202-1Identification and Biological Nitrogen Fixation of Bacteria in the Phyllosphere of the Brazilian Atlantic Forest
Autores:Alice Cassetari (ESALQ - Escola Superior de Agricultura Luiz de Queiroz) ; Marcio Rodrigues Lambais (ESALQ - Escola Superior de Agricultura Luiz de Queiroz)


Brazil has one of the greatest biodiversity of the species on the planet, and the Atlantic Forest is one of the major "hotspots" of diversity for prospecting new microbial species and enzymes. The process of biological nitrogen fixation (BNF) is the main form of N input into several ecosystems and it is mediated by several diazotrophic microorganisms. Previous work on the microbial communities in the Brazilian Atlantic Forest has shown high abundance of epiphytic diazothrophic bacteria on bamboo leaves and litter, which could be used for biotechnological purposes. The aim of this study was to quantify the BNF and identify the diazotrophic species associated with Atlantic Forest bamboo (Merostachys neesii) leaves. Leaf samples from bamboo and litter, were collected at “Serra do Mar State Park”, São Paulo, Brazil and diazotrophic bacteria were isolated in nitrogen-free semi-solid and solid JNFb medium. Nitrogenase activities of the bacterial strains were estimated using the acetylene reduction assay (ARA). The amount of ethylene formed at 28°C in 1 h was determined by gas chromatography. Protein concentration in microbial biomass was also determined. Diazotrophic strains (ARA-positive) were identified based on 16S rRNA gene sequences. 100 strains were isolated, 50 from leaves and 50 from litter. In vitro nitrogenase activity in the different strais ranged from 4 to 41 nmol C2H4 h-1 mg-1 protein. Taxonomic affiliation of all ARA positive strains, based on 16S rRNA gene sequences, revealed the higher abundance of Gammaproteobacteria (93%), whereas Betaproteobacteria (7%) was less abundant. Our data showed that some of the bamboo associated diazotrophic strains are highly efficient in fixing N, as compared to other known bacteria, and could be used for biotechnological purposes.

Palavras-chave:  Biological nitrogen fixation, Acetylene reduction assay, Phyllosphere, Atlantic Forest,, Diazotrophs