Ammonium-excreting Azospirillum sp. become intracellularly established in maize (Zea mays) para-nodules

Maize seedlings develop nodule-like tumour knots (para-nodules) along primary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Inoculated NH ₄ ⁺ -excreting Azospirillum brasilense cells were shown to colonize these tumours, mostly intracellularly, promoting a high level of N₂ fixation when microaerophilic conditions were imposed. The nitrogenase activity inside the para-nodules was less sensitive to free O₂ than in non-para-nodulating roots. Both light and electron microscopy showed a dense bacterial population inside intact tumour cells, with the major part of the cell infection along a central tumour tissue. The bacteria colonized the cytoplasm with a close attachment to inner cell membranes. In an auxin-free growth medium, young 2,4-D-induced para-nodules grew further to become mature differentiated root organs in which introduced bacteria survived with a stable population. These results provide evidence that gramineous plants are potentially able to create a symbiosis with diazotrophic bacteria in which the NH ₄ ⁺ -excreting symbiont will colonize para-nodule tissue intracellularly, thus becoming well protected.