Routes to roots: direct evidence of water transport by arbuscular mycorrhizal fungi to host plants.

Arbuscular mycorrhizal fungi (AMF) can help mitigate plant responses to water stress, but it is unclear whether AMF do so by indirect mechanisms, direct water transport to roots, or a combination of the two. Here, we investigated if and how the AMF Rhizophagus intraradices transported water to the host plant Avena barbata, wild oat. We used two-compartment microcosms, isotopically labeled water, and a fluorescent dye to directly track and quantify water transport by AMF across an air gap to host plants. Plants grown with AMF that had access to a physically separated compartment containing 18 O-labeled water transpired almost twice as much as plants with AMF excluded from that compartment. Using an isotopic mixing model, we estimated that water transported by AMF across the air gap accounted for 34.6% of the water transpired by host plants. In addition, a fluorescent dye indicated that hyphae were able to transport some water via an extracytoplasmic pathway. Our study provides direct evidence that AMF can act as extensions of the root system along the soil-plant-air continuum of water movement, with plant transpiration driving water flow along hyphae outside of the hyphal cell membrane.

Stable isotope informed genome-resolved metagenomics reveals that Saccharibacteria utilize microbially-processed plant-derived carbon.

BACKGROUND: The transformation of plant photosynthate into soil organic carbon and its recycling to CO2 by soil microorganisms is one of the central components of the terrestrial carbon cycle. There are currently large knowledge gaps related to which soil-associated microorganisms take up plant carbon in the rhizosphere and the fate of that carbon. RESULTS: We conducted an experiment in which common wild oats (Avena fatua) were grown in a 13CO2 atmosphere and the rhizosphere and non-rhizosphere soil was sampled for genomic analyses. Density gradient centrifugation of DNA extracted from soil samples enabled distinction of microbes that did and did not incorporate the 13C into their DNA. A 1.45-Mbp genome of a Saccharibacteria (TM7) was identified and, despite the microbial complexity of rhizosphere soil, curated to completion. The genome lacks many biosynthetic pathways, including genes required to synthesize DNA de novo. Rather, it requires externally derived nucleotides for DNA and RNA synthesis. Given this, we conclude that rhizosphere-associated Saccharibacteria recycle DNA from bacteria that live off plant exudates and/or phage that acquired 13C because they preyed upon these bacteria and/or directly from the labeled plant DNA. Isotopic labeling indicates that the population was replicating during the 6-week period of plant growth. Interestingly, the genome is ~ 30% larger than other complete Saccharibacteria genomes from non-soil environments, largely due to more genes for complex carbon utilization and amino acid metabolism. Given the ability to degrade cellulose, hemicellulose, pectin, starch, and 1,3-ß-glucan, we predict that this Saccharibacteria generates energy by fermentation of soil necromass and plant root exudates to acetate and lactate. The genome also encodes a linear electron transport chain featuring a terminal oxidase, suggesting that this Saccharibacteria may respire aerobically. The genome encodes a hydrolase that could breakdown salicylic acid, a plant defense signaling molecule, and genes to interconvert a variety of isoprenoids, including the plant hormone zeatin. CONCLUSIONS: Rhizosphere Saccharibacteria likely depend on other bacteria for basic cellular building blocks. We propose that isotopically labeled CO2 is incorporated into plant-derived carbon and then into the DNA of rhizosphere organisms capable of nucleotide synthesis, and the nucleotides are recycled into Saccharibacterial genomes.

Successional Trajectories of Rhizosphere Bacterial Communities over Consecutive Seasons.

