Environmental influences on dinitrogen fixation by bacteria associated with roots of maize and sorghum

Abstract

Nitrogen-fixing bacteria in the rhizosphere of cereal grasses may reduce the fertilizer nitrogen requirements of grass crops by providing them with fixed nitrogen. This study evaluated the effects of oxygen partial pressure, combined nitrogen, and organic substrates on nitrogen-fixation (acetylene reduction and ¹⁵N₂ incorporation) by bacteria associated with the roots of intact maize and sorghum plants, and investigated the possibility of enhancing associative nitrogen-fixation by inoculating soil with Azospirillum. Acetylene reduction (AR) was greatest when roots of intact plants were exposed to pO₂ between 1.3 and 2.1 kPa. Field-grown and greenhouse-grown plants supported similar levels of activity. Incorporation of ¹⁵N₂ was 200 times greater at 2 kPa O₂ than at 10 kPa O₂. Respiration inhibitors (2,4-dinitrophenol and sodium azide) eliminated AR at 2 kPa O₂, whereas a fermentation inhibitor (sodium fluoride) only partially reduced the activity. Root growth, but not root respiration (CO₂ evolution) was impaired by pO₂ below 6 kPa. The plants supported increasing rates of AR until the beginning of anthesis. Thereafter the activity associated with plants exposed to 2 kPa O₂ decreased, while that associated with plants exposed to 10 kPa O₂ remained the same or increased slightly. Despite these different trends, rates of AR remained higher at 2 kPa O₂ than at 10 kPa O₂ until the plants reached phenological maturity. Acetylene reduction was rapidly (1-3 h) inhibited by NH₄⁺, NO₃⁻, and NO₂⁻ at concentrations of 4-20 mg N l⁻¹ Incorporation of ¹⁵N₂ decreased by 25% following treatment with 10 mg N l⁻¹ as (NH₄)SO₄ Root-associated AR activity increased with increasing rates of fertilization up to 12 mg N pot⁻¹ wk⁻¹ (equivalent to 161 kg N ha⁻¹) in a greenhouse experiment, but decreased at a higher rate (20 mg N pot⁻¹ wk⁻¹). Rates of AR varied substantially among individual plants in each experiment and between experiments. Amendment with any of several organic substrates increased AR activity when rates were low, suggesting that the lack of activity was caused by a shortage of available carbon in the rhizosphere. Bacterial growth inhibitors (chloramphenicol and nalidixic acid) prevented increases in AR activity in response to the added carbon, indicating that a proliferation of nitrogen-fixing bacteria on the roots was responsible for the increases in activity rather than greater specific activity by the existing population. Inoculation with Azospirillum failed to increase rates of AR or ¹⁵N₂-fixation associated with maize plants. In several experiments the indigenous bacteria associated with uninoculated plants exhibited greater activity than the bacteria associated with inoculated plants.