Search

Agricultural soils are increasingly undergoing inadvertent and purposeful exposures to engineered CeO 2 nanoparticles (NPs), which can impact crops and root-associated microbial communities. However, interactions between NP concentration and exposure duration on plant-mediated responses of root-associated bacterial communities are not well understood. Soybeans seedlings were grown in soil with uncoated NPs added at concentrations of 0, 1 or 100 mg kg -1 . Total soil exposure durations were either 190 days, starting 106 days before planting or 84 days with NP amendments coinciding with planting. We assessed plant development, bacterial diversity, differential abundance and inferred functional changes across rhizosphere, rhizoplane, and root tissue compartments. Plant non-monotonic dose responses were mirrored in bacterial communities. Most notably, effects were magnified in the rhizoplane under low-dose, short-exposures. Enriched metabolic pathways were primarily related to biosynthesis and degradation/utilization/assimilation, rather than responses to metals or oxidative stress. Our results indicate that plant-mediated bacterial responses were greater than direct NP impacts. Also, we identify needs for modeling non-monotonic legume stress responses that account for coinfection with mutualistic and parasitic bacteroids. Our findings provide new insights regarding effects of applications of soil amendments such as biosolids containing NPs or nano-enabled formulations used in cultivation of legumes and other crops., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

It is well known that exposure to ambient O 3 can decrease growth in many tree species in the United States (US). Our study reports experimental data from outdoor open-top chamber (OTC) studies that quantify total biomass response changes for seedlings of 16 species native to western and eastern North America, which were exposed to several levels of elevated O 3 for one or more years. The primary objective of this study is to establish a reference set of parameters for these seedling exposure-response relationships using a 3-month (92 day) 12-hr W126 O 3 metric used by US Environmental Protection Agency and other agencies to assess risk to trees from O 3 exposure. We classified the 16 species according to their sensitivity, based on the biomass loss response functions to protect from a 5% biomass loss. The three-month 12-h W126 estimated to result in a 5% biomass loss was 2.5-9.2 ppm-h for sensitive species, 20.8-25.2 ppm-h for intermediate species, and > 28.7 ppm-h for insensitive species. The most sensitive tree species include black cherry, ponderosa pine, quaking aspen, red alder, American sycamore, tulip poplar and winged sumac. These species are ecologically important and widespread across US. The effects of O 3 on whole-plant biomass depended on exposure duration and dynamics and on the number of successive years of exposure. These species-specific exposure-response relationships will allow US agencies and other groups to better estimate biomass losses based on ozone exposures in North America and can be used in risk assessment and scenario analyses.

Micronized copper (Cu) azole (MCA) wood preservative formulations include Cu in nano form, and relatively little is known about longer term effects of Cu leached from MCA into wetland ecosystems. We tested the hypothesis that changes in soil microbiomes within reconstructed freshwater wetlands will be associated with exposure to elevated Cu concentrations originating from immersed MCA-treated wood stakes. Eight replicate communities were assembled with Willamette Valley (OR, USA) flood plain soil and clonally propagated wetland plants within mesocosms. Inundated communities were equilibrated for 5 months before installation of MCA or control southern yellow pine stakes (n = 4 communities/experimental group). Soil samples were collected for 16S and internal transcribed spacer amplicon sequencing to quantify responses in prokaryotes and eukaryotes, respectively, at 15 time points, spanning two simulated seasonal dry downs, for up to 678 days. Physiochemical properties of water and soil were monitored at 20 and 12 time points respectively, over the same period. For both taxonomic groups of organisms, phylogenetic diversity increased and was positively correlated with elapsed days. Furthermore, there was significant divergence among eukaryotes during the second year based on experimental group. Although the composition of taxa underwent succession over time, there was significantly reduced relative abundance of sequence variants from Gomphonema diatoms and Scutellinia fungi in communities where MCA wood stakes were present compared with the controls. These focused microbiome shifts were positively correlated with surface water Cu and soil Cu concentrations, which were significantly elevated in treated communities. The reconstructed communities were effective systems for assessing potential impacts to wetland microbiomes after exposure to released copper. The results further inform postcommercialization risk assessments on MCA-treated wood. Environ Toxicol Chem 2021;40:3351-3368. Published 2021. This article is a U.S. Government work and is in the public domain in the USA., (Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

Micronized Cu (μ-Cu) is used as a wood preservative, replacing toxic chromated copper arsenate (CCA). Micronized Cu is malachite [Cu 2 CO 3 (OH) 2 ] that has been milled to micron/submicron particles, with many particle diameters less than 100 nm, mixed with biocides and then used to treat wood. In addition to concerns about the fate of the Cu from μ-Cu, there is interest in the fate of the nano-Cu (n-Cu) constituents. We examined movement of Cu from μ-Cu-treated wood after placing treated-wood stakes into model wetland ecosystems. Release of Cu into surface and subsurface water was monitored. Surface water Cu reached maximum levels 3 days after stake installation and remained elevated if the systems remained inundated. Subsurface water Cu levels were 10% of surface water levels at day 3 and increased gradually thereafter. Sequential filtering indicated that a large portion of the Cu in solution was associating with soluble organics, but there was no evidence for n-Cu in solution. After 4 months, Cu in thin-sections of treated wood and adjacent soil were characterized with micro X-ray absorption fine structure spectroscopy (μ-XAFS). Localization and speciation of Cu in the wood and adjacent soil using μ-XAFS clearly indicated that Cu concentrations decreased over time in the treated wood and increased in the adjacent soil. However, n-Cu from the treated wood was not found in the adjacent soil or plant roots. The results of this study indicate that Cu in the μ-Cu-treated wood dissolves and migrates into adjacent soil and waters primarily in ionic form (i.e., Cu 2+ ) and not as nano-sized Cu particles. A reduced form of Cu (Cu 2 S) was identified in deep soil proximal to the treated wood, indicating strong reducing conditions. The formation of the insoluble Cu 2 S effectively removes some portion of dissolved Cu from solution, reducing movement of Cu 2+ to the water column and diminishing exposure., (Published by Elsevier Ltd.)

