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.

The association between grass roots and Azospirillum brasilense Sp 7 was investigated by the Fahraeus slide technique, using nitrogen-free medium. Young inoculated roots of pearl millet and guinea grass produced more mucilaginous sheath (mucigel), root hairs, and lateral roots than did uninoculated sterile controls. The bacteria were found within the mucigel that accumulated on the root cap and along the root axes. Adherent bacteria were associated with granular material on root hairs and fibrillar material on undifferentiated epidermal cells. Significantly fewer numbers of azospirilla attached to millet root hairs when the roots were grown in culture medium supplemented with 5 mM potassium nitrate. Under these growth conditions, bacterial attachment to undifferentiated epidermal cells was unaffected. Aseptically collected root exudate from pearl millet contained substances which bound to azospirilla and promoted their adsorption to the root hairs. This activity was associated with nondialyzable and proteasesensitive substances in root exudate. Millet root hairs adsorbed azospirilla in significantly higher numbers than cells of Rhizobium, Pseudomonas, Azotobacter, Klebsiella, or Escherichia. Pectolytic activities, including pectin transeliminase and endopolygalacturonase, were detected in pure cultures of A. brasilense when this species was grown in a medium containing pectin. These studies describe colonization of grass root surfaces by A. brasilense and provide a possible explanation for the limited colonization of intercellular spaces of the outer root cortex.

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Field-grown pearl millet (Pennisetum americanum) and guinea grass (Panicum maximum), lightly fertilized and inoculated with Spirillum lipoferum, produced significantly higher yields of dry matter than did uninoculated controls. Up to 42 and 39 kilograms of nitrogen per hectare were replaced by inoculation for pearl millet and guinea grass, respectively. The data demonstrate that nitrogen fixation by these grass-Spirillum systems is efficient and is achieved at a reasonable energy cost to the plant.