Influence of Azospirillum spp. on the nitrogen supply of a gramineous host

Abstract

The main objectives of this study were to identify factors that control the behaviour of Azospirillum in the rhizosphere of a gramineous plant in order to be able to optimize the association between the bacteria and the host plants in terms of nitrogen supply to the host.Plant produced growth substances such as the auxines indole-acetic-acid (IAA) and 2,4-dichlorphenoxy-acetic-acid (2,4 D) or gibberilic acid enhance the acetylene reduction activity of a pure A.brasilense culture. IAA and the cytocinine 6-benzyl-aminopurine also stimulated bacterial growth. It should be pointed out, that Azospirillum also produces IAA itself, which is often mentioned to be the reason of its plant-growth stimulating activity.When associated with living roots, the nitrogen fixation (acetylene reduction) activity of Azospirillum brasilense is much less sensitive to the repressive influence of free oxygen and mineral nitrogen, i.e. NO3-and NH4+, than in the absence of an active growing root. Potential acetylene reduction rates varied from 10 to 550 nmol C 2 H 4 h -1plant -1depending on environmental conditions.In order to be able to determine the fate of introduced Azospirillum in a soil and in the root environment A.brasilense strains were marked by a transposon (Tn5) insertion into its genome so that reisolation upon double resistance against kanamycin and rifampicin was possible. A.brasilense ::Tn5 established in the rhizosphere of an axenically grown spring wheat to cell numbers as high as 10 6cells per gram dry rhizosphere soil and 10 5cells per gram dry root, respectively. In the rhizosphere of a non-sterile grown plant the number of A.brasilense ::Tn5 was much lower, i.e. approximately 10 4cells per gram dry rhizosphere soil and 10 3cells per gram dry root. The number of A.brasilense ::Tn5 cells was 10 to 100 times higher in the soils closely attached to the roots than in root-free soils. A.brasilense could not be reisolated from inner root-tissue after a root- surface sterilization with 1% chloramine T. When introduced to plants in an early stage of plant growth either by seedling inoculation or by a seed-coating, A.brasilense was able to develop with the growing root and to establish a strong population all over the root.Most intensive root colonization of introduced A.brasilense and highest acetylene reduction rates were observed when plants were treated with Azospirillum cells immediately after seedling emergence as compared to the colonization of roots after inoculation at a later stage of growth. Subsequent inoculations during plant development after an initial addition did neither stimulate root colonization nor acetylene reduction activity.When comparing wheat and sorghum cultivars with different levels of aluminium tolerance a larger rhizosphere acetylene reduction activity was observed when Azospirillum was introduced to roots of aluminium-tolerant cultivars than to roots of Al- sensitive cultivars. The amount of fixed nitrogen, transferred from Azospirillum to the host as calculated by the 15N dilution technique was also significantly higher in case of Al-tolerant cultivars. Aluminium-tolerant plants appeared to exudate significantly larger amounts of total organic carbon than Al-sensitive plants. Not only the quantity but also the quality of the exudates differed in the sense, that higher concentrations of low molecular dicarbonic acids such as succinic, malic and oxalic acid were observed at root-exudates of aluminium-tolerant wheat plants. These organic acids are known to be preferable carbon substrates for Azospirillum spp, what might explain the more intensive colonization and higher nitrogen fixation capacity in the rhizosphere of Al-tolerant plants.Although Azospirillum develops considerable activities in the rhizosphere of host plants the transfer of fixed nitrogen to the host as determined with the 15N-dilution technique appeared to be rather low. Only approximately 3% of the root nitrogen and approximately 2% of the shoot nitrogen was calculated to be derived from the N 2 -fixation activity of the Azospirillum cells.In order to enhance the transfer of nitrogen to the host A.brasilense was selected on ethylenediamine, yielding mutant strains which lack their ammonia transport system across cell membranes and which excrete substantial amounts of NH4+, to the environment. Two of these mutant strains fixed nitrogen in the presence of high concentrations (20 mM) of NH4+. Nitrogenase activity of the NIH4+-excreting mutants was two to three times as high as that of the wild type. The mutant strains colonized the roots of axenically grown wheat to high cell numbers and developed rhizosphere acetylene reduction activities comparable to that of the wild type. Both mutant strains caused a significant increase of dry matter production and of total plant N- accumulation as compared to wild type treated plants or to non-inoculated controls. When exposed to a 15[N] 2 enriched atmosphere the A.brasilense mutant strains transfered higher amounts of 15N to their hosts than the wild type did. 15N- enrichment and nitrogen balance studies both indicated that NH4+-excreting A.brasilense support the nitrogen supply of a wheat host