The effect of combined and separate inoculation of alfalfa plants with <Emphasis Type="Italic">Azospirillum lipoferum</Emphasis> and <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> 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 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 · 24 h) and low denitrification activity (1.8 μmol N₂O/flask · 24 h). These results are useful in applied developments aimed at the use of bacterial and mineral fertilizers for leguminous plants.     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of amino acids on nitrogen fixation ability and growth of Azospirillum spp.

The utilization of amino acids for growth and their effects on nitrogen fixation differ greatly among the several strains of each species of Azospirillum spp. that were examined. A. brasiliense grew poorly or not at all on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources. Nitrogen fixation by most A. brasiliense strains was inhibited only slightly even by 10 mM concentrations of these amino acids. In contrast, A. lipoferum and A. amazonense grew very well on glutamate, aspartate, serine, or histidine as the sole nitrogen and carbon sources; nitrogen fixation, which was measured in the presence of malate or sucrose, was severely inhibited by these amino acids. It was concluded that growth on histidine as the sole source of nitrogen, carbon, and energy may be used for the taxonomic characterization of Azospirillum spp. and for the selective isolation of A. lipoferum. The different utilization of various amino acids by Azospirillum spp. may be important for their establishment in the rhizosphere and for their associative nitrogen fixation with plants. The physiological basis for the different utilization of glutamate by Azospirillum spp. was investigated further. A. brasiliense and A. lipoferum exhibited a high affinity for glutamate uptake (Km values for uptake were 8 and 40 microM, respectively); the Vmax was 6 times higher in A. lipoferum than in A. brasiliense. At high substrate concentrations (10 mM), the nonsaturable component of glutamate uptake was most active in A. lipoferum and A. amazonense.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between survival rates of associative nitrogen-fixers on roots and yield response of plants to inoculation

Abstract: The population dynamics of associative nitrogen-fixers Azospirillum lipoferum 137, Arthrobacter mysorens 7, Flavobacterium sp. L30 and phosphate-solubilizing strain Agrobacterium radiobacter 10 in soil and the rhizoplane of inoculated plants was studied in pot and field experiments. All of the present strains were able to actively colonize the rhizoplane of barley, wheat, oat, tomatoes, rape, and alfalfa. For the most part the population size and dynamics of introduced bacteria depended only slightly on the plant genotype and soil conditions. The overall pictures of survival of the strains in soil and on plant roots were similar. The reliable effect of inoculation on plants was observed only in individual cases. No correlation was established between survival of introduced bacteria and their effect on plant development. It was concluded that the influence of plants on survival of bacteria was not specific. In contrast, the plant response to inoculation was conditioned to a greater extent by the plant genotype.     

Influence of different Sinorhizobium meliloti inocula on abundance of genes involved in nitrogen transformations in the rhizosphere of alfalfa (Medicago sativa L.)

Inoculation of leguminous seeds with selected rhizobial strains is practised in agriculture to ameliorate the plant yield by enhanced root nodulation and nitrogen uptake of the plant. However, effective symbiosis between legumes and rhizobia does not only depend on the capacity of nitrogen fixation but also on the entire nitrogen turnover in the rhizosphere. We investigated the influence of seed inoculation with two indigenous Sinorhizobium meliloti strains exhibiting different efficiency concerning plant growth promotion on nitrogen turnover processes in the rhizosphere during the growth of alfalfa. Quantification of six target genes (bacterial amoA, nirK, nirS, nosZ, nifH and archaeal amoA) within the nitrogen cycle was performed in rhizosphere samples before nodule formation, at bud development and at the late flowering stage. The results clearly demonstrated that effectiveness of rhizobial inocula is related to abundance of nifH genes in the late flowering phase of alfalfa. Moreover, other genes involved in nitrogen turnover had been affected by the inocula, e.g. higher numbers of amoA copies were observed during flowering when the more effective strain had been inoculated. However, the respective gene abundances differed overall to a greater extent between the three plant development stages than between the inoculation variants.     