This study investigated the effects of third generation exposure to cerium oxide nanoparticles (CeO 2 -NPs) on biomass, elemental and 15 N uptake, and fatty acid contents of wheat (Triticum aestivum). At low or high nitrogen treatment (48 or 112 mg N), seeds exposed for two generations to 0 or 500 mg CeO 2 -NPs per kg soil treatment were cultivated for third year in soil amended with 0 or 500 mg CeO 2 -NPs per kg soil. The results showed that parental and current exposures to CeO 2 -NPs increased the root biomass in daughter plants with greater magnitude of increase at low N than high N. When wheat received CeO 2 -NPs in year 3, root elemental contents increased primarily at low N, suggesting an important role of soil N availability in altering root nutrient acquisition. The δ 15 N ratios, previously shown to be altered by CeO 2 -NPs, were only affected by current and not parental exposure, indicating effects on N uptake and/or metabolism are not transferred from one generation to the next. Seed fatty acid composition was also influenced both by prior and current exposure to CeO 2 -NPs. The results suggest that risk assessments of NP exposure may need to include longer-term, transgenerational effects on growth and grain quality of agronomic crops., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It is important to understand molecular effects on plants exposed to compounds released from use of products containing engineered nanomaterials. Here, we present mRNA sequencing data on transcriptome impacts to Douglas-fir following 2 weeks of sublethal exposure to 30:1 diluted airborne emissions released from combustion of diesel fuel containing engineered CeO 2 nanoparticle catalysts (DECe). Our hypothesis was that chamber exposure to DECe would induce distinct transcriptome changes in seedling needles compared with responses to conventional diesel exhaust (DE) or filtered DECe Gas Phase. Significantly increased uptake/binding of Ce in needles of DECe treated seedlings was 2.7X above background levels and was associated with altered gene expression patterns. All 225 Blast2GO gene ontologies (GOs) enriched by up-regulated DECe transcripts were nested within GOs for DE, however, 29 of 31 enriched GOs for down-regulated DECe transcripts were unique. MapMan analysis also identified three pathways enriched with DECe down-regulated transcripts. There was prominent representation of genes with attenuated expression in transferase, transporter, RNA regulation and protein degradation GOs and pathways. CeO 2 nanoparticle additive decreased and shifted molecular impact of diesel emissions. Wide-spread use of such products and chronic environmental exposure to DECe may adversely affect plant physiology and development.

The effects of cerium oxide nanoparticles (CeO 2 -NPs) on 15 N/ 14 N ratio (δ 15 N) in wheat and barley were investigated. Seedlings were exposed to 0 and 500 mg CeO 2 -NPs/L (Ce-0 and Ce-500, respectively) in hydroponic suspension supplied with NH 4 NO 3 , NH 4 + , or NO 3 - . N uptake and δ 15 N discrimination (i.e. differences in δ 15 N of plant and δ 15 N of N source) were measured. Results showed that N content and 15 N abundance decreased in wheat but increased in barley. Ce-500 only induced whole-plant δ 15 N discrimination (-1.48‰, P ≤ 0.10) with a simultaneous decrease (P ≤ 0.05) in whole-plant δ 15 N (-3.24‰) compared to Ce-0 (-2.74‰) in wheat in NH 4 + . Ce-500 decreased (P ≤ 0.01) root δ 15 N of wheat in NH 4 NO 3 and NH 4 + (3.23 and -2.25‰, respectively) compared to Ce-0 (4.96 and -1.27‰, respectively), but increased (P ≤ 0.05) root δ 15 N of wheat in NO 3 - (3.27‰) compared to Ce-0 (2.60‰). Synchrotron micro-XRF revealed the presence of CeO 2 -NPs in shoots of wheat and barley regardless of N source. Although the longer-term consequences of CeO 2 -NP exposure on N uptake and metabolism are unknown, the results clearly show the potential for ENMs to interfere with plant metabolism of critical plant nutrients such as N even when toxicity is not observed.

We followed soil C fluxes in a subalpine grassland system exposed to experimentally increased atmospheric N deposition for 7 years. Earlier we found that, different from the plant productivity response, the bulk soil C stock increase was highest at the medium, not the high N input as hypothesized. This implies that a smaller N-deposition rate has a greater potential to favor the biological greenhouse gas-sink. To help elucidate the mechanisms controlling those changes in SOC in response to N deposition, we produced four soil density fractions and analyzed soil organic C concentration [SOC], as well as δ 13 C signatures (δ 13 C SOC ) of SOC components. Soil respired CO 2 (δ 13 C CO2 ) was analyzed to better distinguish seasonal short term dynamics from N-deposition effects and to identify the predominant substrate of soil respiration. Both at the start of the experiment and after 7 years we found a strong, negative correlation between [SOC] and δ 13 C SOC of the soil density fractions in the control treatment, consistent with an advanced stage of microbial processing of SOC in fractions of higher density. During the experiment the [SOC] increased in the two lighter density fractions, but decreased in the two heavier fractions, suggesting a possible priming effect that accelerated decomposition of formerly recalcitrant (heavy) organic matter pools. The seasonal pattern of soil δ 13 C CO2 was affected by weather and canopy development, and δ 13 C CO2 values for the different N treatment levels indicated that soil respiration originated primarily from the lightest density fractions. Surprisingly, [SOC] increases were significantly higher under medium N deposition in the <1.8 fraction and in bulk soil, compared to the high N treatment. Analogously, the depletion of δ 13 C SOC was significantly higher in the medium compared to the high N treatment in the three lighter fractions. Thus, medium N deposition favored the highest C sequestration potential, compared to the low N control and the high N treatment. Clearly, our results show that it is inappropriate to use plant productivity N response as an indicator for shifts in SOC content in grassland ecosystems. Here, isotopic techniques illustrated why atmospheric N deposition of 14 kg N ha -1 yr -1 is below, and 54 kg N ha -1 yr -1 is above a threshold that tips the balance between new, assimilative gains and respiratory losses towards a net loss of [SOC] for certain soil fractions in the subalpine grassland.

