Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, was investigated to establish the effect on plant growth of growth substances produced by the bacteria. Thin-layer chromatography, highpressure liquid chromatography, and bioassay were used to separate and identify plant growth substances produced by the bacteria in liquid culture. Indole acetic acid and indole lactic acid were produced by A. brasilense from tryptophan. Indole acetic acid production increased with increasing tryptophan concentration from 1 to 100 ,ig/ml. Indole acetic acid concentration also increased with the age of the culture until bacteria reached the stationary phase. Shaking favored the production of indole acetic acid, especially in a medium containing nitrogen. A small but biologically significant amount of gibberellin was detected in the culture medium. Also at least three cytokinin-like substances, equivalent to about 0.001 ,tg of kinetin per ml, were present. The morphology of pearl millet roots changed when plants in solution culture were inoculated. The number of lateral roots was increased, and all lateral roots were densely covered with root hairs. Experiments with pure plant hormones showed that gibberellin causes increased production of lateral roots. Cytokinin stimulated root hair formation, but reduced lateral root production and elongation of the main root. Combinations of indole acetic acid, gibberellin, and kinetin produced changes in root morphology of pearl millet similar to those produced by inoculation with A. brasilense.

Polygalacturonic acid transeliminase (PATE) was produced by all of six Azospirillum strains studied. Characteristics were similar to those of PATE from other bacteria: activity was maximal at pH 8.0 and was stimulated by CaCl2. Polygalacturonic acid was used more readily than pectin as a substrate. Polygalacturonic acid in the medium stimulated PATE production by several but not all strains. In all cases some of the PATE produced in cultures remained bound to cell walls. In one strain, most remained cell wall bound. When nitrogen was supplied as amino acids rather than ammonium salts, the ratio of free to bound enzyme was increased. The strains studied varied considerably to response to nutrient amendments and in maximum PATE activity.