Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize

Recent studies suggest a high diversity of diazotrophic bacteria in maize. However, none of these works have been based on a sufficient number of samples to provide reasonable quantitative estimates of diazotrophic bacterial diversity. Here we present the use of molecular tools and statistical inference to assess diazotrophic bacterial diversity within rhizosphere soils, roots and stems of field grown maize. DNA was isolated from the latter collected from six maize growing regions within the southern most state in Brazil, Rio Grande do Sul. Using conserved primers, nifH Cluster I gene fragments were amplified from each of the three zones, and the products cloned and sequenced. The majority of the sequences were classified within the Proteobacteria with the alpha-proteobacteria and beta-proteobacteria being the most abundant in the rhizosphere soil and stem samples. The gamma-proteobacteria were most abundant in rhizosphere soils, less so in roots, and least in the stem samples. According to three different diversity measures, the rhizosphere soil samples possessed greater diazotrophic bacterial diversity than the roots and stems of the maize plants. Only two genera, Azospirillum and Azotobacter, were found in virtually all samples at an abundance of over 1% of the total nifH sequences obtained. Other genera were largely restricted to soil (Methylocystis, Beijerinckia, Geobacter, Rhodovulum, Methylobacterium, Gluconacetobacter, Methylocella, and Delftia), roots (Dechloromonas), or stems (Methylosinus, Raoultella, and Rhizobium). Three genera, Herbaspirillum, Ideonella, and Klebsiella, appeared to dominate in the interior of the plant but were much rarer in soil.