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Azospirillum brasilense is a diazotrophic bacterium and one of the best studied plant-growth-promoting bacterium living in close association with several agronomically important crops. The production of plant-growth-regulating substances is a main mechanism of plant growth stimulation, although other mechanisms have also been proposed. Nitrogen transfer from the bacterium to the plant is one among the other possible mechanisms of plant growth stimulation. In this study, we investigated, by means of a greenhouse trial with winter wheat inoculation, the effect of a point mutation in the ammonium binding site of the A. brasilense glutamine synthetase. The glutamine synthetase is one of the main ammonium-assimilating enzymes and mutations in this enzyme generally result in the release of ammonium from the bacterium to its environment. The ammonium-excreting mutant used in this study was shown to perform better than the wild-type A. brasilense strain with respect to wheat growth parameters and yield. In the greenhouse conditions used, this effect was independent of the way fertilizer was applied.

The production of phytohormones by plant-growth promoting rhizobacteria is considered to be an important mechanism by which these bacteria promote plant growth. In this study the importance of indole-3-acetic acid (IAA) produced by Azospirillum brasilense Sp245 in the observed plant growth stimulation was investigated by using Sp245 strains genetically modified in IAA production. Firstly wild-type A. brasilense Sp245 and an ipdC knock-out mutant which produces only 10% of wild-type IAA levels (Vande Broek et al., J Bacteriol 181:1338–1342, 1999) were compared in a greenhouse inoculation experiment for a number of plant parameters, thereby clearly demonstrating the IAA effect in plant growth promotion. Secondly, the question was addressed whether altering expression of the ipdC gene, encoding the key enzyme for IAA biosynthesis in A. brasilense, could also contribute to plant growth promotion. For that purpose, the endogenous promoter of the ipdC gene was replaced by either a constitutive or a plant-inducible promoter and both constructs were introduced into the wild-type strain. Based on a greenhouse inoculation experiment it was found that the introduction of these recombinant ipdC constructs could further improve the plant-growth promoting effect of A. brasilense. These data support the possibility of constructing Azospirillum strains with better performance in plant growth promotion.

Several plant growth-promoting rhizobacteria (PGPR) have shown potential to enhance nodulation of legumes when coinoculated with Rhizobium. To optimize the efficiency of these Rhizobium-PGPR-host plant interactions, unravelling the underlying mechanisms and analyzing the influence of specific environmental conditions is crucial. In this work the effect of four PGPR strains on the symbiotic interaction between Rhizobium and common bean (Phaseolus vulgaris) was studied under deficient versus sufficient phosphorus supply. It was observed that the effect on nodulation of three out of four PGPR tested was strongly dependent on P nutrition. Further, the use of specific PGPR mutant strains indicated that bacterial indole-3-acetic-acid production (IAA) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity play an important role in the host nodulation response, particularly under low P conditions. Moreover, it was shown that the differential response to PGPR under low versus high P conditions was associated with changes in the host hormone sensitivity for nodulation induced under P deficiency. These findings contribute to the understanding of the interplay between Rhizobium, PGPR and the plant host under different environmental settings.

Sulfate modification on Rhizobium Nod factor signaling molecules is not a prerequisite for successful symbiosis with the common bean (Phaseolus vulgaris L.). However, many bean-nodulating rhizobia, including the broad host strain Sinorhizobium sp. BR816, produce sulfated Nod factors. Here, we show that the nodH gene, encoding a sulfotransferase, is responsible for the transfer of sulfate to the Nod factor backbone in Sinorhizobium sp. BR816, as was shown for other rhizobia. Interestingly, inactivation of nodH enables inoculated bean plants to fix significantly more nitrogen under different experimental setups. Our studies show that nodH in the wild-type strain is still expressed during the later stages of symbiosis. This is the first report on enhanced nitrogen fixation by blocking Nod factor sulfation.

