NH 4 + -Excreting Azospirillum brasilense Mutants Enhance the Nitrogen Supply of a Wheat Host

Abstract

Spontaneous ethylenediamine-resistant mutants of Azospirillum brasilense were selected on the basis of their excretion of NH 4 + . Two mutants exhibited no repression of their nitrogenase enzyme systems in the presence of high (20 mM) concentrations of NH 4 + . The nitrogenase activities of these mutants on nitrogen-free minimal medium were two to three times higher than the nitrogenase activity of the wild type. The mutants excreted substantial amounts of ammonia when they were grown either under oxygen-limiting conditions (1 kPa of O 2 ) or aerobically on nitrate or glutamate. The mutants grew well on glutamate as a sole nitrogen source but only poorly on NH 4 Cl. Both mutants failed to incorporate [ 14 C]methylamine. We demonstrated that nitrite ammonification occurs in the mutants. Wild-type A. brasilense , as well as the mutants, became established in the rhizospheres of axenically grown wheat plants at levels of > 10 7 cells per g of root. The rhizosphere acetylene reduction activity was highest in the preparations containing the mutants. When plants were grown on a nitrogen-free nutritional medium, both mutants were responsible for significant increases in root and shoot dry matter compared with wild-type-treated plants or with noninoculated controls. Total plant nitrogen accumulation increased as well. When they were exposed to a 15 N 2 -enriched atmosphere, both A. brasilense mutants incorporated significantly higher amounts of 15 N inside root and shoot material than the wild type did. The results of our nitrogen balance and 15 N enrichment studies indicated that NH 4 + -excreting A. brasilense strains potentially support the nitrogen supply of the host plants.