Engineered nanomaterials (ENM) are a growing aspect of the global economy, and their safe and sustainable development, use, and eventual disposal requires the capability to forecast and avoid potential problems. This review provides a framework to evaluate the health and safety implications of ENM releases into the environment, including purposeful releases such as for antimicrobial sprays or nano-enabled pesticides, and inadvertent releases as a consequence of other intended applications. Considerations encompass product life cycles, environmental media, exposed populations, and possible adverse outcomes. This framework is presented as a series of compartmental flow diagrams that serve as a basis to help derive future quantitative predictive models, guide research, and support development of tools for making risk-based decisions. After use, ENM are not expected to remain in their original form due to reactivity and/or propensity for hetero-agglomeration in environmental media. Therefore, emphasis is placed on characterizing ENM as they occur in environmental or biological matrices. In addition, predicting the activity of ENM in the environment is difficult due to the multiple dynamic interactions between the physical/chemical aspects of ENM and similarly complex environmental conditions. Others have proposed the use of simple predictive functional assays as an intermediate step to address the challenge of using physical/chemical properties to predict environmental fate and behavior of ENM. The nodes and interactions of the framework presented here reflect phase transitions that could be targets for development of such assays to estimate kinetic reaction rates and simplify model predictions. Application, refinement, and demonstration of this framework, along with an associated knowledgebase that includes targeted functional assay data, will allow better de novo predictions of potential exposures and adverse outcomes.

The intergenerational impact of engineered nanomaterials in plants is a key knowledge gap in the literature. A soil microcosm study was performed to assess the effects of multi-generational exposure of wheat ( Triticum aestivum L.) to cerium oxide nanoparticles (CeO 2 -NPs). Seeds from plants that were exposed to 0, 125, and 500 mg CeO 2 -NPs/kg soil (Ce-0, Ce-125 or Ce-500, respectively) in first generation (S1) were cultivated in factorial combinations of Ce-0, Ce-125 or Ce-500 to produce second generation (S2) plants. The factorial combinations for first/second generation treatments in Ce-125 were S1-Ce-0/S2-Ce-0, S1-Ce-0/S2-Ce-125, S1-Ce-125/S2-Ce-0 and S1-Ce-125/S2-Ce-125, and in Ce-500 were S1-Ce-0/S2-Ce-0, S1-Ce-0/S2-Ce-500, S1-Ce-500/S2-Ce-0 and S1-Ce-500/S2-Ce-500. Agronomic, elemental, isotopic, and synchrotron X-ray fluorescence (XRF) and X-ray absorption near-edge spectroscopy (XANES) data were collected on second generation plants. Results showed that plants treated during the first generation only with either Ce-125 or Ce-500 (e.g. S1-Ce-125/S2-Ce-0 or S1-Ce-500/S2-Ce-0) had reduced accumulation of Ce (61 or 50%), Fe (49 or 58%) and Mn (34 or 41%) in roots, and δ 15 N (11 or 8%) in grains compared to the plants not treated in both generations (i.e. S1-Ce-0/S2-Ce-0). Plants treated in both generations with Ce-125 (i.e. S1-Ce-125/S2-Ce-125) produced grains that had lower Mn, Ca, K, Mg and P relative to plants treated in the second generation only (i.e. S1-Ce-0/S2-Ce-125). In addition, synchrotron XRF elemental chemistry maps of soil/plant thin-sections revealed limited transformation of CeO 2 -NPs with no evidence of plant uptake or accumulation. The findings demonstrated that first generation exposure of wheat to CeO 2 -NPs affects the physiology and nutrient profile of the second generation plants. However, the lack of concentration-dependent responses indicate that complex physiological processes are involved which alter uptake and metabolism of CeO 2 -NPs in wheat.

Changes in tissue transcriptomes and productivity of Arabidopsis thaliana were investigated during exposure of plants to 2 widely used engineered metal oxide nanoparticles, titanium dioxide (nano-titania) and cerium dioxide (nano-ceria). Microarray analyses confirmed that exposure to either nanoparticle altered the transcriptomes of rosette leaves and roots, with comparatively larger numbers of differentially expressed genes found under nano-titania exposure. Nano-titania induced more differentially expressed genes in rosette leaves, whereas roots had more differentially expressed genes under nano-ceria exposure. MapMan analyses indicated that although nano-titania up-regulated overall metabolism in both tissues, metabolic processes under nano-ceria remained mostly unchanged. Gene enrichment analysis indicated that both nanoparticles mainly enriched ontology groups such as responses to stress (abiotic and biotic), and defense responses (pathogens), and responses to endogenous stimuli (hormones). Nano-titania specifically induced genes associated with photosynthesis, whereas nano-ceria induced expression of genes related to activating transcription factors, most notably those belonging to the ethylene responsive element binding protein family. Interestingly, there were also increased numbers of rosette leaves and plant biomass under nano-ceria exposure, but not under nano-titania. Other transcriptomic responses did not clearly relate to responses observed at the organism level, possibly because of functional and genomic redundancy in Arabidopsis, which may mask expression of morphological changes, despite discernable responses at the transcriptome level. In addition, transcriptomic changes often relate to transgenerational phenotypic development, and hence it may be productive to direct further experimental work to integrate high-throughput genomic results with longer term changes in subsequent generations. Environ Toxicol Chem 2017;36:71-82. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Ten agronomic plant species were exposed to different concentrations of nano-titanium dioxide (nTiO2 ) or nano-cerium oxide (nCeO2 ) (0 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL) to examine potential effects on germination and early seedling development. The authors modified a standard test protocol developed for soluble chemicals (OPPTS 850.4200) to determine if such an approach might be useful for screening engineered nanomaterials (ENMs) and whether there were differences in response across a range of commercially important plant species to 2 common metal oxide ENMs. Eight of 10 species responded to nTiO2 , and 5 species responded to nCeO2 . Overall, it appeared that early root growth may be a more sensitive indicator of potential effects from ENM exposure than germination. The observed effects did not always relate to the exposure concentration, indicating that mass-based concentration may not fully explain the developmental effects of these 2 ENMs. The results suggest that nTiO2 and nCeO2 have different effects on early plant growth of agronomic species, with unknown effects at later stages of the life cycle. In addition, standard germination tests, which are commonly used for toxicity screening of new materials, may not detect the subtle but potentially more important changes associated with early growth and development in terrestrial plants. Environ Toxicol Chem 2016;35:2223-2229. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.)

