Identification of the rhizospheric microbe and metabolites that led by the continuous cropping of ramie (Boehmeria nivea L. Gaud).

Continuous cropping lowers the production and quality of ramie (Boehmeria nivea L. Gaud). This study aimed to reveal the metagenomic and metabolomic changes between the healthy- and obstacle-plant after a long period of continuous cropping. After 10 years of continuous cropping, ramie planted in some portions of the land exhibited weak growth and low yield (Obstacle-group), whereas, ramie planted in the other portion of the land grew healthy (Health-group). We collected rhizosphere soil and root samples from which measurements of soil chemical and plant physiochemical properties were taken. All samples were subjected to non-targeted gas chromatograph-mass spectrometer (GS/MS) metabolome analysis. Further, metagenomics was performed to analyze the functional genes in rhizospheric soil organisms. Based on the findings, ramie in Obstacle-group were characterized by shorter plant height, smaller stem diameter, and lower fiber production than that in Health-group. Besides, the Obstacle-group showed a lower relative abundance of Rhizobiaceae, Lysobacter antibioticus, and Bradyrhizobium japonicum, but a higher relative abundance of Azospirillum lipoferum and A. brasilense compared to the Health-group. Metabolomic analysis results implicated cysteinylglycine (Cys-Gly), uracil, malonate, and glycerol as the key differential metabolites between the Health- and Obstacle-group. Notably, this work revealed that bacteria such as Rhizobia potentially synthesize IAA and are likely to reduce the biotic stress of ramie. L. antibioticus also exerts a positive effect on plants in the fight against biotic stress and is mediated by metabolites including orthophosphate, uracil, and Cys-Gly, which may serve as markers for disease risk. These bacterial effects can play a key role in plant resistance to biotic stress via metabolic and methionine metabolism pathways.

Structural characterization and applications of a novel polysaccharide produced by Azospirillum lipoferum MTCC 2306.

Azospirillum lipoferum MTCC 2306, a free-living nitrogen fixing bacteria, has a doubling time of 1.7 h in MPSS media. At the end of 28 h at a pH of 7 and temperature of 30 °C it produces 1.8 ± 0.013 g/L biomass and 2.1 ± 0.018 g/L of cyclic beta glucan (CßG) in MPSS medium with a yield coefficient (YP/S) of 2.1. This novel polysaccharide is a water-soluble cyclic biopolymer and is generally produced by Rhizobiaceae and predominantly made up of glucose. The CßG has a degree of polymerisation varying between 10 and 13 and has both α- and ß-glycosidic linkages. It is not substituted with any functional groups such as acetates or succinates. Its ability to bind to aniline blue suggests that it can be a potential candidate for being used as carrier in medical imaging as well as in reducing toxicity of textile effluents. It is able to encapsulate rifampicin, a hydrophobic drug and increase its aqueous solubility by 71%. So, CßG appears to have promising applications in the field of drug, food, cosmetic and nutraceutical industries.

Field-based assessment of the mechanism of maize yield enhancement by Azospirillum lipoferum CRT1.

Plant Growth-Promoting Bacteria (PGPB) of the genus Azospirillum are known to enhance root growth and yield in many plant species including cereals. To probe the underlying mechanisms, correlations between modifications of yield and 6-leaf plantlet characteristics were estimated on maize in four fields with contrasting soil properties over two consecutive years using the commercial isolate A. lipoferum CRT1. In both years, plantlet metabolome, photosynthetic potential and organ morphology were found to display field- and inoculation-specific signatures. Metabolomic analyses revealed that A. lipoferum CRT1 mostly affected sugar metabolism with no suggested impact on N and P assimilation. Mineral nitrogen feeding increased yield but did not affect yield enhancement by the bacterial partner. However, greater improvements of leaf photosynthetic potential correlated with yield diminutions and larger plantlets in all of their proportions correlated with yield enhancements. Bacterial inoculation restored proper seed-to-adult plant ratio when it accidentally dropped below 80%. Only in these cases did it raise yield. All in all, securing mature plant density is hypothesized as being the primary driver of A. lipoferum CRT1-mediated yield enhancement in maize fields.

[Effect of flavonoids on the composition of surface glycopolymers in Azospirillum lipoferum Sp59b].

Cultivation of the type strain Azospirillum lipoferum Sp59b in the presence of the flavonoid quercetin induced modification of the structure of the bacterial lipopolysaccharide. Cultivation in the presence of the flavonoid was shown to result in altered serological characteristics of the bacteria, increased heterogeneity of the outer membrane lipopolysaccharide pool, as well as in modified composition and fatty acid ratio of lipid A. The flavonoid was shown to induce the synthesis of the O-specific polysaccharide with the repeating structure represented by a tetrasaccharide consisting of a linear trisaccharide fragment of α-L-Rhap residues in the main chain and the terminal ß-D-Glcp residue. The structure of this O-specific polysaccharide was identical to the previously determined structure of the capsular polysaccharide of these bacteria grown without quercetin. Modifications in the structural composition of the capsular polysaccharide induced by cultivation in the presence of quercetin were revealed.

A quorum-quenching approach to identify quorum-sensing-regulated functions in Azospirillum lipoferum.

