Infection with a Shoot-Specific Fungal Endophyte (Epichloë) Alters Tall Fescue Soil Microbial Communities.

Tall fescue (Schedonorus arundinaceus) is a widespread grass that can form a symbiotic relationship with a shoot-specific fungal endophyte (Epichloë coenophiala). While the effects of fungal endophyte infection on fescue physiology and ecology have been relatively well studied, less attention has been given to how this relationship may impact the soil microbial community. We used high-throughput DNA sequencing and phospholipid fatty acid analysis to determine the structure and biomass of microbial communities in both bulk and rhizosphere soils from tall fescue stands that were either uninfected with E. coenophiala or were infected with the common toxic strain or one of several novel strains of the endophyte. We found that rhizosphere and bulk soils harbored distinct microbial communities. Endophyte presence, regardless of strain, significantly influenced soil fungal communities, but endophyte effects were less pronounced in prokaryotic communities. E. coenophiala presence did not change total fungal biomass but caused a shift in soil and rhizosphere fungal community composition, increasing the relative abundance of taxa within the Glomeromycota phylum and decreasing the relative abundance of genera in the Ascomycota phylum, including Lecanicillium, Volutella, Lipomyces, Pochonia, and Rhizoctonia. Our data suggests that tripartite interactions exist between the shoot endophyte E. coenophiala, tall fescue, and soil fungi that may have important implications for the functioning of soils, such as carbon storage, in fescue-dominated grasslands.

Toxicogenomic Responses of the Model Legume Medicago truncatula to Aged Biosolids Containing a Mixture of Nanomaterials (TiO2, Ag, and ZnO) from a Pilot Wastewater Treatment Plant.

Toxicogenomic responses in Medicago truncatula A17 were monitored following exposure to biosolids-amended soils. Treatments included biosolids produced using a pilot wastewater treatment plant with either no metal introduced into the influent (control); bulk/ionic TiO2, ZnO, and AgNO3 added to influent (bulk/dissolved treatment); or Ag, ZnO, and TiO2 engineered nanomaterials added to influent (ENM treatment) and then added to soil, which was aged in the field for 6 months. In our companion study, we found inhibition of nodulation in the ENM but not in the bulk/dissolved treatment. Gene expression profiling revealed highly distinct profiles with more than 10-fold down-regulation in 239 genes in M. truncatula roots from the ENM treatment, while gene expression patterns were similar between bulk/dissolved and control treatments. In response to ENM exposure, many of the identified biological pathways, gene ontologies, and individual genes are associated with nitrogen metabolism, nodulation, metal homeostasis, and stress responses. Expression levels of nine genes were independently confirmed with qRT-PCR. Exposure to ENMs induced unique shifts in expression profiles and biological pathways compared with bulk/dissolved treatment, despite the lack of difference in bioavailable metal fractions, metal oxidation state, and coordination environment between ENM and bulk/dissolved biosolids. As populations of Sinorhizobium meliloti Rm2011 were similar in bulk/dissolved and ENM treatments, our results suggest that inhibition of nodulation in the ENM treatment was primarily due to phytotoxicity, likely caused by enhanced bioavailability of Zn ions.

Nanomaterials in Biosolids Inhibit Nodulation, Shift Microbial Community Composition, and Result in Increased Metal Uptake Relative to Bulk/Dissolved Metals.