UNLABELLED: It is well known that rhizosphere microbiomes differ from those of surrounding soil, and yet we know little about how these root-associated microbial communities change through the growing season and between seasons. We analyzed the response of soil bacteria to roots of the common annual grass Avena fatua over two growing seasons using high-throughput sequencing of 16S rRNA genes. Over the two periods of growth, the rhizosphere bacterial communities followed consistent successional patterns as plants grew, although the starting communities were distinct. Succession in the rhizosphere was characterized by a significant decrease in both taxonomic and phylogenetic diversity relative to background soil communities, driven by reductions in both richness and evenness of the bacterial communities. Plant roots selectively stimulated the relative abundance of Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes but reduced the abundance of Acidobacteria, Actinobacteria, and Firmicutes. Taxa that increased in relative abundance in the rhizosphere soil displayed phylogenetic clustering, suggesting some conservation and an evolutionary basis for the response of complex soil bacterial communities to the presence of plant roots. The reproducibility of rhizosphere succession and the apparent phylogenetic conservation of rhizosphere competence traits suggest adaptation of the indigenous bacterial community to this common grass over the many decades of its presence. IMPORTANCE: We document the successional patterns of rhizosphere bacterial communities associated with a "wild" annual grass, Avena fatua, which is commonly a dominant plant in Mediterranean-type annual grasslands around the world; the plant was grown in its grassland soil. Most studies documenting rhizosphere microbiomes address "domesticated" plants growing in soils to which they are introduced. Rhizosphere bacterial communities exhibited a pattern of temporal succession that was consistent and repeatable over two growing seasons. There are few studies assessing the reproducibility over multiple seasons. Through the growing season, the rhizosphere community became progressively less diverse, likely reflecting root homogenization of soil microniches. Phylogenetic clustering of the rhizosphere dynamic taxa suggests evolutionary adaptation to Avena roots. The reproducibility of rhizosphere succession and the apparent phylogenetic conservation of rhizosphere competence traits suggest adaptation of the indigenous bacterial community to this common grass over the many decades of its presence.

Compartmentalized microbial composition, oxygen gradients and nitrogen fixation in the gut of Odontotaenius disjunctus.

Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of insects have evolved to take advantage of. These insects are ecologically important and represent useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches combined with microelectrode measurements of oxygen, we have characterized the gut microbiome and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United States. We hypothesized that morphological and physiological differences among gut regions would correspond to distinct microbial populations and activities. In fact, significantly different communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG. Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the AHG, and also the sole region where methanogenic Archaea were detected. Although each gut region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen concentration in all regions. Nitrogen fixation was confirmed by (15)N2 incorporation, and nitrogenase gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most abundant transcript as related to Ni-Fe nitrogenase of a Bacteroidetes species, Paludibacter propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism to occur within the same regions in close proximity. We provide evidence for the microbial fixation of N2 that is important for this beetle to subsist on woody biomass.

Nitrogen deposition, plant carbon allocation, and soil microbes: changing interactions due to enrichment.

PREMISE OF THE STUDY: Nitrogen (N) inputs to the terrestrial environment have doubled worldwide during the past century. N negatively impacts plant diversity, but it is unknown why some species are more susceptible than others. While it is often assumed that competition drives species decline, N enrichment also strongly affects soil microbial communities. Can these changes affect plant-microbe interactions in ways that differentially influence success of plant species? Furthermore, can altered plant-microbe interactions lead to carbon (C) limitation in plants? METHODS: We focused on a species that increases (Deschampsia cespitosa) and one that decreases (Geum rossii) in abundance in N-fertilized plots in alpine tundra. We measured soil microbes using phospholipid fatty acids, and C limitation and transfer using a (13)C tracer experiment, C:N ratios, nonstructural carbohydrates, and leaf preformation. KEY RESULTS: While N profoundly influenced microbial communities, this change occurred similarly in association with both plant species. N addition did not alter total C allocation to microbes in either species, but it changed patterns of microbial C acquisition more in Geum, specifically in gram-negative bacteria. Geum showed evidence of C limitation: it allocated less C to storage organs, had lower C:N and carbohydrate stores, and fewer preformed leaves in N plots. CONCLUSIONS: Carbon limitation may explain why some species decline with N enrichment, and the decline may be due to physiological responses of plants to N rather than to altered plant-microbe interactions. Global change will alter many processes important in structuring plant communities; noncompetitive mechanisms of species decline may be more widespread than previously thought.

An arbuscular mycorrhizal fungus significantly modifies the soil bacterial community and nitrogen cycling during litter decomposition.