Isolation and symbiotic characterization of transposon Tn5-induced arginine auxotrophs of Sinorhizobium meliloti

Seventeen arginine auxotrophic mutants of Sinorhizobium meliloti Rmd201 were isolated by random transposon Tn5 mutagenesis using Tn5 delivery vector pGS9. Based on intermediate feeding studies, these mutants were designated as argA/argB/argC/argD/argE (ornithine auxotrophs), argF/argI, argG and argH mutants. The ornithine auxotrophs induced ineffective nodules whereas all other arginine auxotrophs induced fully effective nodules on alfalfa plants. In comparison to the parental strain induced nodule, only a few nodule cells infected with rhizobia were seen in the nitrogen fixation zone of the nodule induced by the ornithine auxotroph. TEM studies showed that the bacteroids in the nitrogen fixation zone of ornithine auxotroph induced nodule were mostly spherical or oval unlike the elongated bacteroids in the nitrogen fixation zone of the parental strain induced nodule. These results indicate that ornithine or an intermediate of ornithine biosynthesis, or a chemical factor derived from one of these compounds is required for the normal development of nitrogen fixation zone and transformation of rhizobial bacteria into bacteroids during symbiosis of S. meliloti with alfalfa plants.     

Attenuation of Symbiotic Effectiveness by Rhizobium meliloti SAF22 Related to the Presence of a Cryptic Plasmid

Several wild-type strains of Rhizobium meliloti isolated from alfalfa nodules exhibited different plasmid profiles, yet did not differ in growth rate in yeast-mannitol medium, utilization of 43 different carbon sources, intrinsic resistance to 14 antibiotics, or detection of 16 enzyme activities. In contrast, three measures of effectiveness in symbiotic nitrogen fixation with alfalfa (shoot length, dry weight, and nitrogen content) indicated that R. meliloti SAF22, whose plasmid profile differs from those of the other strains tested, is significantly less effective than other wild-type strains in symbiotic nitrogen fixation. Light microscopy of nodules infected with strain SAF22 showed an abnormal center of nitrogen fixation zone III, with bacteria occupying a smaller portion of the infected host cells and vacuoles occupying a significantly larger portion of adjacent uninfected host cells. In contrast, the effective nodules infected with other wild types or plasmid pRmSAF22c-cured segregants of SAF22 did not display this cytological abnormality.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

Growth of Spirillum lipoferum at constant partial pressures of oxygen, and the properties of its nitrogenase in cell-free extracts.

Spirillum lipoferum, an N2-fixing organism, was grown at constant concentrations of dissolved O2. When supplied with NH4+ aerobically, its doubling time was 1 h; when it fixed N2 microaerophilically, its doubling time was 5-5 to 7 h and the optimal PO2 for growth was 0-005 to 0-007 atm. At its optimal PO2 for growth on N2, S. lipoferum assimilated 8 to 10 mg nitrogen/g carbon substrate used; its efficiency was less at higher PO2 levels. Nitrogenase in cell-free extracts required Mg2+ and Mn2+, and the Fe-protein was activated by Rhodospirillum rubrum activating factor. The nitrogenase had an optimal pH of 7-1 to 7-4 and an apparent Km for acetylene of 0-0036 atm. Extracts of S. lipoferum lost their nitrogenase activity on storage at -18 degrees C, and activity was restored by adding purified Fe-protein from other N2-fixing bacteria.     

Symbiotic nitrogen fixation by a nifA deletion mutant of Rhizobium meliloti: the role of an unusual ntrC allele.

In the N2-fixing alfalfa symbiont Rhizobium meliloti, the three sigma 54 (NTRA)-dependent positively acting regulatory proteins NIFA, NTRC, and DCTD are required for activation of promoters involved in N2 fixation (pnifHDKE and pfixABCX), nitrogen assimilation (pglnII), and C4-dicarboxylate transport (pdctA), respectively. Here, we describe an allele of ntrC which results in the constitutive activation of the above NTRC-, NIFA-, and DCTD-regulated promoters. The expression and activation of wild-type NTRC occur in response to nitrogen availability, whereas in cells carrying the ntrC283 allele, the NTRC283 protein appears constitutively active and is constitutively expressed. The ntrC283 allele was shown to carry a single mutation resulting in the replacement of an Asp by a Tyr residue in the helix-turn-helix motif of ntrC283. Introduction of the ntrC283 allele into a nifA deletion mutant restores the N2-fixation ability to 70 to 80% of the wild-type level. Thus, the nifA gene is dispensable for symbiotic N2 fixation.     