We examined the effects of amending soil with biosolids produced from a pilot-scale wastewater treatment plant containing a mixture of metal-based engineered nanomaterials (ENMs) on the growth of Medicago truncatula, its symbiosis with Sinorhizobium meliloti, and on soil microbial community structure. Treatments consisted of soils amended with biosolids generated with (1) Ag, ZnO, and TiO2 ENMs introduced into the influent wastewater (ENM biosolids), (2) AgNO3, Zn(SO4)2, and micron-sized TiO2 (dissolved/bulk metal biosolids) introduced into the influent wastewater stream, or (3) no metal added to influent wastewater (control). Soils were amended with biosolids to simulate 20 years of metal loading, which resulted in nominal metal concentrations of 1450, 100, and 2400 mg kg(-1) of Zn, Ag, and Ti, respectively, in the dissolved/bulk and ENM treatments. Tissue Zn concentrations were significantly higher in the plants grown in the ENM treatment (182 mg kg(-1)) compared to those from the bulk treatment (103 mg kg(-1)). Large reductions in nodulation frequency, plant growth, and significant shifts in soil microbial community composition were found for the ENM treatment compared to the bulk/dissolved metal treatment. These results suggest differences in metal bioavailability and toxicity between ENMs and bulk/dissolved metals at concentrations relevant to regulatory limits.

Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products.

BACKGROUND: The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana. RESULTS: This study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3'-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase, secondary wall-associated NAC domain protein1, MYB58 and MYB63 resulting in higher accumulation of lignin content compared to the control. CONCLUSIONS: These results indicate that the additions of microbially based soil additives have a perceptible influence on phenylpropanoid pathway gene regulation and its production of secondary metabolites. These findings open an avenue of research to investigate the mode of action of microbially-based soil additives which may assist in the sustainable production of food, feed, fuel and fiber.

The chromium detoxification pathway in the multimetal accumulator Silene vulgaris.

Phytomanagement could be a viable alternative in areas polluted with wastes from chromium-using industries. This study investigated the ability of Silene vulgaris to take up Cr(III) and Cr(VI) with special attention on the mechanism used by this species to tolerate high doses of Cr(VI). Plants were grown semihydroponically with different concentrations of either Cr(III) or Cr(VI). A combination of synchrotron X-ray spectroscopic techniques, scanning electron and light microscopy and infrared spectroscopy were used to determine the distribution and speciation of Cr. S. vulgaris accumulated more Cr when grown with Cr(VI) resulting in an overall reduction in biomass. Starch accumulation in leaves may be attributed to an impartment between carbon utilization and assimilation resulted from stunted plant growth but not the complete inhibition of photosynthesis indicating that S. vulgaris possess tolerance mechanisms that allows it to survive in Cr(VI) rich environments. These primary tolerance mechanisms are (a) the total reduction of Cr(VI) to Cr(III) in the rhizosphere or just after uptake in the fine lateral root tips and (b) chelation of Cr(III) to the cell wall both of which reduce metal interference with critical cell functions. These mechanisms make S. vulgaris suitable for in situ remediation of Cr polluted soils.

Evidence for avoidance of Ag nanoparticles by earthworms (Eisenia fetida).

Silver nanoparticles have been incorporated into a wide variety of consumer products, ideally acting as antimicrobial agents. Silver exposure has long been known to cause toxic effects to a wide variety of organisms, making large scale production of silver nanoparticles a potential hazard to environmental systems. Here we describe the first evidence that an organism may be able to sense manufactured nanoparticles in a complex, environmentally relevant exposure and that the presence of nanoparticles alters the organism's behavior. We found that earthworms (Eisenia fetida) consistently avoid soils containing silver nanoparticles and AgNO(3) at similar concentrations of Ag. However, avoidance of silver nanoparticles occurred over 48 h, while avoidance of AgNO(3) was immediate. It was determined that avoidance of silver nanoparticles could not be explained by release of silver ions or any changes in microbial communities caused by the introduction of Ag. This leads us to conclude that the earthworms were in some way sensing the presence of nanoparticles over the course of a 48 h exposure and choosing to avoid exposure to them. Our results demonstrate that nanoparticle interactions with organisms may be unpredictable and that these interactions may result in ecologically significant effects on behavior at environmentally relevant concentrations.

Chromated copper arsenate-treated fence posts in the agronomic landscape: soil properties controlling arsenic speciation and spatial distribution.

