Evaluating the genetic and functional diversity of major microorganisms in nitrogen-fixing sugar cane-microbe communities

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Despite the crucial importance of nitrogen availability on the functioning of ecosystems, information on important diazotrophs in many natural environments is still very poor, since most bacteria defy cultivation. Direct targeting of nifH mRNA is the tool of choice to identify primary N2-fixing organisms in habitats where major diazotrophs are inconspicious. Although sugar cane shows a high input from biological N2 fixation (BNF), current data based on cultivated diazotrophic bacteria from sugar cane plants cannot explain it. Furthermore, most active sites and diazotrophs are not well characterized without the bias of cultivation. Therefore, culture-independent methods will be applied. NifH mRNA levels are estimated in different parts of roots and shoots of cane varieties which are known to get large or almost negligible inputs by BNF. To obtain nifH sequences from the most active N2-fixing bacteria, nifH clone libraries are constructed and evaluated by comparative sequence analyses from plant tissues having the highest nifH expression levels. These data will be used for DNA/mRNA profiling with microarrays and DGGE analyses. These profiling studies aim to detect stable microbial communities which are very active in nitrogen fixation, and aim to assess the influence of different soil types and N-fertilization on them.