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.     

A phosphate transport system is required for symbiotic nitrogen fixation by Rhizobium meliloti.

The bacterium Rhizobium meliloti forms N2-fixing root nodules on alfalfa plants. The ndvF locus, located on the 1,700-kb pEXO megaplasmid of R. meliloti, is required for nodule invasion and N2 fixation. Here we report that ndvF contains four genes, phoCDET, which encode an ABC-type transport system for the uptake of Pi into the bacteria. The PhoC and PhoD proteins are homologous to the Escherichia coli phosphonate transport proteins PhnC and PhnD. The PhoT and PhoE proteins are homologous to each other and to the E. coli phosphonate transport protein PhnE. We show that the R. meliloti phoD and phoE genes are induced in response to phosphate starvation and that the phoC promoter contains two elements which are similar in sequence to the PHO boxes present in E. coli phosphate-regulated promoters. The R. meliloti ndvF mutants grow poorly at a phosphate concentration of 2 mM, and we hypothesize that their symbiotic phenotype results from their failure to grow during the nodule infection process. Presumably, the PhoCDET transport system is employed by the bacteria in the soil environment, where the concentration of available phosphate is normally 0.1 to 1 microM.     

Nitrogen fixation by reductively dechlorinating bacteria

Significant advances in the ecology, physiology and genetics of reductively dechlorinating bacteria have revealed their important environmental roles in bioremediation and in the global chlorine cycle. N2 fixation has been widely observed in symbiotic, associative and free-living bacteria. Here we show physiological and molecular evidence that reductively dechlorinating bacteria are capable of fixing atmospheric nitrogen. Furthermore, N2 fixation in some of these dechlorinating bacteria stimulated reductive dechlorination, which should help predict and regulate the environmental function of dechlorinating bacteria in in situ bioremediation of chlorinated pollutants. These results imply that N2 fixation in dechlorinating bacteria interacts with other biogeochemical cycles to control the nitrogen status of the anaerobic ecosystem.     

Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations

The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp. L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2. Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants. Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings. The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd. Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly. The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A. lipoferum 137. Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots. The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements.     

Relevance of ammonium oxidation within biological soil crust communities

Thin, vertically structured topsoil communities that become ecologically important in arid regions (biological soil crusts or BSCs) are responsible for much of the nitrogen inputs into pristine arid lands. We studied N(2) fixation and ammonium oxidation (AO) at subcentimetre resolution within BSCs from the Colorado Plateau. Pools of dissolved porewater nitrate/nitrite, ammonium and organic nitrogen in wetted BSCs were high in comparison with those typical of aridosoils. They remained stable during incubations, indicating that input and output processes were of similar magnitude. Areal N(2) fixation rates (6.5-48 micromol C(2)H(2) m(-2) h(-1)) were high, the vertical distribution of N(2) fixation peaking close to the surface if populations of heterocystous cyanobacteria were present, but in the subsurface if they were absent. Areal AO rates (19-46 micromol N m(-2) h(-1)) were commensurate with N(2) fixation inputs. When considering oxygen availability, AO activity invariably peaked 2-3 mm deep and was limited by oxygen (not ammonium) supply. Most probable number (MPN)-enumerated ammonia-oxidizing bacteria (6.7-7.9 x 10(3) cells g(-1) on average) clearly peaked at 2-3 mm depth. Thus, AO (hence nitrification) is a spatially restricted but important process in the nitrogen cycling of BSC, turning much of the biologically fixed nitrogen into oxidized forms, the fate of which remains to be determined.     

Analysis of infection thread development using Gfp- and DsRed-expressing Sinorhizobium meliloti

Sinorhizobium meliloti growth inside infection threads was monitored after inoculation of alfalfa with red- or green-tagged bacteria. Most threads were populated with single bacterial types. Mixed infections were present but gave mixed nodules less often than expected. These patterns are explained by a model describing bacterial growth during infection.     

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.