A novel intracellular nitrogen‐fixing symbiosis made by U stilago maydis and B acillus spp.

Summary We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)‐free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N 2 . Because only prokaryotic organisms have the capacity to reduce N 2 , we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N 2 by use of 15 N 2 . Presence of an intracellular N 2 ‐fixing bacterium was analyzed by PCR amplification of bacterial 16S r RNA and nifH genes, and by microscopic observations. Nitrogenase activity and 15 N incorporation into the cells proved that U. maydis fixed N 2 . Light and electron microscopy, and fluorescence in situ hybridization ( FISH ) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus , as evidenced by sequencing of the PCR ‐amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N 2 ‐fixing association involving a fungus and a bacterium.