Under the controlled conditions of the greenhouse and by varying some biotic and abiotic factors, we tried to identify some of the factors critical to obtain successful Azospirillum inoculation. Spring wheat and grain maize were inoculated with different concentrations of the wild type strains A. brasilense Sp245 and A. irakense KBC1, and grown in a substrate with varying concentrations of organic matter (OM) and N fertiliser. The inoculum concentration was one of the factors that influenced most the outcome of an inoculation experiment on wheat, with lower inoculum concentrations (105–106 cfu plant–1) stimulating root development and plant dry weight and higher inoculum concentrations (107–108 cfu plant–1) having no effect or sometimes even inhibiting root development. The effect of inoculation was most pronounced at low to intermediate N fertilisation levels, while the OM content of the substrate had no effect. Inoculation was found to affect early plant and root development, plant and root dry weight, grain yield and the N-uptake efficiency of plants. However, inoculation did not change the N concentration in plants or grains. In addition, a difference in the ability of both strains to stimulate plant growth and N uptake of wheat and maize was observed, with A. brasilense Sp245 having most effect on spring wheat and A. irakense KBC1 being more effective on grain maize. The significance of the obtained results for agriculture is discussed.

Auxin production by Azospirillum is believed to play a major role in the observed plant growth promoting effect. By using different genetically modified strains, the contribution of auxin biosynthesis by A. brasilense in altering root morphology was evaluated in a plate assay. Inoculation with the wild type strains A. brasilense Sp245 and Sp7 resulted in a strong decrease in root length and increase in root hair formation. This effect was abolished when inoculating with an ipdC mutant of A. brasilense. The ipdC gene encodes a key enzyme in the IPyA pathway of IAA synthesis by A. brasilense. On the other hand, the observed auxin effect was further enhanced by adding tryptophan, a precursor of IAA, to the plates and could be mimicked by replacing the Azospirillum cells by a particular concentration of IAA. Furthermore, particular mutants (rpoN, scrp) and transconjugants (extra copy of ipdC) of A. brasilense were tested in the plate assay. Together, these results confirm the important role of IAA produced by Azospirillum in altering root morphology and illustrate the power of combining genetic tools and bioassays to elucidate the mechanism of a beneficial Azospirillum-plant interaction.

© 2007 Springer. All rights reserved. Several plant growth-promoting rhizobacteria (PGPR) have shown potential to enhance nodulation of legumes when coinoculated with Rhizobium. To optimize the efficiency of these Rhizobium-PGPR-host plant interactions, unravelling the underlying mechanisms and analyzing the influence of specific environmental conditions is crucial. In this work the effect of four PGPR strains on the symbiotic interaction between Rhizobium and common bean (Phaseolus vulgaris) was studied under deficient versus sufficient phosphorus supply. It was observed that the effect on nodulation of three out of four PGPR tested was strongly dependent on P nutrition. Further, the use of specific PGPR mutant strains indicated that bacterial indole-3-acetic-acid production (IAA) and l-aminocyclopropane-l-carboxylate (ACC) deaminase activity play an important role in the host nodulation response, particularly under low P conditions. Moreover, it was shown that the differential response to PGPR under low versus high P conditions was associated with changes in the host hormone sensitivity for nodulation induced under P deficiency. These findings contribute to the understanding of the interplay between Rhizobium, PGPR and the plant host under different environmental settings. ispartof: New Perspectives and Approaches in Plant Growth-Promoting Rhizobacteria Research pages:341-351 ispartof: pages:341-351 status: published

This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Azospirilla are free-living rhizobacteria that are able to promote plant growth and increase yields in many crops of agronomic importance. It is assumed that the bacteria affect plant growth mainly by the production of plant growth promoting substances, which leads to an improvement in root development and an increase in the rate of water and mineral uptake. In the present review, we discuss the physiological responses of the plant roots to inoculation with Azospirillum, and report on field and greenhouse experiments carried out with these bacteria during 1994–2001 in Belgium, Uruguay, Mexico and Israel.