Soils adjacent to chromated copper arsenate (CCA)-treated fence posts along a fence line transecting different soil series, parent material, drainage classes, and slope were used to determine which soil properties had the most influence on As spatial distribution and speciation. Metal distribution was evaluated at macroscopic (total metal concentration contour maps) and microscopic scales (micro-synchrotron X-ray fluorescence maps), As speciation was determined using extended X-ray absorption fine structure spectroscopy, and redox status and a myriad of other basic soil properties were elucidated. All geochemical parameters measured point to a condition in which the mobilization of As becomes more favorable moving down the topographic gradient, likely resulting through competition (Meh-P, SOM), neutral or slightly basic pH, and redox conditions that are favorable for As mobilization (higher Fe(II) and total-Fe concentrations in water extracts). On the landscape scale, with hundreds of kilometers of fence, the arsenic loading into the soil can be substantial (∼8-12 kg km). Although a significant amount of the As is stable, extended use of CCA-treated wood has resulted in elevated As concentrations in the local environment, increasing the risk of exposure and ecosystem perturbation. Therefore, a move toward arsenic-free alternatives in agricultural applications for which it is currently permitted should be considered.

Nanotoxicity of engineered nanomaterials (ENMs) to environmentally relevant beneficial soil bacteria - a critical review.

Deposition of engineered nanomaterials (ENMs) in various environmental compartments is projected to continue rising exponentially. Terrestrial environments are expected to be the largest repository for environmentally released ENMs. Because ENMs are enriched in biosolids during wastewater treatment, agriculturally applied biosolids facilitate ENM exposure of key soil micro-organisms, such as plant growth-promoting rhizobacteria (PGPR). The ecological ramifications of increasing levels of ENM exposure of terrestrial micro-organisms are not clearly understood, but a growing body of research has investigated the toxicity of ENMs to various soil bacteria using a myriad of toxicity end-points and experimental procedures. This review explores what is known regarding ENM toxicity to important soil bacteria, with a focus on ENMs which are expected to accumulate in terrestrial ecosystems at the highest concentrations and pose the greatest potential threat to soil micro-organisms having potential indirect detrimental effects on plant growth. Knowledge gaps in the fundamental understanding of nanotoxicity to bacteria are identified, including the role of physicochemical properties of ENMs in toxicity responses, particularly in agriculturally relevant micro-organisms. Strategies for improving the impact of future research through the implementation of in-depth ENM characterization and use of necessary experimental controls are proposed. The future of nanotoxicological research employing microbial ecoreceptors is also explored, highlighting the need for continued research utilizing bacterial isolates while concurrently expanding efforts to study ENM-bacteria interactions in more complex environmentally relevant media, e.g. soil. Additionally, the particular importance of future work to extensively examine nanotoxicity in the context of bacterial ecosystem function, especially of plant growth-promoting agents, is proposed.

Silver engineered nanomaterials and ions elicit species-specific O2 consumption responses in plant growth promoting rhizobacteria.

Metal containing engineered nanomaterials (ENMs) are now commonly used in various industrial and commercial applications. Many of these materials can be transformed during waste water treatment and ultimately enter terrestrial ecosystems via agriculturally applied biosolids. It is unclear how agriculturally important soil microbes will be affected by exposure to environmentally relevant, sublethal concentrations of ENMs and their transformation products (i.e., ions, aggregates, etc.). A method was developed, which puts O2 consumption responses in terms of viability, and tested by examining the toxic effects of Ag+, Zn2+, and Ni2+ ions on the plant growth promoting rhizobacterium (PGPR) Bacillus amyloliquefaciens GB03. The method was then used to examine the toxicity of Ag+, as-synthesized polyvinylpyrrolidone-coated silver ENM (PVP-AgENMs), and 100% sulfidized AgENM on B. amyloliquefaciens GB03, and two additional PGPRs Sinorhizobium meliloti 2011, and Pseudomonas putida UW4. S. meliloti was found to have the highest LC50 for Ag+ and PVP-AgENMs (6.6 and 207 µM, respectively), while B. amyloliquefaciens and P. putida exhibited LC50's for Ag+ and PVP-AgENMs roughly half those observed for S. meliloti. The authors observed species-specific O2 consumption responses to ENM and ion exposure. PVP-AgENMs were less toxic than ions on a molar basis, and abiotic dissolution likely explains a significant portion of the observed toxic responses. Our results suggest microbes may exhibit distinct metabolic responses to metal and ENM exposure, even when similar LC50's are observed. These findings together illustrate the importance of understanding species-specific toxic responses and the utility of examining O2 consumption for doing so.

