A limitation of this is that PCR amplification of genomic DNA is inherently biased by primer design [24


A limitation of this is that PCR amplification of genomic DNA is inherently biased by primer design 24,25 and generally only identifies the target organisms. Complex environments are inhabited by organisms from all domains of life. Eukaryotes, including fungi, protozoa, oomycetes and nematodes, are ubiquitous in soils and can be important plant pathogens or symbionts, whereas others are bacterial grazers. The archaea carry out important biochemical reactions, particularly in agricultural soils, such as ammonia oxidation 26 and methanogenesis 13. Viruses too are abundant and widespread and can affect the metabolism and population dynamics of their hosts 27. Microbes in a community interact with each other and the host plant 28, so it is important to capture as much of the diversity of a microbiome as possible. To do so requires the use of global analyses such as metagenomics, metatranscriptomics and metaproteomics, which allow simultaneous assessment and comparison of microbial populations across all domains of life. Metagenomics can reveal the functional potential of a microbiome (the abundance of genes involved in particular metabolic processes), whereas metatranscriptomics and metaproteomics provide snapshots of community-wide gene expression and protein abundance, respectively. Metatranscriptomics has revealed kingdom-level changes in the structure of crop-plant rhizosphere microbiomes 29. The relative abundance of eukaryotes in pea and oat rhizospheres was five-fold higher than in plant-free soil or the rhizosphere of modern hexaploid wheat. The pea rhizosphere in particular was highly enriched with fungi. Additional molecular techniques can complement such approaches. For example, stable isotope probing allows organisms metabolizing a particular labeled substrate to be identified 30. This has been used in studies of rhizosphere microbiomes where 13CO2 was fed to plants and fixed by photosynthesis, revealing that a subset of the microbial community actively metabolized plant-derived carbon 31,32. Combining these techniques with culture-based approaches should improve our understanding of plant-microbe interactions at the systems level. The rhizosphere environment The rhizosphere is the region of soil influenced by plant roots through rhizodeposition of exudates, mucilage and sloughed cells. Root exudates contain a variety of compounds, predominately organic acids and sugars, but also amino acids, fatty acids, vitamins, growth factors, hormones and antimicrobial compounds 33. Root exudates are key determinants of rhizosphere microbiome structure 34?37.
The composition of root exudates can vary between plant species and cultivars 38,39, and with plant age and developmental stage 40-42. Also, the microbiome influences root exudates, as axenically grown (sterile) plants have markedly different exudate compositions from those influenced by microbes. Some accessions of A. thaliana have been shown to have different root exudate compositions and correspondingly different rhizosphere bacterial communities 38, whereas the rhizosphere bacterial communities of other accessions have shown high similarity 43,44, although root exudates were not analyzed in the latter two studies. Root exudates are not the only component of rhizodeposition. The sloughing of root cells and the release of mucilage deposits a large amount of material into the rhizosphere, including plant cell wall polymers such as cellulose and pectin 45. Cellulose degradation is widespread among microbial residents of high-organic-matter soils 46,47. The decomposition of pectin releases methanol 10, which can be used as a carbon source by other microbes, and active metabolism of methanol in the rhizosphere has been observed 48. As well as providing a carbon source to rhizosphere microbes, plant roots also provide a structure on which microbes can attach. Supporting this is the observation of significant overlap between bacteria attaching to a root and to an inert wooden structure 44. Studies of rhizosphere microbiomes have revealed remarkably similar distributions of microbial phyla 29,43,44. Differences between plant cultivars become apparent when comparing microbial species and strains 49,50. The Proteobacteria usually dominate samples, particularly those of the ? and ? classes. Other major groups include Actinobacteria, Firmicutes, Bacteroidetes, Planctomycetes, Verrucomicrobia and Acidobacteria. Of particular interest in the rhizosphere are plant growth promoting rhizobacteria, which act through a variety of mechanisms 14. Nitrogen-fixing bacteria, including those that are free-living (such as Azotobacter Turner et al. Genome Biology 2013, and symbiotic (such as root-nodulating Rhizobium spp.), provide a source of fixed nitrogen for the plant, and many bacteria can solubilize phosphorous-containing minerals, increasing its bioavailability. Microbial manipulation of plant hormones, particularly auxins, gibberellins and ethylene, can also lead to growth promotion or stress tolerance. Many plant-growth-promoting rhizobacteria act antagonistically towards plant pathogens by producing antimicrobials or by interfering with virulence factors via effectors delivered by type 3 secretion systems (T3SSs) 51. Actinomycetes, in particular, are known to produce a wide array of compounds with antibacterial, antifungal, antiviral, nematicidal and insecticidal properties. They are often found as one of the most abundant bacterial classes in soil and rhizospheres, and are notably enriched in endophytic communities.