Isolation of Azospirillum lipoferum from the rhizosphere of rice by a new, simple method

A new, simple method for isolating Azospirillum strains from the roots and the rhizosphere of rice is described. The method is based on the capacity of Azospirillum, a nitrogen-fixing bacterial genus, to grow in nutrient-deficient liquid media such as distilled water, KCl (8.5 g/L), or soil extract medium. The enrichment efficiency of the deficient media was compared with classical N-free malate medium. Serial dilutions from 10−1 to 10−10 of rice root macerates and rhizosphere soil were incubated at 28 °C in these media without agitation for 15 days. Pellicle formation was regularly examined under a microscope to detect the spiral motility of Azospirillum cells. Tubes with putative azospirilla were plated on nutrient agar. Most probable numbers of Azospirillum in root macerates were 2 × 103/g dry root weight in KCl, 2 × 104/g dry root weight in H2O, 2 × 106/g dry root weight in malate, and 3 × 106/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial popul...