Tall fescue cultivar and fungal endophyte combinations influence plant growth and root exudate composition.

Tall fescue [Lolium arundinaceum (Schreb.)] is a cool-season perennial grass used in pastures throughout the Southeastern United States. The grass can harbor a shoot-specific fungal endophyte (Epichloë coenophiala) thought to provide the plant with enhanced resistance to biotic and abiotic stresses. Because alkaloids produced by the common variety of the endophyte cause severe animal health issues, focus has been on replacing the common-toxic strain with novel varieties that do not produce the mammal-toxic alkaloids but maintain abiotic and biotic stress tolerance benefits. Little attention has been given to the influence of the plant-fungal symbiosis on rhizosphere processes. Therefore, our objective was to study the influence of this relationship on plant biomass production and root exudate composition in tall fescue cultivars PDF and 97TF1, which were either not infected with the endophyte (E-), infected with the common toxic endophyte (CTE+) strain or with one of two novel endophytes (AR542E+, AR584E+). Plants were grown sterile for 3 weeks after which plant biomass, total organic carbon, total phenolic content and detailed chemical composition of root exudates were determined. Plant biomass production and exudate phenolic and organic carbon content were influenced by endophyte status, tall fescue cultivar, and their interaction. GC-TOF MS identified 132 compounds, including lipids, carbohydrates and carboxylic acids. Cluster analysis showed that the interaction between endophyte and cultivar resulted in unique exudate profiles. This is the first detailed study to assess how endophyte infection, notably with novel endophytes, and tall fescue cultivar interact to influence root exudate composition. Our results illustrate that tall fescue cultivar and endophyte status can influence plant growth and root exudate composition, which may help explain the observed influence of this symbiosis on rhizosphere biogeochemical processes.

Morphological and genetic changes induced by excess Zn in roots of Medicago truncatula A17 and a Zn accumulating mutant.

BACKGROUND: Nutrient fluxes associated with legume-rhizobia symbioses are poorly understood and little is known regarding the influence of abiotic stresses on development and maintenance of N-fixing nodules and root system architecture (RSA). We examined effects of Zn on nodule development and structure, root architecture, and expression of nodulation-related miRNAs in Medicago truncatula and the mutant, raz (requires additional Zn). FINDINGS: Excess Zn increased root and shoot associated Zn in both genotypes, however, raz plants had lower root associated Zn than WT plants. Roots of raz plants exposed to excess Zn had less volume, surface area, and total length compared to WT plants. Raz plants had lower lateral root number than WT plants. Excess Zn was found to increase root diameter in both genotypes. The Mn Translocation Factor (TfMn) increased in response to Zn in both genotypes; this was more pronounced in raz plants. TfZn was higher in raz plants and reduced in both genotypes in response to Zn. Nodulation was not influenced by Zn treatment or plant genotype. MicroRNA166 was upregulated under excess Zn in WT plants. CONCLUSIONS: Neither the raz mutation nor Zn treatment affected nodulation, however, raz plants had altered RSA compared with WT and responded differently to Zn, implying the mutation potentially modulates RSA responses to Zn but doesn't play a direct role in nodulation. MicroRNA166 was significantly induced in WT plants by excess Zn, warranting further investigation into the potential role it plays in controlling RSA.

Exploring the structural basis for selenium/mercury antagonism in Allium fistulosum.

