Treatment of wheat seedlings with the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), induced nodule-like structures or tumours (termed para-nodules) where lateral roots would normally emerge.The formation of these structures promoted increased rates of acetylene reduction at reduced oxygen pressure (0.02-0.04 atm) in seedling inoculated with Azospirillum brasilense, compared to seedlings inoculated without auxin treatment. Fluorescent microscopy, laser scanning confocal microscopy and direct bacterial counts all showed that the 2,4-D treatment stimulated internal colonization of the root system with azospirilla, particularly in the basal region of the nodular structures. Both colonization with azospirilla and acetylene-reducing activity were further stimulated by simultaneous treatment with another synthetic auxin, naphthaleneacetic acid (NAA) and, less reliably, with indoleacetic acid (IAA) and indolebutyric acid (IBA). These auxins produced shortening of many initiated lateral roots, although 20 times the concentration of NAA was required to achieve rounded structures similar to those obtained with 2,4-D. Treatment with NAA, IAA or IBA alone also stimulated colonization with azospirilla and acetylene reduction rates at 0.02 atm oxygen, but less effectively than by treatment with 2,4-D. Such exogenous treatments of wheat seedlings with synthetic growth regulators provide an effective laboratory model for studies on the development of a N2-fixing system in cereals.

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The characteristics of C2H4 formation and H2 evolution associated with 2,4-D induced root structures on wheat seedlings inoculated with Azospirillum brasilense Sp7 have been investigated. C2H4 formation and H2 evolution with 2,4-D treatment is several times greater than that by wheat seedlings inoculated with Azospirillum alone, as the result of the formation of a niche for azospirilla protected from O2. This increased C2H4 production in 2,4-D treated seedlings was proportional to C2H2 concentration and it was strongly inhibited by nitrite and ammonia. The inhibition increased with greater ammonia and nitrite concentration, but was reversible. The increased rate of C2H2 reduction activity was correlated with 15N enrichment in 2,4-D treated seedlings. These results confirm that the C2H4 formation was C2H2 dependent and represented the nitrogenase activity of A. brasilense associated with the 2,4-D induced root structures of wheat seedlings rather than C2H2 produced by plant tissues.

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