a QQ activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report confirming by unequivocal methodology the ability of A. brasilense, one of the most agriculturally used benefic bacteria around the world, to degrade AHLs by a QQ mechanism.

Restoration of polar-flagellum motility and biofilm-forming capacity in the mmsB1 mutant of the alphaproteobacterium Azospirillum brasilense Sp245 points to a new role for a homologue of 3-hydroxyisobutyrate dehydrogenase.

The bacterium Azospirillum brasilense can swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella, respectively. They also form biofilms on various interfaces. Experimental data on flagellar assembly and social behaviours in these bacteria are scarce. Here, for the first time, the chromosomal coding sequence mmsB1 for a homologue of 3-hydroxyisobutyrate dehydrogenase (protein accession Nos. ADT80774 and E7CWE2) was shown to play a role in the assembly of motile Fla and in biofilm biomass accumulation. In the previously obtained mutant SK039 of A. brasilense Sp245, an Omegon-Km insertion in mmsB1 was concurrent with changes in cell-surface properties and with suppression of Fla assembly (partial) and Fla-dependent motility (complete). Here, the immotile leaky Fla- mutant SK039 was complemented with the expression vector pRK415-borne mmsB1 gene of Sp245. In the complemented mutant, the elevated relative cell hydrophobicity and changed relative membrane fluidity of SK039 returned to the wild-type levels; also, biofilm biomass accumulation increased and even reached Sp245's levels under nutritionally rich conditions. In strain SK039 (pRK415-mmsB1), the percentage of cells with Fla became significantly higher than that in mutant SK039, and the Fla-driven swimming velocity was equal to that in strain Sp245.

Quorum sensing communication: Bradyrhizobium-Azospirillum interaction via N-acyl-homoserine lactones in the promotion of soybean symbiosis.

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.

Functioning of plant-bacterial associations under osmotic stress in vitro.

The search for effective plant-growth-promoting strains of rhizospheric bacteria that would ensure the resistance of plant-microbial associations to environmental stressors is essential for the design of environmentally friendly agrobiotechnologies. We investigated the interaction of potato (cv. Nevsky) microplants with the plant-growth-promoting bacteria Azospirillum brasilense Sp245 and Ochrobactrum cytisi IPA7.2 under osmotic stress in vitro. The bacteria improved the physiological and biochemical variables of the microplants, significantly increasing shoot length and root number (1.3-fold, on average). Inoculation also led a more effective recovery of the plants after stress. During repair, inoculation contributed to a decreased leaf content of malonic dialdehyde. With A. brasilense Sp245, the decrease was 1.75-fold; with O. cytisi IPA7.2, it was 1.4-fold. During repair, the shoot length, node number, and root number of the inoculated plants were greater than the control values by an average of 1.3-fold with A. brasilense Sp245 and by an average of 1.6-fold with O. cytisi IPA7.2. O. cytisi IPA7.2, previously isolated from the potato rhizosphere, protected the physiological and biochemical processes in the plants under stress and repair better than did A. brasilense Sp245. Specifically, root weight increased fivefold during repair, as compared to the noninoculated plants, while chlorophyll a content remained at the level found in the nonstressed controls. The results indicate that these bacteria can be used as components of biofertilizers. A. brasilense Sp245 has favorable prospects for use in temperate latitudes, whereas O. cytisi IPA7.2 can be successfully used in saline and drought-stressed environments.

Revealing strategies of quorum sensing in Azospirillum brasilense strains Ab-V5 and Ab-V6.

Azospirillum brasilense is an important plant-growth promoting bacterium (PGPB) that requires several critical steps for root colonization, including biofilm and exopolysaccharide (EPS) synthesis and cell motility. In several bacteria these mechanisms are mediated by quorum sensing (QS) systems that regulate the expression of specific genes mediated by the autoinducers N-acyl-homoserine lactones (AHLs). We investigated QS mechanisms in strains Ab-V5 and Ab-V6 of A. brasilense, which are broadly used in commercial inoculants in Brazil. Neither of these strains carries a luxI gene, but there are several luxR solos that might perceive AHL molecules. By adding external AHLs we verified that biofilm and EPS production and cell motility (swimming and swarming) were regulated via QS in Ab-V5, but not in Ab-V6. Differences were observed not only between strains, but also in the specificity of LuxR-type receptors to AHL molecules. However, Ab-V6 was outstanding in indole acetic acid (IAA) synthesis and this molecule might mimic AHL signals. We also applied the quorum quenching (QQ) strategy, obtaining transconjugants of Ab-V5 and Ab-V6 carrying a plasmid with acyl-homoserine lactonase. When maize (Zea mays L.) was inoculated with the wild-type and transconjugant strains, plant growth was decreased with the transconjugant of Ab-V5-confirming the importance of an AHL-mediated QS system-but did not affect plant growth promotion by Ab-V6.

Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger c-di-GMP to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect the metabolic status of the cells to the sensory activity of chemotaxis receptors.