While continuing efforts are devoted to studying the mutually protective effect of mercury and selenium in mammals, few studies have investigated the mercury-selenium antagonism in plants. In this study, we report the metabolic fate of mercury and selenium in Allium fistulosum (green onion) after supplementation with sodium selenite and mercuric chloride. Analysis of homogenized root extracts via capillary reversed phase chromatography coupled with inductively coupled plasma mass spectrometry (capRPLC-ICP-MS) suggests the formation of a mercury-selenium containing compound. Micro-focused synchrotron X-ray fluorescence mapping of freshly excised roots show Hg sequestered on the root surface and outlining individual root cells, while Se is more evenly distributed throughout the root. There are also discrete Hg-only, Se-only regions and an overall strong correlation between Hg and Se throughout the root. Analysis of the X-ray absorption near edge structure (XANES) spectra show a "background" of methylselenocysteine within the root with discrete spots of SeO(3)(2-), Se(0) and solid HgSe on the root surface. Mercury outlining individual root cells is possibly binding to sulfhydryl groups or plasma membrane or cell wall proteins, and in some places reacting with reduced selenium in the rhizosphere to form a mercury(ii) selenide species. Together with the formation of the root-bound mercury(ii) selenide species, we also report on the formation of cinnabar (HgS) and Hg(0) in the rhizosphere. The results presented herein shed light on the intricate chemical and biological processes occurring within the rhizosphere that influence Hg and Se bioavailability and will be instrumental in predicting the fate and assisting in the remediation of these metals in the environment and informing whether or not fruit and vegetable food selection from aerial plant compartments or roots from plants grown in Hg contaminated soils, are safe for consumption.

The hyperaccumulator Alyssum murale uses complexation with nitrogen and oxygen donor ligands for Ni transport and storage.

The Kotodesh genotype of the nickel (Ni) hyperaccumulator Alyssum murale was examined to determine the compartmentalization and internal speciation of Ni, and other elements, in an effort to ascertain the mechanism used by this plant to tolerate extremely high shoot (stem and leaf) Ni concentrations. Plants were grown either hydroponically or in Ni enriched soils from an area surrounding an historic Ni refinery in Port Colborne, Ontario, Canada. Electron probe micro-analysis (EPMA) and synchrotron based micro X-ray fluorescence (micro-SXRF) spectroscopy were used to determine the metal distribution and co-localization and synchrotron X-ray and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopies were used to determine the Ni speciation in plant parts and extracted sap. Nickel is concentrated in the dermal leaf and stem tissues of A. murale bound primarily to malate along with other low molecular weight organic ligands and possibly counter anions (e.g., sulfate). Ni is present in the plant sap and vasculature bound to histidine, malate and other low molecular weight compounds. The data presented herein supports a model in which Ni is transported from the roots to the shoots complexed with histidine and stored within the plant leaf dermal tissues complexed with malate, and other low molecular weight organic acids or counter-ions.

Application of quantitative fluorescence and absorption-edge computed microtomography to image metal compartmentalization in Alyssum murale.

This paper shows that synchrotron-based fluorescence and absorption-edge computed microtomographies (CMT) are well-suited for determining the compartmentalization and concentration of metals in hyperaccumulating plant tissues. Fluorescence CMT of intact leaf, stem, and root samples revealed that Ni concentrated in stem and leaf dermal tissues and, together with Mn, in distinct regions associated with the Ca-rich trichomes on the leaf surface of the nickel hyperaccumulator Alyssum murale "Kotodesh". Metal enrichment was also observed within the vascular system of the finer roots, stem, and leaves but absent from the coarser root, which had a well-correlated metal coating. Absorption-edge CMT showed the three-dimensional distribution of the highest metal concentrations and verified that epidermal localization and Ni and Mn co-localization at the trichome base are phenomena that occurred throughout the entire leaf and may contribute significantly to metal detoxification and storage. Ni was also observed in the leaf tips, possibly resulting from release of excess Ni with guttation fluids. These results are consistent with a transport model where Ni is removed from the soil by the finer roots, carried to the leaves through the stem xylem, and distributed throughout the leaf by the veins to the dermal tissues, trichome bases, and in some cases the leaf tips.