Degradation of transgenic Bt-rice straw incorporated with two different paddy soils.

Transgenic Bt-rice is rice that has been genetically modified to produce insecticidal proteins (Cry1Ab/Ac) within the plant. Rice straw is incorporated into paddy soils after harvest for fertilization or to improve the soil structure. The incorporation of straw from transgenic Bt-rice may pose risks to the paddy soil system. The decomposition of Bt-rice straw and degradation of Cry1Ab/Ac proteins from the straw were investigated under laboratory conditions. In addition, effects of the incorporation with chopped rice straw on microbial communities in differently textured paddy soils were studied. The results indicated that the incorporation of straw from transgenic Bt-rice might have a slight influence on soil respiration and CH4 emissions in two paddy soils, i.e. the Silt Loam soil and the Silty Clay soil. Differences were also observed in the cumulative emissions of CO2 between the two amended paddy soils in addition to the well-known increase in emissions of both CO2 and CH4 due to straw incorporation. The Cry1Ab/Ac proteins from straw of transgenic Bt-rice were degraded in paddy soils. The rate of decline in the concentration of Cry1Ab/Ac proteins was different in the two soils. After 29 d of incubation, 61% and 42% of initial Cry1Ab/Ac proteins were detected in the silt loam and silty clay, respectively. As a result of the presence of the rice straw, the abundance of bacteria, archaea, and total cells were increased in two soils. The numbers of bacteria and total cells were 6.4% and 11.5% higher in the silt loam amended with straw of Bt-rice than non-Bt-rice, respectively. The silty clay displayed a similar trend as the silt loam.

Rates and Microbial Players of Iron-Driven Anaerobic Oxidation of Methane in Methanic Marine Sediments.

The flux of methane, a potent greenhouse gas, from the seabed is largely controlled by anaerobic oxidation of methane (AOM) coupled to sulfate reduction (S-AOM) in the sulfate methane transition (SMT). S-AOM is estimated to oxidize 90% of the methane produced in marine sediments and is mediated by a consortium of anaerobic methanotrophic archaea (ANME) and sulfate reducing bacteria. An additional methane sink, i.e., iron oxide coupled AOM (Fe-AOM), has been suggested to be active in the methanic zone of marine sediments. Geochemical signatures below the SMT such as high dissolved iron, low to undetectable sulfate and high methane concentrations, together with the presence of iron oxides are taken as prerequisites for this process. So far, Fe-AOM has neither been proven in marine sediments nor have the governing key microorganisms been identified. Here, using a multidisciplinary approach, we show that Fe-AOM occurs in iron oxide-rich methanic sediments of the Helgoland Mud Area (North Sea). When sulfate reduction was inhibited, different iron oxides facilitated AOM in long-term sediment slurry incubations but manganese oxide did not. Especially magnetite triggered substantial Fe-AOM activity and caused an enrichment of ANME-2a archaea. Methane oxidation rates of 0.095 ± 0.03 nmol cm-3 d-1 attributable to Fe-AOM were obtained in short-term radiotracer experiments. The decoupling of AOM from sulfate reduction in the methanic zone further corroborated that AOM was iron oxide-driven below the SMT. Thus, our findings prove that Fe-AOM occurs in methanic marine sediments containing mineral-bound ferric iron and is a previously overlooked but likely important component in the global methane budget. This process has the potential to sustain microbial life in the deep biosphere.

Correlative Imaging Reveals Holistic View of Soil Microenvironments.

The microenvironmental conditions in soil exert a major control on many ecosystem functions of soil. Their investigation in intact soil samples is impaired by methodological challenges in the joint investigation of structural heterogeneity that defines pathways for matter fluxes and biogeochemical heterogeneity that governs reaction patterns and microhabitats. Here we demonstrate how these challenges can be overcome with a novel protocol for correlative imaging based on image registration to combine three-dimensional microstructure analysis of X-ray tomography data with biogeochemical microscopic data of various modalities and scales (light microscopy, fluorescence microscopy, electron microscopy, secondary ion mass spectrometry). Correlative imaging of a microcosm study shows that the majority (75%) of bacteria are located in mesopores (<10 µm). Furthermore, they have a preference to forage near macropore surfaces and near fresh particulate organic matter. Ignoring the structural complexity coming from the third dimension is justified for metrics based on size and distances but leads to a substantial bias for metrics based on continuity. This versatile combination of imaging modalities with freely available software and protocols may open up completely new avenues for the investigation of many important biogeochemical and physical processes in structured soils.