The effects of exposure to nanoparticles of titanium dioxide (nano-titanium) and cerium oxide (nano-cerium) on gene expression and growth in Arabidopsis thaliana germinants were studied by using microarrays and quantitative real-time polymerase chain reaction (qPCR), and by evaluating germinant phenotypic plasticity. Exposure to 12 d of either nano-titania or nano-ceria altered the regulation of 204 and 142 genes, respectively. Genes induced by the nanoparticles mainly include ontology groups annotated as stimuli responsive, including both abiotic (oxidative stress, salt stress, water transport) and biotic (respiratory burst as a defense against pathogens) stimuli. Further analysis of the differentially expressed genes indicates that both nanoparticles affected a range of metabolic processes (deoxyribonucleic acid [DNA] metabolism, hormone metabolism, tetrapyrrole synthesis, and photosynthesis). Individual exposures to the nanoparticles increased percentages of seeds with emergent radicles, early development of hypocotyls and cotyledons, and those with fully grown leaves. Although there were distinct differences between the nanoparticles in their affect on molecular mechanisms attributable to enhancing germinant growth, both particles altered similar suites of genes related to various pathways and processes related to enhanced growth., (Published 2014 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Elucidating mechanistic interactions between monovalent cations (Na(+)/K(+)) and engineered nanoparticle surfaces to alter particle stability in polar media have received little attention. We investigated relative preferential interaction of Na(+) and K(+) with carboxylate-functionalized silver nanoparticles (carboxylate-AgNPs) to determine if interaction preference followed the Hofmeister series (Na(+)>K(+)). We hypothesized that Na(+) will show greater affinity than K(+) to pair with carboxylates on AgNP surfaces, thereby destabilizing the colloidal system. Destabilization upon Na(+) or K(+) interacting with carboxylate-AgNPs was evaluated probing changes in multiple physicochemical characteristics: surface plasmon resonance/optical absorbance, electrical conductivity, pH, hydrodynamic diameter, electrophoretic mobility, surface charge, amount of Na(+)/K(+) directly associated with AgNPs, and Ag(+) dissociation kinetics. We show that Na(+) and K(+) react differently, indicating local Na(+) pairing with carboxylates on AgNP surfaces is kinetically faster and remarkably favored over K(+), thus supporting Hofmeister ordering. Our results suggest that AgNPs may transform into micron-size aggregates upon release into aqueous environments and that the fate of such aggregates may need consideration when assessing environmental risk., (Copyright © 2014 Elsevier B.V. All rights reserved.)

While conducting toxicity tests with nano titanium dioxide, the authors found that test suspensions were being contaminated with aluminum and titanium from tip erosion during direct sonication. The contaminating alloy particles had a measurable size distribution and zeta potential using dynamic light scattering, which changed the measured characteristics of the suspensions. Caution should be used when employing direct sonication for preparing test suspensions due to potential interferences of these particles in toxicological assessments., (Copyright © 2013 SETAC.)

Canopies of adult European beech (Fagus sylvatica) and Norway spruce (Picea abies) were labeled with CO(2) depleted in (13)C to evaluate carbon allocation belowground. One-half the trees were exposed to elevated O(3) for 6 yrs prior to and during the experiment. Soil-gas sampling wells were placed at 8 and 15 cm and soil CO(2) was sampled during labeling in mid-late August, 2006. In beech, delta(13)CO(2) at both depths decreased approximately 50 h after labeling, reflecting rapid translocation of fixed C to roots and release through respiration. In spruce, label was detected in fine-root tissue, but there was no evidence of label in delta(13)CO(2). The results show that C fixed in the canopy rapidly reaches respiratory pools in beech roots, and suggest that spruce may allocate very little of recently-fixed carbon into root respiration during late summer. A change in carbon allocation belowground due to long-term O(3) exposure was not observed.

The effects of experimentally elevated O(3) on soil respiration rates, standing fine-root biomass, fine-root production and delta(13)C signature of newly produced fine roots were investigated in an adult European beech/Norway spruce forest in Germany during two subsequent years with contrasting rainfall patterns. During humid 2002, soil respiration rate was enhanced under elevated O(3) under beech and spruce, and was related to O(3)-stimulated fine-root production only in beech. During dry 2003, the stimulating effect of O(3) on soil respiration rate vanished under spruce, which was correlated with decreased fine-root production in spruce under drought, irrespective of the O(3) regime. delta(13)C signature of newly formed fine-roots was consistent with the differing g(s) of beech and spruce, and indicated stomatal limitation by O(3) in beech and by drought in spruce. Our study showed that drought can override the stimulating O(3) effects on fine-root dynamics and soil respiration in mature beech and spruce forests., (2009 Elsevier Ltd. All rights reserved.)

