Metagenomic analysis of nitrogen fixing bacterial populations from soils with different agricultural management from the humid pampa region

BERGOTTINI VM; COLLAVINO MM; GRASSO DH; AGUILAR O.M

Córdoba

Congreso; VI Congreso argentino de microbiología general-SAMIGE; 2009

SAMIGE

In this study we investigated the nitrogen fixing populations of bacteria from soils with different agricultural management from The Humid Pampa region by metagenomic analysis. Soil samples were collected at two field sites located in Monte Buey (Córdoba) and Viale (Entre Ríos). At each location, we took samples from soils corresponding to different agricultural management such as 1) accepted good agricultural practices with records of no-tillage management within the aim to accomplish sustainable agriculture such as crop rotation and nutrient reposition, 2) contrarily, mono-cropping without reposition, which is definitely unsustainable, and 3) undisturbed natural environment, as reference soil . In the beginning of this study, we tested two degenerated pair of primers, Zf/Zr and 19F/407R (Zehr et al., 1989; Ueda et al., 1995), to amplify the nifH gene of diverse representative nitrogen fixing bacteria. Both pair of primers resulted in successful amplification. Next, DNA was directly extracted from the soils and used as template to amplify nifH sequences using the nifH primers. In order to reveal diversity among strains and soils, RFLP assays was performed. Several endonucleases were assessed such as HaeIII, Sau3AI and MnlI which resulted in the best profiles to discriminate nifH diversity, resulting in differences in strain at the species level. The representative N2-fixing strains were all discriminated by RFLP, indicating that the technique was suitable to study diversity of environmental samples. Analysis of soil provided a specific profile for each DNA sample. In particular, the restriction patterns obtained from soils samples under the different agricultural management showed distinct restriction patterns in Monte Buey and Viale. These preliminary results confirm that agriculture practices may affect the N2-fixing community in soils from sites with very different edafic conditions. Considering these results, we approached construction of DNA libraries with the amplified nifH fragments of soils samples to determine microbial structure by DNA sequencing clones and use data to perform a phylogenetic analysis to identify some of the N2-fixing bacteria predominating in each sample. A comparative and limited comparison of some of our sequences will be shown. Our results indicated the capacity of our analysis to reveal differences in the structure of nitrogen fixing population in soils that underwent different agricultural practices.