Emergent Properties of Microbial Activity in Heterogeneous Soil Microenvironments: Different Research Approaches Are Slowly Converging, Yet Major Challenges Remain.

Over the last 60 years, soil microbiologists have accumulated a wealth of experimental data showing that the bulk, macroscopic parameters (e.g., granulometry, pH, soil organic matter, and biomass contents) commonly used to characterize soils provide insufficient information to describe quantitatively the activity of soil microorganisms and some of its outcomes, like the emission of greenhouse gasses. Clearly, new, more appropriate macroscopic parameters are needed, which reflect better the spatial heterogeneity of soils at the microscale (i.e., the pore scale) that is commensurate with the habitat of many microorganisms. For a long time, spectroscopic and microscopic tools were lacking to quantify processes at that scale, but major technological advances over the last 15 years have made suitable equipment available to researchers. In this context, the objective of the present article is to review progress achieved to date in the significant research program that has ensued. This program can be rationalized as a sequence of steps, namely the quantification and modeling of the physical-, (bio)chemical-, and microbiological properties of soils, the integration of these different perspectives into a unified theory, its upscaling to the macroscopic scale, and, eventually, the development of new approaches to measure macroscopic soil characteristics. At this stage, significant progress has been achieved on the physical front, and to a lesser extent on the (bio)chemical one as well, both in terms of experiments and modeling. With regard to the microbial aspects, although a lot of work has been devoted to the modeling of bacterial and fungal activity in soils at the pore scale, the appropriateness of model assumptions cannot be readily assessed because of the scarcity of relevant experimental data. For significant progress to be made, it is crucial to make sure that research on the microbial components of soil systems does not keep lagging behind the work on the physical and (bio)chemical characteristics. Concerning the subsequent steps in the program, very little integration of the various disciplinary perspectives has occurred so far, and, as a result, researchers have not yet been able to tackle the scaling up to the macroscopic level. Many challenges, some of them daunting, remain on the path ahead. Fortunately, a number of these challenges may be resolved by brand new measuring equipment that will become commercially available in the very near future.

Microbial life on a sand grain: from bulk sediment to single grains.

Globally, marine surface sediments constitute a habitat for estimated 1.7 × 1028 prokaryotes. For benthic microbial community analysis, usually, several grams of sediment are processed. In this study, we made the step from bulk sediments to single sand grains to address the microbial community directly in its micro-habitat: the individual bacterial diversity on 17 sand grains was analyzed by 16S ribosomal RNA gene sequencing and visualized on sand grains using catalyzed reporter deposition fluorescence in situ hybridization. In all, 104-105 cells were present on grains from 202 to 635 µm diameter. Colonization was patchy, with exposed areas largely devoid of any epi-growth (mean cell-cell distance 4.5±5.9 µm) and protected areas more densely populated (0.5±0.7 µm). Mean cell-cell distances were 100-fold shorter compared with the water column. In general, growth occurred in monolayers. Each sand grain harbors a highly diverse bacterial community as shown by several thousand species-level operational taxonomic units (OTU)0.97. Only 4-8 single grains are needed to cover 50% of OTU0.97 richness found in bulk sediment. Although bacterial communities differed between sand grains, a core community accounting for >50% of all cells was present on each sand grain. The communities between sediment grains are more similar than between soil macroaggregates.

Evaluation of Strategies to Separate Root-Associated Microbial Communities: A Crucial Choice in Rhizobiome Research.