We demonstrate an approach for evaluating the level of protection attained using a variety of forms and levels of past, current, and proposed Air Quality Standards (AQSs). The U.S. Clean Air Act requires the establishment of ambient air quality standards to protect health and public welfare. However, determination of attainment of these standards is based on ambient pollutant concentrations rather than prevention of adverse effects. To determine if a given AQS protected against adverse effects on vegetation, hourly ozone concentrations were adjusted to create exposure levels that "just attain" a given standard. These exposures were used in combination with a physiologically-based tree growth model to account for the interactions of climate and ozone. In the evaluation, we used ozone concentrations from two 6-year time periods from the San Bernardino Mountains in California. There were clear differences in the level of vegetation protection achieved with the various AQSs. Based on modeled plant growth, the most effective standards were the California 8-hr average maximum of 70 ppb and a seasonal, cumulative, concentration-weighted index (SUM06), which if attained, resulted in annual growth reductions of 1% or less. Least effective was the 1-hr maximum of 120 ppb which resulted in a 7% annual reduction. We conclude that combining climate, exposure scenarios, and a process-based plant growth simulator was a useful approach for evaluating effectiveness of current or proposed air quality standards, or evaluating the form and/or level of a standard based on preventing adverse growth effects.

Root respiration has been shown to increase with temperature, but less is known about how this relationship is affected by the fungal partner in mycorrhizal root systems. In order to test respiratory temperature dependence, in particular Q (10) of mycorrhizal and non-mycorrhizal root systems, seedlings of PICEA ABIES (L.) Karst. (Norway spruce) were inoculated with the ectomycorrhizal fungus PILODERMA CROCEUM (Eriksson and Hjortstam, SR430; synonym: PILODERMA FALLAX: [Libert] Stalpers) and planted in soil respiration cuvettes (mycocosms). Temperature dependence of hyphal respiration in sterile cultures was determined and compared with respiration of mycorrhizal roots. Respiration rates of mycorrhizal and non-mycorrhizal root systems as well as sterile cultures were sensitive to temperature. Q (10) of mycorrhizal root systems of 3.0 +/- 0.1 was significantly higher than that of non-mycorrhizal systems (2.5 +/- 0.2). Q (10) of P. CROCEUM in sterile cultures (older than 2 months) was similar to that of mycorrhizal root systems, suggesting that mycorrhizae may have a large influence on the temperature sensitivity of roots in spite of their small biomass. Our results stress the importance of considering mycorrhization when modeling the temperature sensitivity of spruce roots.

•  Respiration of intact ponderosa pine (Pinus ponderosa) fine roots (< 2.5 mm) was measured to determine the role of recently fixed carbohydrate in maintaining root metabolism of growing white (WR) and recently suberized brown roots (BR). •  The CO 2 efflux and O 2 uptake of individual roots were followed continuously over 24 h after carbohydrate supply was altered by exposing shoots to light/dark treatments and by root excision. •  In situ respiration of individual WR and BR averaged 86.0 ± 2.6 and 21.1 ± 1.5 mol CO 2  g -1  h -1 , respectively. Growth respiration was estimated to be approximately two-thirds the rate of WR respiration. Attached WR and BR respiration did not decline significantly over 24 h under continuous light. The WR respiration significantly decreased during a dark period. All roots maintained relatively constant respiration rates for at least 6 h after excision. Respiratory quotient (RQ; CO 2  : O 2 ) was not different between attached (0.84 ± 0.014) and detached (0.85 ± 0.017) roots. CO 2 environment of the cuvette did not influence WR or BR respiration. •  The WR appear to be more sensitive to supply of current photosynthate than BR. Shoot light environment needs to be considered when measuring root and soil CO 2 efflux.

Research aimed at understanding the response of plants to ozone has been conducted for over four decades but little of it has addressed intact natural systems. Even so, there is sufficient scientific information at this time to support air quality standards that will protect natural terrestrial ecosystems from ozone. What is unknown is the risk associated with continued exposure of natural systems, including both above- and below-ground components, in combination with other stresses including changing temperature and precipitation, elevated carbon dioxide, pests and pathogens, invasive species, and other activities that may fragment the landscape. Research to support an assessment of the ecological risk associated with ozone as it exists, in a milieu of stresses, must include endpoints beyond those addressed in the past, primarily productivity and species composition. To estimate the risk to society of ozone impacts on natural systems, endpoints such as the integrity of soil food webs, the quantity and quality of water supplied from terrestrial ecosystems, wildlife and recreational values, and the transfer and fate of carbon, nutrients, and water within the systems must be quantified. Not only will this research provide the basis for a sound estimate of risk, but also it will improve our understanding of fundamental ecosystem processes.

The role of tropospheric ozone in altering plant growth and development has been the subject of thousands of publications over the last several decades. Still, there is limited understanding regarding the possible effects of ozone on soil processes. In this review, the effects of ozone are discussed using the flow of carbon from the atmosphere, through the plant to soils, and back to the atmosphere as a framework. A conceptual model based on carbohydrate signaling is used to illustrate physiological changes in response to ozone, and to discuss possible feedbacks that may occur. Despite past emphasis on above-ground effects, ozone has the potential to alter below-ground processes and hence ecosystem characteristics in ways that are not currently being considered. Contents Summary 213 I. Introduction 213 II. Source-sink model: carbohydrate signaling 214 III. Effect of ozone on above-ground sources and sinks 216 IV. Decreased allocation below ground 218 V. Carbon flux to soils 220 VI. Soil food web 223 VII. Summary, conclusions and future research 223 Acknowledgements 223 References 223.

