Comparative molecular analysis of Herbaspirillum strains by RAPD, RFLP, and 16S rDNA sequencing

Herbaspirillum spp. are endophytic diazotrophic bacteria associated with important agricultural crops. In this work, we analyzed six strains of H. seropedicae (Z78, M2, ZA69, ZA95, Z152, and Z67) and one strain of H. rubrisubalbicans (M4) by restriction fragment length polymorphism (RFLP) using HindIII or DraI restriction endonucleases, random amplified polymorphic DNA (RAPD), and partial sequencing of 16S rDNA. The results of these analyses ascribed the strains studied to three distinct groups: group I, consisting of M2 and M4; group II, of ZA69; and group III, of ZA95, Z78, Z67, and Z152. RAPD fingerprinting showed a higher variability than the other methods, and each strain had a unique electrophoretic pattern with five of the six primers used. Interestingly, H. seropedicae M2 was found by all analyses to be genetically very close to H. rubrisubalbicans M4. Our results show that RAPD can distinguish between all Herbaspirillum strains tested.

Herbaspirillum-like bacteria in banana plants

Diazotrophic bacteria isolated from banana plants were characterized by morphological and physiological aspects. Three different groups of these plant-bacteria could be established. Two of them showed similarity to species of the Herbaspirillum genus. The third one was different because used only a few carbon substrates and produced water diffusible compounds that fluoresced under UV light. All three bacterial groups were thin rods with mono or bipolar flagella, presented negative reaction in Gram stain, showed catalase activity, were able to reduce nitrate and grew better in semi-solid JNFb medium at 31§C. The nitrogenase activity was detected in semi-solid N-free JNFb medium and expressed higher values when pH ranged from 6.5 to 7.0 (groups I and II) and 6.0 to 6.5 (group III). The diazotrophs isolated from banana plants were distinct from species of Herbaspirillum previously identified in gramineous plants.