Molecular microbial ecology approaches applied to diazotrophs associated with nonlegumes

Species- and genus-specific oligonucleotide probes, as well as monoclonal antibodies and polyclonal antisera, were used to study the taxonomic identity and ecology of diazotrophic bacteria associated with non-legumes. Species-specific oligonucleotide probes directed against variable regions of the ribosomal rRNAs of five Azospirillum spp., Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans. Acetobacter diazotrophicus and new Burkholderia spp. isolates, as well as genus-specific Azospirillum and Azoarcus probes, have been developed and applied to prove the identification of new isolates. In addition, different approaches to identify bacteria on the basis of restriction fragment length polymorphism (RFLP) and PCR-fingerprinting techniques allow the characterization of the diversity of populations even at strain level. On the other hand, strain-specific monoclonal antibodies directed against different Azospirillum brasilense strains enabled us to localize and quantify inoculant bacteria in different parts of the rhizosphere. In addition, antibodies were used successfully to enrich and (re)isolate specific bacteria from different sources The application of confocal laser scanning microscopy offers high resolution images of diazotrophic bacteria specifically labelled with fluorescent probes in plant tissues.