A quorum-quenching approach was exploited in order to identify functions regulated by quorum-sensing (QS) in the plant growth-promoting bacterium Azospirillum lipoferum. The AttM lactonase from Agrobacterium tumefaciens was shown to enzymatically inactivate N-acyl homoserine lactones (AHLs) produced by two A. lipoferum strains. The targeted analysis of several phenotypes revealed that in strain B518, a rice endophyte, AHL inactivation abolished pectinase activity, increased siderophore synthesis and reduced indoleacetic acid production (in stationary phase) but no effect was observed on cellulase activity or on swimming and swarming motilities. None of the tested phenotypes appeared to be under QS regulation in strain TVV3 isolated from the rice rhizosphere. Moreover, AHL inactivation had no deleterious effect on the phytostimulatory effect of the two strains in vitro. A global proteomic approach revealed little modification of protein patterns when comparing attM-expressing TVV3 and the wild-type strain, but numerous proteins appeared to be regulated by the AHL-mediated QS system in strain B518. Several proteins identified by MS-MS analysis were revealed to be implicated in transport (such as OmaA) and chemotaxis (ChvE). Altogether, the results indicate that in A. lipoferum, QS regulation is strain-specific and is dedicated to regulating functions linked to rhizosphere competence and adaptation to plant roots.

[The effect of combined and separate inoculation of alfalfa plants with Azospirillum lipoferum and Sinorhizobium meliloti on denitrification and nitrogen-fixing activities].

The effects of associative nitrogen fixer Azospirillum lipoferum strain 137 and root nodule bacteria Sinorhizobium meliloti after combined and separate inoculation of alfalfa seedlings on the background of mineral nitrogen applied at various times were studied. It was demonstrated that exudates of the alfalfa seedlings with the first pair of cotyledonary leaves already provide a high activity of these bacteria in the rhizosphere. To 74.6% of the introduced nitrate was transformed into N2O when the binary preparation of these bacteria was used. In an extended experiment (30 days), an active reduction of nitrates to N2O (11 micromol N2O/pot x 24 h) with inhibition of nitrogen fixation was observed in all of the experimental variants during the formation of legume-rhizobial and associative symbioses and simultaneous introduction of nitrates and bacteria. The most active enzyme fixation was observed in the case of a late (after 14 days) application of nitrates in the variants with both separate inoculations and inoculation with the binary preparation of A. lipoferum and S. meliloti. Separation in time of the application of bacterial preparations and mineral nitrogen assisted its preservation in all of the experimental variants. The variant of alfalfa inoculation with the binary preparation of A. lipoferum and S. meliloti and application of nitrates 2 weeks after inoculation was optimal for active nitrogen fixation (224.7 C2H4 nmol/flask x 24 h) and low denitrification activity (1.8 x micromol N2O/flask x 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.

Growth dynamics of Azospirillum lipoferum at steady and transitory states in the presence of NH.

AIMS: The objective of this paper was to study the adaptation dynamics and biochemical response of Azospirillum lipoferum grown in a continuous culture at various environmental shifts. METHODS AND RESULTS: The kinetics of A. lipoferum Sp 59b grown at steady states in a microaerobic chemostatic environment deviated from a typical Monod kinetics in both low and high dilution rates (D) due to several metabolic shifts that occurred in the microbial cell. When NH4Cl was exhausted (at low D), the microbial cell partitioned carbon flow in order to sustain growth, nitrogen fixation and assimilation processes (occurred via the glutamate synthase reaction). Increasing D the specific activities of the enzymes involved in the tricarboxylic acid cycle and the respiration rate were decreased. At transitory states, under optimal for nitrogen fixation dissolved oxygen (DO) concentrations, ammonium nitrogen negatively affected, besides nitrogen fixing activity, the bacterial growth. At sub-optimal for nitrogen fixation DO concentration (i.e. 1.56 microM) and 0.1 g l(-1) NH4Cl in the fed medium, the activities of citrate synthase and succinate dehydrogenase were significantly reduced. CONCLUSIONS: Important shifts in both carbon and nitrogen metabolism occur in A. lipoferum grown in the presence of the ammonium nitrogen, while the boundaries of ammonium nitrogen concentration in which A. lipoferum can be adapted depend on the DO concentration in the growth environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on growth dynamics and physiology of A. lipoferum, grown in experimental model systems, can contribute to an efficient application of these bacteria as plant-growth-promoting-agents.

Metabolic activities in Azospirillum lipoferum grown in the presence of NH4+.

The utilization of some agro-industrial wastes as soil conditioners to provide free-living nitrogen-fixing bacterial populations (e.g. Azospirillum spp.) with carbon and energy sources, may be an interesting perspective for agriculture. However, the presence of ammonium nitrogen in cultivated soils and/or various wastes could inhibit the growth of the nitrogen-fixing populations. The present investigation shows that growth of Azospirillum lipoferum was restricted at a dissolved oxygen (DO) concentration equal to 135 microM, when the initial NH4Cl concentration increased from 0.5 to 0.9 g/l. The activities of both citrate synthase (CS) and isocitrate dehydrogenase were significantly decreased in the presence of 0.9 g/l NH4Cl (e.g., 40% and 66%, respectively, in cells incubated for 95 h), while ammonium assimilation occurred via the glutamate dehydrogenase reaction. Furthermore, growth limitation occurred even in the presence of 0.5 g/l NH4Cl, when the DO concentration decreased from 135 to 30 microM. The activities of both CS and succinate dehydrogenase were dramatically decreased in cells grown at the lower DO concentration (e.g., 90% and 93% respectively, in a 95 h incubation), while ammonium assimilation was limited due to the low activities of both glutamate dehydrogenase and glutamate synthase. It is concluded that the threshold of ammonium concentration at which growth of A. lipoferum is limited, depends on the DO concentration in the medium.