ISOLATION OF AZOSPIRILLUM-LIPOFERUM FROM THE RHIZOSPHERE OF RICE BY ANEW, 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 i n 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 diluti ons from 10(-1) to 10(-10) of rice root macerates and rhizosphere soil were incubated at 28 degrees C in these media without agitation for 1 5 days. Pellicle formation was regularly examined under a microscope t o detect the spiral motility of Azospirillum cells. Tubes with putativ e azospirilla were plated on nutrient agar. Most probable numbers of A zospirillum in root macerates were 2 x 10(3)/g dry root weight in KCl, 2 x 10(4)/g dry root weight in H2O, 2 x 10(6)/g dry root weight in ma late, and 3 x 10(6)/g dry root weight in the soil extract medium. Soil extract medium repressed the most abundant bacterial populations and facilitated isolation of azospirilla from a population representing le ss than 0.001% of the total microflora. The isolates had vibrioid to S -shaped cells with one polar flagellum when grown in liquid medium and fixed nitrogen according to the acetylene reduction assay. Their iden tification as Azospirillum lipoferum was based on biochemical tests an d hybridization with a 16S rRNA probe specific for this species and wa s confirmed by the detection of a 150-MDa plasmid.