Arbuscular mycorrhizal fungi (AMF) perform an important ecosystem service by improving plant nutrient capture from soil, yet little is known about how AMF influence soil microbial communities during nutrient uptake. We tested whether an AMF modifies the soil microbial community and nitrogen cycling during litter decomposition. A two-chamber microcosm system was employed to create a root-free soil environment to control AMF access to (13) C- and (15) N-labelled root litter. Using a 16S rRNA gene microarray, we documented that approximately 10% of the bacterial community responded to the AMF, Glomus hoi. Taxa from the Firmicutes responded positively to AMF, while taxa from the Actinobacteria and Comamonadaceae responded negatively to AMF. Phylogenetic analyses indicate that AMF may influence bacterial community assembly processes. Using nanometre-scale secondary ion mass spectrometry (NanoSIMS) we visualized the location of AMF-transported (13) C and (15) N in plant roots. Bulk isotope ratio mass spectrometry revealed that the AMF exported 4.9% of the litter (15) N to the host plant (Plantago lanceolata L.), and litter-derived (15) N was preferentially exported relative to litter-derived (13) C. Our results suggest that the AMF primarily took up N in the inorganic form, and N export is one mechanism by which AMF could modify the soil microbial community and decomposition processes.

Interactions between an arbuscular mycorrhizal fungus and a soil microbial community mediating litter decomposition.

We investigated arbuscular mycorrhizal fungi (AMF) alteration of microbial mediation of litter decomposition. AMF (Glomus hoi) were either allowed access to or excluded from Plantago lanceolata L. root litter embedded in soil; litter was labeled with either (13) C only or (13) C and (15) N. Plant N uptake was significantly increased if AMF accessed the litter, and (15) N analysis of the plant material indicated that 2-3% of plant N originated from litter. Succession of the soil community mediating decomposition was assessed by phospholipid fatty acids (PLFA) combined with (13) C-PLFA. During the first 21 days of decomposition, saprotrophic fungi and Gram-negative bacteria were the dominant consumers of litter C. As decomposition progressed however, (13) C content of the fungal biomarkers declined substantially, and Gram-negative and Gram-positive bacteria became the primary reservoirs of labeled litter C. The putative PLFA marker for AMF (16:1ω5c) originated primarily from non-AMF sources. In AMF-invaded root litter, Gram-negative, Gram-positive, and 16:1ω5c markers became less (13) C-enriched relative to their counterparts in non-AMF-invaded microcosms during active decomposition. These patterns of (13) C: (12) C enrichment may result from AMF supply of (12) C from the plant to the decomposing soil microbial community; such C inputs could alter the microbial mediation of litter decomposition.

Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska.

Nitrification and denitrification processes are crucial to plant nutrient availability, eutrophication and greenhouse gas production both locally and globally. Unravelling the major environmental predictors for nitrification and denitrification is thus pivotal in order to understand and model environmental nitrogen (N) cycling. Here, we sampled five plant community types characteristic of interior Alaska, including black spruce, bog birch, tussock grass and two fens. We assessed abundance of functional genes affiliated with nitrification (bacterial and archaeal amoA) and denitrification (nirK/S and nosZ) using qPCR, soil characteristics, potential nitrification and denitrification rates (PNR and PDR) and gross mineralization rates. The main chemical and biological predictors for PNR and PDR were assigned through path analysis. The potential N cycling rates varied dramatically between sites, from some of the highest (in fens) to some of the lowest (in black spruce) measured globally. Based on path analysis, functional gene abundances were the most important variables to predict potential rates. PNR was best explained by bacterial amoA gene abundance followed by ammonium content, whereas PDR was best explained directly by nosZ gene abundance and indirectly by nirK/S gene abundance and nitrate. Hence, functional gene abundance is a valuable index that integrates recent environmental history and recent process activity, and therefore is a good predictor of potential rates. The results of this study contribute to our understanding of the relative importance of different biological and chemical factors in driving the potential for nitrification and denitrification across terrestrial ecosystems.

