Land plants interact with microbes primarily at roots. Despite the importance of root microbial communities for health and nutrient uptake, the current understanding of the complex plant-microbe interactions in the rhizosphere is still in its infancy. Roots provide different microhabitats at the soil-root interface: rhizosphere soil, rhizoplane, and endorhizosphere. We discuss technical aspects of their differentiation that are relevant for the functional analysis of their different microbiomes, and we assess PCR (polymerase chain reaction)-based methods to analyze plant-associated bacterial communities. Development of novel primers will allow a less biased and more quantitative view of these global hotspots of microbial activity. Based on comparison of microbiome data for the different root-soil compartments and on knowledge of bacterial functions, a three-step enrichment model for shifts in community structure from bulk soil toward roots is presented. To unravel how plants shape their microbiome, a major research field is likely to be the coupling of reductionist and molecular ecological approaches, particularly for specific plant genotypes and mutants, to clarify causal relationships in complex root communities.

Although some sugarcane cultivars may benefit substantially from biological nitrogen fixation (BNF), the responsible bacteria have been not identified yet. Here, we examined the active diazotrophic bacterial community in sugarcane roots from Africa and America by reverse transcription (RT)-PCR using broad-range nifH-specific primers. Denaturing gradient gel electrophoresis (DGGE) profiles obtained from sugarcane showed a low diversity at all sample locations with one phylotype amounting up to 100% of the nifH transcripts. This major phylotype has 93.9-99.6% DNA identity to the partial nifH sequence from a strain affiliated with Rhizobium rosettiformans. In addition, nifH transcripts of this phylotype were also detected in spruce roots sampled in Germany, where they made up 91% of nifH transcripts detected. In contrast, in control soil or shoot samples two distinct nifH transcript sequences distantly related to nifH from Sulfurospirillum multivorans or Bradyrhizobium elkanii, respectively, were predominant. These results suggest that R. rosettiformans is involved in root-associated nitrogen fixation with sugarcane and spruce, plants that do not form root-nodule symbioses.

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Colophospermum mopane is an indigenous legume tree that grows in Southern Africa and is one of the predominant trees of the woodland vegetation. In order to increase knowledge about its ecology, especially how C. mopane thrives in the nitrogen-poor soils of the region, we analyzed the root-associated bacteria to assess the active diazotrophic diversity and total microbial diversity by culture-dependent and independent techniques. Root nodules were not detected but in some samples the lateral roots showed an outgrowth-like protuberance, that were not likely to have functions related to legume root nodules. The bacterial isolates recovered were related to Actinobacteria, Firmicutes and Proteobacteria. The total microbial diversity was dominated by Actinobacteria-related phylotypes, while the active diazotrophic diversity showed that the majority of the sequences were related to the order Rhizobiales but also to Spirochaetes, Firmicutes, Bacteroidetes and Deltaproteobacteria. Several isolates showed characteristics of plant growth-promoting bacteria. These findings increase the spectrum of possible phylotypes that can be found in legume trees that are typically nodulated by Alpha- and Betaproteobacteria, and reveal for the first time a surprising diversity of nitrogen-fixing bacteria active in legume tree roots.

In order to study the active diazotrophic bacterial community and to capture the majority of its individuals in environmental samples, strategies improving gene detection by increasing sensitivity and efficiency of PCR reactions are highly desirable. Since LNA (locked nucleic acids) modifications might alleviate a low sensitivity and specificity often limiting PCR reactions utilizing degenerate primers, the effect of LNA substituted primers on the detection of nifH transcripts in roots of rice and sugar cane by direct reverse transcription polymerase chain reaction (RT-PCR) was studied. The LNA substitution of the RT primer increased the sensitivity of the RT-PCR up to 26-fold, whereas LNA substitution of the PCR primers decreased specificity. Terminal restriction fragment length polymorphism (T-RFLP) analysis of RT-PCR products showed that LNA substitutions in the RT-primer did not change the pattern of nifH cDNA phylotypes. The use of the LNA-substituted RT-primer allowed the detection of nifH transcripts in sugar cane, where DNA primers alone failed to produce RT-PCR products. These results suggest that similar improvements to PCR detection of nucleic acids can be expected for other environmental samples and genes likewise, when LNA-substituted primers are used.

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