Plants shape distinct, species-specific microbiomes in their rhizospheres. A main premise for evaluating microbial communities associated with root-soil compartments is their successful separation into the rhizosphere (soil-root interface), the rhizoplane (root surface), and the endosphere (inside roots). We evaluated different approaches (washing, sonication, and bleaching) regarding their efficiency to separate microbial cells associated with different root compartments of soil-grown rice using fluorescence microscopy and community fingerprinting of 16S rRNA genes. Vigorous washing detached 45% of the rhizoplane population compared to untreated roots. Additional sonication reduced rhizoplane-attached microorganisms by up to 78% but caused various degrees of root tissue destruction at all sonication intensities tested. Treatment with sodium hypochlorite almost completely (98%) removed rhizoplane-associated microbial cells. Community fingerprinting revealed that microbial communities obtained from untreated, washed, and sonicated roots were not statistically distinguishable. Hypochlorite-treated roots harbored communities significantly different from all other samples, likely representing true endospheric populations. Applying these procedures to other root samples (bean and clover) revealed that treatment efficiencies were strongly affected by root morphological parameters such as root hair density and rigidity of epidermis. Our findings suggest that a careful evaluation of separation strategies prior to molecular community analysis is indispensable, especially when endophytes are the subject of interest.

Detection and quantification of native microbial populations on soil-grown rice roots by catalyzed reporter deposition-fluorescence in situ hybridization.

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) was applied to detect microbial cells on the rhizoplane of wetland rice (Oryza sativa L.). Fluorescent signals of high intensity and specificity allowed for a reliable quantification of selected microbial phyla. Absolute cell numbers of archaea and bacteria were observed to be highest at flowering stage of rice plant development (P < 0.05) showing values of 1.32 and 6.26 × 10(4)  cells mm(-2) rhizoplane, respectively. Highest colonization densities shifted from the root tip toward more mature regions with increasing plant age. Significant differences between cell numbers observed within a short distance (0-15 mm) indicated irregular distribution patterns of microbiota. Root tips, elongation zones, and openings at the base of lateral roots represented preferential areas for microbial colonization, which were often covered with iron coatings and densely colonized with potential iron-oxidizing Betaproteobacteria (59% of bacteria). Methanogenic archaea were abundant on the rhizoplane (up to 0.96 × 10(3)  cells mm(-2) rhizoplane), and the decline of their relative abundance with plant age was also found in the associated rhizosphere soil. Cell numbers of methanotrophic bacteria significantly increased at flowering (6.38 × 10(3)  cells mm(-2) rhizoplane; P < 0.05), indicating their stimulation by root-derived substrates which was less pronounced in the rhizosphere soil.

Evaluation of tyramide solutions for an improved detection and enumeration of single microbial cells in soil by CARD-FISH.

Several tyramide solutions were evaluated for the application of fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) in soil. Fluorescently labeled tyramide solutions were synthesized and compared to commercially available tyramides for the detection and quantification of single microbial cells. Among the tyramide solutions tested, a succinimidyl ester of fluorescein diluted with dimethylformamide containing iodophenolboronic acid (SFX-DMF-IPBA) gave the best results, yielding highly reproducible cell numbers and detection rates of archaea and bacteria along with negligible non-specific signals. The addition of organic and inorganic compounds to the amplification reagents had a positive impact on the detection of prokaryotic cells. The applicability of SFX-DMF-IPBA for CARD-FISH in soil was further evaluated in soils of different texture. Cell numbers and detection rates of bacteria and archaea remained on a high level independent of the clay or organic matter content. Based on the results obtained in this study, the choice of the tyramide solution used for CARD-FISH has a significant influence on the detection and quantification of single microbial cells in soil. Therefore, we suggest the application of the presented tyramide signal amplification procedure including the tyramide solution SFX-DMF-IPBA for comprehensive CARD-FISH studies investigating the abundance and spatial distribution of soil microorganisms.

Defining the core Arabidopsis thaliana root microbiome.

Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surrounding the root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation. Colonization of the root occurs despite a sophisticated plant immune system, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plant-microbe interactions derived from complex soil communities.

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota.

The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. By contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on other cues from metabolically active host cells.

Gold-FISH: a new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy.

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold-streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD-FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells. The gold-FISH protocol was developed and optimised using pure cultures and environmental samples, such as rice roots and marine sediments. The combination of epifluorescence and scanning electron microscopy provides a promising tool for investigating microorganisms at levels of high resolution. Correlative characterisation of physicochemical properties by EDS will allow for the analysis of microbe-surface interactions.