Microbial community response to addition of polylactate compounds to stimulate hexavalent chromium reduction in groundwater.

To evaluate the efficacy of bioimmobilization of Cr(VI) in groundwater at the Department of Energy Hanford site, we conducted a series of microcosm experiments using a range of commercial electron donors with varying degrees of lactate polymerization (polylactate). These experiments were conducted using Hanford Formation sediments (coarse sand and gravel) immersed in Hanford groundwater, which were amended with Cr(VI) and several types of lactate-based electron donors (Hydrogen Release Compound, HRC; primer-HRC, pHRC; extended release HRC) and the polylactate-cysteine form (Metal Remediation Compound, MRC). The results showed that polylactate compounds stimulated an increase in bacterial biomass and activity to a greater extent than sodium lactate when applied at equivalent carbon concentrations. At the same time, concentrations of headspace hydrogen and methane increased and correlated with changes in the microbial community structure. Enrichment of Pseudomonas spp. occurred with all lactate additions, and enrichment of sulfate-reducing Desulfosporosinus spp. occurred with almost complete sulfate reduction. The results of these experiments demonstrate that amendment with the pHRC and MRC forms result in effective removal of Cr(VI) from solution most likely by both direct (enzymatic) and indirect (microbially generated reductant) mechanisms.

Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation.

Reduction of soluble uranium U(VI) to less-soluble uranium U(IV) is a promising approach to minimize migration from contaminated aquifers. It is generally assumed that, under constant reducing conditions, U(IV) is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner.

Plant invasion alters nitrogen cycling by modifying the soil nitrifying community.

Plant invasions have dramatic aboveground effects on plant community composition, but their belowground effects remain largely uncharacterized. Soil microorganisms directly interact with plants and mediate many nutrient transformations in soil. We hypothesized that belowground changes to the soil microbial community provide a mechanistic link between exotic plant invasion and changes to ecosystem nutrient cycling. To examine this possible link, monocultures and mixtures of exotic and native species were maintained for 4 years in a California grassland. Gross rates of nitrogen (N) mineralization and nitrification were quantified with 15 N pool dilution and soil microbial communities were characterized with DNA-based methods. Exotic grasses doubled gross nitrification rates, in part by increasing the abundance and changing the composition of ammonia-oxidizing bacteria in soil. These changes may translate into altered ecosystem N budgets after invasion. Altered soil microbial communities and their resulting effects on ecosystem processes may be an invisible legacy of exotic plant invasions.

In situ reduction of chromium(VI) in heavily contaminated soils through organic carbon amendment.

Chromium has become an important soil contaminant at many sites, and facilitating in situ reduction of toxic Cr(VI) to nontoxic Cr(III) is becoming an attractive remediation strategy. Acceleration of Cr(VI) reduction in soils by addition of organic carbon was tested in columns pretreated with solutions containing 1000 and 10 000 mg L(-1) Cr(VI) to evaluate potential in situ remediation of highly contaminated soils. Solutions containing 0,800, or 4000 mg L(-1) organic carbon in the form of tryptic soy broth or lactate were diffused into the Cr(VI)-contaminated soils. Changes in Cr oxidation state were monitored through periodic micro-XANES analyses of soil columns. Effective first-order reduction rate constants ranged from 1.4 x 10(-8) to 1.5 x 10(-7) s(-1), with higher values obtained for lower levels of initial Cr(VI) and higher levels of organic carbon. Comparisons with sterile soils showed that microbially dependent processes were largely responsible for Cr(VI) reduction, except in the soils initially exposed to 10 000 mg L(-1) Cr(VI) solutions that receive little (800 mg L(-1)) or no organic carbon. However, the microbial populations (< or = 2.1 x 10(5) g(-1)) in the viable soils are probably too low for direct enzymatic Cr(VI) reduction to be important. Thus, synergistic effects sustained in whole soil systems may have accounted for most of the observed reduction. These results show that acceleration of in situ Cr(VI) reduction with addition of organic carbon is possible in even heavily contaminated soils and suggest that microbially dependent reduction pathways can be dominant.