Individual ponderosa pine (Pinus ponderosa Dougl. ex Laws.) seedlings were grown in mesocosms with three densities of blue wild-rye grass (Elymus glaucus Buckl.) (equivalent to 0, 32 or 88 plants m-2) to determine if the presence of a natural competitor alters the response of ponderosa pine seedlings to ozone. After 3 years of ozone exposure, grass presence reduced total ponderosa pine dry mass by nearly 50%, whereas ozone alone had no significant effect on ponderosa pine growth. The combination of ozone and grass further reduced needle, stem and branch dry mass significantly below that induced by grass competition alone. Root:shoot ratios increased in response to the combined grass and ozone treatments. Grass competition significantly reduced soluble sugar concentrations in all ponderosa pine tissue components examined. Starch concentrations were highly variable but did not differ significantly between treatments. Ozone significantly reduced soluble sugar concentrations in fine roots and stems. In the absence of grass, ozone-treated seedlings tended to have higher tissue N concentrations than controls. In the presence of grass, ozone-treated seedlings had lower N concentrations than controls, resulting in a significant interaction between these two stresses in 1- and 2-year-old needles. Needle C:N ratios decreased in response to grass competition, as a result of increased N concentration and no change in C concentration. The opposite response was observed in ozone-treated seedlings as a result of decreased N concentrations, indicating that ozone-treated seedlings were unable to take up or retain as much nitrogen when grown in the presence of grass. We conclude that ponderosa pine seedlings are more susceptible to ozone when grown in competition with blue wild-rye grass.

Seasonal patterns of carbohydrate concentration in coarse and fine roots, stem or bole, and foliage of ponderosa pine (Pinus ponderosa Laws) were described across five tree-age classes from seedlings to mature trees at an atmospherically clean site. Relative to all other tree-age classes, seedlings exhibited greater tissue carbohydrate concentration in stems and foliage, and greater shifts in the time at which maximum and minimum carbohydrate concentration occurred. To determine the effect of environmental stressors on tissue carbohydrate concentration, two tree-age classes (40-year-old and mature) were compared at three sites along a well-established, long-term O3 and N deposition gradient in the San Bernardino Mountains, California. Maximum carbohydrate concentration of 1-year-old needles declined with increasing pollution exposure in both tree-age classes. Maximum fine root monosaccharide concentration was depressed for both 40-year-old and mature trees at the most polluted site. Maximum coarse and fine root starch concentrations were significantly depressed at the most polluted site in mature trees. Maximum bole carbohydrate concentration of 40-year-old trees was greater for the two most polluted sites relative to the cleanest site: the bole appeared to be a storage organ at sites where high O3 and high N deposition decreased root biomass.

Short-term (14)CO(2) pulse and chase experiments were conducted in order to investigate the effect of ozone on below-ground carbon allocation in spring wheat seedlings (Triticum aestivum L. 'ANZA'). Wheat seedlings were grown in a sand-hydroponic system and exposed to either high ozone (38-40 ppm-h) or low ozone (23-31 ppm-h) for 21 days in a series of replicated experiments. Following the ozone exposures, the plants were pulsed with (14)CO(2) and allocation of (14)C-labeled photosynthate was measured in the plant and growth media. Soluble root exudates were measured, without disturbing the plant roots, 24 h after the (14)CO(2) pulse. Shoot biomass was reduced by 17% for the high ozone and 9% for the low ozone exposures, relative to control treatments. Root biomass was reduced by 9% for the high ozone exposures, but was not significantly different than the controls for the low ozone. The amount of (14)C activity in the shoot and root tissue 24 h after the (14)CO(2) pulse, normalized to tissue weight, total (14)CO(2) uptake, or the total (14)C retention in each plant, was not affected by either high or low ozone exposures. The amount of (14)C activity measured in the growth media solution surrounding the roots increased 9% for the high ozone exposures, and after normalizing to root size or root (14)C activity, the growth media solution (14)C activity increased 29 and 40%, respectively. Total respiration of (14)CO(2) from the ozone-treated plants decreased, but the decrease was not statistically significant. Our results suggest that soluble root exudation of (14)C activity to the surrounding rhizosphere increases in response to ozone. Increased root exudation to the rhizosphere in response to ozone is contrary to reports of decreased carbon allocation below ground and suggests that rhizosphere microbial activity may be initially stimulated by plant exposure to ozone.

Although soil is a component of terrestrial ecosystems, it is comprised of a complex web of interacting organisms, and therefore can be considered itself as an ecosystem. Soil microflora and fauna derive energy from plants and plant residues and serve important functions in maintaining soil physical and chemical properties, thereby affecting net primary productivity (NPP), and in the case of contained environments, the quality of the life support system. We have been using 3 controlled-environment facilities (CEF's) that incorporate different levels of soil biological complexity and environmental control, and differ in their resemblance to natural ecosystems, to study relationships among plant physiology, soil ecology, fluxes of minerals and nutrients, and overall ecosystem function. The simplest system utilizes growth chambers and specialized root chambers with organic-less media to study the physiology of plant-mycorrhizal associations. A second system incorporates natural soil in open-top chambers to study soil bacterial and fungal population response to stress. The most complex CEF incorporates reconstructed soil profiles in a "constructed" ecosystem, enabling close examination of the soil foodweb. Our results show that closed ecosystem research is important for understanding mechanisms of response to ecosystem stresses. In addition, responses observed at one level of biological complexity may not allow prediction of response at a different level of biological complexity. In closed life support systems, incorporating soil foodwebs will require less artificial manipulation to maintain system stability and sustainability.

