Evaluating the genetic and functional diversity of major microorganisms in nitrogen-fixing sugar cane-microbe communities
Final Report Abstract
To date, sugarcane is the most important model for beneficial plant-associated biological nitrogen fixation in non-leguminous field crops. This process is poorly understood. For a better understanding of this nitrogen-fixing system as well as for its application and optimization, it is instrumental to identify the active diazotrophs providing nitrogen to the host. Up to now the responsible bacteria have been not identified yet. By using a nested RT-PCR strategy, where nifH was applied as marker for profiling diazotrophic bacteria with two sets of gene-specific, highly degenerate primers (nifH coverage >90%), distinct nifH-transcribing microbial communities were identified in shoots and roots of different sugarcane lines from Africa and America. NifH transcripts of the potential key nitrogen-fixing bacterium in roots from all lines, including one known to derive large quantities of N from BNF, could be assigned with a high degree of certainty to Rhizobium rosettiformans. NifH transcription of R. rosettiformans appeared to be abundant in all samples in spite of different fertilization regimes. This suggested that the composition of bacterial communities in sugarcane roots active in nitrogen fixation is quite stable. Besides in sugarcane, nifH transcription of this bacterium was probably prevalent also in Norway spruce. The prevalence of a distinct, globally distributed clade of nifH transcript sequences in sugarcane and spruce indicates a tight interaction between the corresponding nitrogen-fixing bacteria and their host. Consistent with the only other cultivation independent study which targeted nifH with broadrange primers in sugarcane stems we obtained evidence for the preponderance of nifH sequences homologous to those of Bradyrhizobium there. Also Fischer et al., (2012) found nifH sequences related to Bradyrhizobium and Rhizobium in their sugarcane line studied. The wide occurence of nifH expression by members of the order Rhizobiales in field-grown sugarcane (and spruce roots) is quite surprising, since a search of the EMBL database revealed that within this order nifH transcription in non-host environments was largely unknown. Since nodulation genes (nodA, nodC and nodD) could not be detected in R. rosettiformans, the anticipated interaction between R. rosettiformans and these plants is probably not dependent on nod genes, which is consistent with the absence of symbiotic structures in sugarcane and spruce. The cultivation independent broad-range detection of nifH transcribing microbial communities in sugarcane proved to be challenging. In course of this project a new method was developed which improves the low sensitivity and efficiency of broad-range PCR with highly degenerate oligonucleotide primers by LNA- modification of the RT-primer.
Publications
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2010. LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples. Environ. Microbiol. Rep. 2, 251–257
Burbano, C.S., Reinhold-Hurek, B., Hurek, T.
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Functional diversity of root associated diazotrophic bacteria in sugarcane microbe communities. 9th European Nitrogen Fixation in Geneva, Switzerland from 6-10 September 2010
Burbano et al.
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Widespread transcription of nifH genes by a novel Rhizobium species in sugarcane. 12th International Symposium on Biological Nitrogen Fixation (BNF) with Non-Legumes, Buzios, Rio de Janeiro, Brazil from 3-8 October 2010
T. Hurek et al.
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2011. Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field grown sugarcane plants and in Norway spruce. Environ. Microbiol. Rep. 3, 383–389
Burbano, C.S., Liu, Y., Rösner, K., Reis, V., Caballero-Mellado, J., Reinhold-Hurek, B., Hurek, T.