Ozone exposure decreases belowground carbon allocation and root growth of plants; however, the extent to which these effects persist and the cumulative impact of ozone stress on plant growth are poorly understood. To evaluate the potential for plant compensation, we followed the progression of ozone effects, with particular emphasis on the development of new roots. Ponderosa pine (Pinus ponderosa Dougl. ex Laws.) seedlings were exposed to ozone for 2 years. Following removal of the seedlings from ozone, root growth was assessed to characterize the carry-over effects on new root production, and carbohydrate concentrations were measured to determine if allocation strategies differed among ozone treatments. Four months after removal from ozone, dormant seedlings had significantly lower starch concentrations in stems, coarse roots and fine roots than control seedlings. Following root flushing, starch concentrations in all seedlings decreased, with ozone-treated seedlings containing significantly less starch, sucrose, fructose, glucose and total monosaccharides than control seedlings. There was some evidence that stem starch was mobilized to compensate partially for the lower concentrations of root starch in ozone-treated seedlings; however, there was significantly less new root production in seedlings previously exposed to ozone for 2 years than in control seedlings. Early senescence of older needle age classes, perhaps resulting in inadequate available photosynthate, may be responsible for the reduction in new root production during the year following exposure to ozone. Stored carbohydrate reserves, which were depleted in seedlings previously exposed to ozone, were insufficient to compensate for the ozone-induced reduction in canopy photosynthate. We conclude that there are carry-over effects of ozone exposure on ponderosa pine seedlings, including an enhanced potential for seedling susceptibility to other stresses even in respite years when ozone concentrations are low.

Exposure to ozone (O 3 ) has been shown to decrease the allocation of carbon to tree roots. Decreased allocation of carbon to roots might disrupt root metabolism and rhizosphere organisms. The effects of soil type and shoot O 3 exposure on below-ground respiration and soil microbial populations were investigated using container-grown ponderosa pine (Pinus ponderosa Laws.) growing in a low-nutrient soil, or a fertilizer-amended organic potting media, and exposed to one of three levels of O 3 for two growing seasons in open-top exposure chambers. A closed system, designed to measure below-ground respiratory activity (CO 2 production, O 2 consumption and RQ-Respiration Quotient; (CO 2 :0 2 ) of plants growing in pots, was used monthly to monitor below-ground respiration of 3-yr-old ponderosa pine. Although seasonal differences were detected, CO 2 production (μmol h -1 g -1 total root d. wt), O 2 consumption (μmol h -1 g -1 total root d. wt) and RQ (CO 2 :O 2 ) increased with increasing O 3 exposure level. Seasonal patterns showed increased respiration rates during periods of rapid root growth in spring and early fall. Respiration quotient tended to decrease during known periods of active root growth in control seedlings, but a similar response was not observed in O 3 -treated seedlings. Responses to O3 were greatest in the soil-grown plants, which had a lower fertility level than media-grown plants. Although root d. wt was decreased, root: shoot ratios did not change in response to O 3 . Soil-grown plants had higher root-shoot ratios than media-grown plants, reflecting the lower fertility of the soil. Plant exposure to O 3 was found to affect both active and total populations of soil organisms. In both organic potting media and in soil, biomass of active soil fungi, and the ratio of active-fungal to active-bacterial biomass increased with increasing plant exposure to O 3 . The effect of O 3 on total fungal and bacterial biomass was not linear: at low O 3 levels, total fungal and bacterial biomass increased; at the high O 3 level, total fungal and bacterial biomass decreased compared with those of controls. Our results show that O 3 exposure to shoots significantly disrupts CO 2 production and O 2 consumption of soil and roots of ponderosa pine seedlings. Below-ground respiratory differences were thought to be a result of changes in respiratory substrates, carbon refixation within the plant and soil microbial activity. Ozone also changes below-ground RQ, suggesting that O 3 substantially disrupts root metabolism and interactions with rhizosphere organisms. Ozone exposure of ponderosa pine grown in different soil types can disrupt below-ground respiration and influence populations of soil organisms without alteration of biomass partitioning between above- and below-ground plant components. Collectively, the effect of O 3 on the below-ground system is of concern since it is likely that these changes are accompanied by a change in the ability of root systems to acquire nutrient and water resources and possibly to synthesize amino acids and proteins necessary for normal plant function.

Exposure to ozone (O(3)) and changes in soil fertility influence both the metabolism of plant roots and their interaction with rhizosphere organisms. Because one indication of altered root metabolism is a change in belowground respiratory activity, we used specially designed measurement chambers to assess the effects of O(3) and nutrient availability on belowground respiratory activity of potted three-year-old ponderosa pine (Pinus ponderosa Dougl. ex Laws.). Seedlings were exposed to a factorial combination of three O(3) treatments and three fertilization treatments in open-top O(3) exposure chambers. Ozone exposure decreased and high nutrient supply increased total plant dry weight, but root/shoot ratios were not affected. In general, exposure to O(3) increased rates of belowground O(2) uptake and CO(2) release and the respiratory quotient (RQ, CO(2)/O(2)), although seasonal differences were detected. In October, following the second season of O(3) exposure, rates of belowground O(2) uptake and CO(2) release and RQ were increased in trees in the high-O(3) exposure treatment by 22, 73 and 32%, respectively, over values in control trees in charcoal-filtered air. Increasing nutrient supply resulted in decreasing rates of belowground O(2) uptake and CO(2) release but it had little effect on RQ. In the high-nutrient supply treatment, rates of belowground O(2) uptake and CO(2) release were decreased by 38 and 39%, respectively, compared with rates in the low-nutrient supply treatment. At the end of the second growing season, the high-nutrient supply treatment had decreased lateral root total nonstructural carbohydrates by 22% compared with the low-nutrient supply treatment. Nutrient availability altered the belowground respiratory response to O(3), such that the response to O(3) was greatest in the low-nutrient supply treatment. Significant O(3) effects on belowground respiratory activity were apparent before any reduction in total plant growth was found, suggesting that roots and rhizosphere organisms may be early indicators of physiological dysfunction in stressed seedlings.

The effect of ozone on tree growth and metabolism has been studied widely. Despite the research emphasis, relatively little is known about how the below-ground component responds when shoots are exposed to ozone, even though evidence suggests that ozone can affect roots more than shoots. Undemanding how ozone affects carbohydrate allocation throughout the plant is essential to understanding the mechanisms of response to ozone. The purpose of this study was to follow the allocation and metabolism of carbon in a Pinus Ponderosa Laws.-Hebeloma crustuliniforme (Bull.: St. Amans) Quel seedling system under ozone stress. The hypothesis that ozone affects carbon transport below ground and overall sink strength of roots. similarly in mycorrhizal and non-mycorrhizal seedlings was tested. To test the hypothesis, a unique culturing system was used to quantify carbon movement to all components of the symbiosis and to construct an overall budget for carbon for both mycorrhizal and non-mycorrhizal seedlings. Fluxes of CO 2 and carbon allocation were followed by measuring instantaneous CO 2 flux and by 14 C labelling. Two experiments were conducted that differed in their total ozone exposure (39.3 ppm h in expt 1, and 58.1 ppm h in expt 2). Mycorrhizal inoculation significantly increased CO., assimilation rates (A) and A/R (R = shoot respiration) ratios in both experiments compared with non-mycorrhizal seedlings. Ozone exposure in expt 2 significantly decreased the A/R ratio (P < 0.003) in both mycorrhizal treatments. Below-ground respiration was significantly greater in mycorrhizal than in non-mycorrhizal seedlings in both experiments, and was not affected by ozone exposure, Intact, extramatrical hyphal respiration was lower by 33% in seedlings exposed to ozone, but differences were not statistically significant (P ≤ (0.167). Mycorrhizal seedling roots reached maximum respiratory 14 CO 2 release rates c. 5 h and < 20 h earlier than non-mycorrhizal seedlings in expts 1 and 2, respectively, suggesting accelerated transport of 14 C below ground in mycorrhizal seedlings. Mycorrhizal seedlings also exhibited greater rates of 14 C release below ground than non-mycorrhizal controls. The maximum rate of respiratory release of 14 CO 2 below ground was significantly reduced by exposure to ozone in both mycorrhizal and non-mycorrhizal treatments. Ozone significantly reduced 14 C activity in the fungus of mycorrhizal plants. This constitutes the first report of an ozone-induced reduction in carbon allocation to the fungal symbiont in a mycorrhizal association. The results suggest a substantial impact of ozone on the carbon balance of the mycorrhiza: however, there was no evidence to suggest that mycorrhizal and non-mycorrhizal ponderosa pine seedlings responded differently to ozone stress.

Seasonal changes in water relations of current-year shoots of red spruce (Picea rubens Sarg.) were examined in relation to climatic conditions in trees growing at elevations of 1720 and 1935 m on Clingman's Dome, Tennessee, USA, where increment core data have shown that red spruce decline increases with elevation. Relative height growth of trees at 1720 m was 68% greater than in trees at 1935 m. Following two weeks in July with only traces of precipitation, trees at both sites showed decreased saturated osmotic potentials. The magnitude of the reduction was greater in trees at the high elevation site than in trees at the low elevation site. However, during August and September, shoot water relations of trees at both sites were similar. Precipitation patterns and water relations measurements suggested that, at both sites, trees experienced water stress only briefly during the growing season and to a degree that could not account for the lower growth rates of trees at the high elevation site. During the period of cold hardening in October and November, trees at the low elevation site exhibited saturated osmotic potentials that were lower by 0.2 MPa and solute accumulation (osmol kg(dw) (-1)) that was 48% greater than in trees at the high elevation site.

In this paper, a framework is presented for studying responses of mycorrhiza to external stresses, including possible feedback effects which are likely to occur. The authors review recent literature linking carbon allocation and host/fungal response under natural and anthropogenic stress, and present a conceptual model to discuss how carbon may be involved in singular and multiple stress interactions of mycorrhizal seedlings. Due to an integral integral role in metabollic processes, characterizing carbon allocation in controlled laboratory environments could be useful for understanding host/fungal responses to a variety of natural and anthropogenic stresses. Carbon allocation at the whole-plant level reflects an integrated response which links photosynthesis to growth and maintenance processes. A root-mycocosm system is described which permits spatial separation of a portion of extramatrical hyphae growing in association with seedling roots. Using this system, it is shown that root/hyphal respiratory release of pulse-labeled 14C follows a sigmoidal pattern, with typical lag, exponential and saturation phases. Total respiratory release of 14C per mg root and the fraction respired of total 14C allocated to the root is greater in ponderosa pine inoculated with Hebeloma crustuliniforme than in noninoculated controls. Results illustrate the nature of information than can be obtained using this system. Current projects using the mycocosms include characterizing the dynamics of carbon allocation under ozone stress, and following the fate of organic pollutants. The authors believe that the system could be used to differentiate fungal- and host-mediated responses to a large number of other stresses and to study a variety of physiological processes in mycorrhizal plants.

On the basis of routine diagnostic examinations of 12424 bovine faeces samples for larvae of Dictyocaulus viviparus, performed during the period 1963 through 1977, some calculations and reflections have been made on the incidence and epidemiology of lungworm infection in cattle in Denmark. It seems justified to conclude that dictyocaulosis is an important disease in heifers and cows as well as in calves. A distinct seasonal variation in the incidence of lungworm infection is apparent, in that more than 85% of the faeces samples were submitted within the period July through October. Lungworm larvae could be demonstrated in faeces samples from cattle during the winter and spring- This is considered to be of great importance with a view to re-establishment of the infection in the following grazing season. Overwintering of larvae on pastures seems possible. too, since patent infections among calves were demonstrated already in May and June. A comparison of meteorological data with totals of samples submitted and with relative numbers of positive samples seems to confirm, that both the level and the spread of lungworm infections are influenced by the amount of rainfall during the period June through August.