Transformation and release of micronized Cu used as a wood preservative in treated wood in wetland soil.

Micronized Cu (µ-Cu) is used as a wood preservative, replacing toxic chromated copper arsenate (CCA). Micronized Cu is malachite [Cu2CO3(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., Cu2+) and not as nano-sized Cu particles. A reduced form of Cu (Cu2S) was identified in deep soil proximal to the treated wood, indicating strong reducing conditions. The formation of the insoluble Cu2S effectively removes some portion of dissolved Cu from solution, reducing movement of Cu2+ to the water column and diminishing exposure.

Chemistry and long-term decomposition of roots of Douglas-fir grown under elevated atmospheric carbon dioxide and warming conditions.

Elevated atmospheric CO(2) concentrations and warming may affect the quality of litters of forest plants and their subsequent decomposition in ecosystems, thereby potentially affecting the global carbon cycle. However, few data on root tissues are available to test this feedback to the atmosphere. In this study, we used fine (diameter < or = 2 mm) and small (2-10 mm) roots of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings that were grown for 4 yr in a 2 x 2 factorial experiment: ambient or elevated (+ 180 ppm) atmospheric CO(2) concentrations, and ambient or elevated (+3.8 degrees C) atmospheric temperature. Exposure to elevated CO(2) significantly increased water-soluble extractives concentration (%WSE), but had little effect on the concentration of N, cellulose, and lignin of roots. Elevated temperature had no effect on substrate quality except for increasing %WSE and decreasing the %lignin content of fine roots. No significant interaction was found between CO(2) and temperature treatments on substrate quality, except for %WSE of the fine roots. Short-term (< or = 9 mo) root decomposition in the field indicated that the roots from the ambient CO(2) and ambient temperature treatment had the slowest rate. However, over a longer period of incubation (9-36 mo) the influence of initial substrate quality on root decomposition diminished. Instead, the location of the field incubation sites exhibited significant control on decomposition. Roots at the warmer, low elevation site decomposed significantly faster than the ones at the cooler, high elevation site. This study indicates that short-term decomposition and long-term responses are not similar. It also suggests that increasing atmospheric CO(2) had little effect on the carbon storage of Douglas-fir old-growth forests of the Pacific Northwest.

Elevated CO(2) and elevated temperature have no effect on Douglas-fir fine-root dynamics in nitrogen-poor soil.

Here, we investigate fine-root production, mortality and standing crop of Douglas-fir (Pseudotsuga menziesii) seedlings exposed to elevated atmospheric CO(2) and elevated air temperature. We hypothesized that these treatments would increase fine-root production, but that mortality would be greater under elevated temperature, leading to a smaller increase in standing crop. Seedlings were grown in outdoor, sun-lit controlled-environment chambers containing native soil. They were exposed in a factorial design to two levels of atmospheric CO(2) and two levels of air temperature. Minirhizotron methods were used to measure fine-root length production, mortality and standing crop every 4 wk for 36 months. Neither elevated atmospheric CO(2) nor elevated air temperature affected fine-root production, mortality, or standing crop. Fine roots appeared to root deeper in the soil profile under elevated CO(2) and elevated temperature. Low soil nitrogen (N) levels apparently limited root responses to the treatments. This suggests that forests on nutrient-poor soils may exhibit limited fine-root responses to elevated atmospheric CO(2) and elevated air temperature.

Does soil CO2 efflux acclimatize to elevated temperature and CO2 during long-term treatment of Douglas-fir seedlings?

We investigated the effects of elevated soil temperature and atmospheric CO2 on soil CO2 efflux (SCE) during the third and fourth years of study. We hypothesized that elevated temperature would stimulate SCE, and elevated CO2 would also stimulate SCE with the stimulation being greater at higher temperatures. The study was conducted in sun-lit controlled-environment chambers using Douglas-fir (Pseudotsuga menziesii) seedlings grown in reconstructed litter-soil systems. We used a randomized design with two soil temperature and two atmospheric CO2 treatments. The SCE was measured every 4 wk for 18 months. Neither elevated temperature nor CO2 stimulated SCE. Elevated CO2 increased the temperature sensitivity of SCE. During the winter, the relationship between SCE and soil moisture was negative but it was positive during the summer. The seasonal patterns in SCE were associated with seasonal changes in photosynthesis and above-ground plant growth. SCE acclimatized in the high-temperature treatment, probably because of a loss of labile soil carbon. Elevated CO2 treatment increased the temperature sensitivity of SCE, probably through an increase in substrate availability.

Foliar nitrogen concentrations and natural abundance of (15)N suggest nitrogen allocation patterns of Douglas-fir and mycorrhizal fungi during development in elevated carbon dioxide concentration and temperature.

Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) seedlings were grown in a 2 x 2 factorial design in enclosed mesocosms at ambient temperature or 3.5 degrees C above ambient, and at ambient CO2 concentration ([CO2]) or 179 ppm above ambient. Two additional mesocosms were maintained as open controls. We measured the extent of mycorrhizal infection, foliar nitrogen (N) concentrations on both a weight basis (%N) and area basis (Narea), and foliar delta15N signatures (15N/14N ratios) from summer 1993 through summer 1997. Mycorrhizal fungi had colonized nearly all root tips across all treatments by spring 1994. Elevated [CO2] lowered foliar %N but did not affect N(area), whereas elevated temperature increased both foliar %N and Narea. Foliar delta15N was initially -1 per thousand and dropped by the final harvest to between -4 and -5 per thousand in the enclosed mesocosms, probably because of transfer of isotopically depleted N from mycorrhizal fungi. Based on the similarity in foliar delta15N among treatments, we conclude that mycorrhizal fungi had similar N allocation patterns across CO2 and temperature treatments. We combined isotopic and Narea data for 1993-94 to calculate fluxes of N for second- and third-year needles. Yearly N influxes were higher in second-year needles than in third-year needles (about 160 and 50% of initial leaf N, respectively), indicating greater sink strength in the younger needles. Influxes of N in second-year needles increased in response to elevated temperature, suggesting increased N supply from soil relative to plant N demands. In the elevated temperature treatments, N effluxes from third-year needles were higher in seedlings in elevated [CO2] than in ambient [CO2], probably because of increased N allocation below ground. We conclude that N allocation patterns shifted in response to the elevated temperature and [CO2] treatments in the seedlings but not in their fungal symbionts.

Soil temperature and plant growth stage influence nitrogen uptake and amino acid concentration of apple during early spring growth.

In spring, nitrogen (N) uptake by apple roots begins about 3 weeks after bud break. We used 1-year-old 'Fuji' Malus domestica Borkh on M26 bare-root apple trees to determine whether the onset of N uptake in spring is dependent solely on the growth stage of the plant or is a function of soil temperature. Five times during early season growth, N uptake and total amino acid concentration were measured in trees growing at aboveground day/night temperatures of 23/15 degrees C and belowground temperatures of 8, 12, 16 or 20 degrees C. We used (15NH4)(15NO3) to measure total N uptake and rate of uptake and found that both were significantly influenced by both soil temperature and plant growth stage. Rate of uptake of 15N increased with increasing soil temperature and changed with plant growth stage. Before bud break, 15N was not detected in trees growing in the 8 degrees C soil treatment, whereas 15N uptake increased with increasing soil temperatures between 12 and 20 degrees C. Ten days after bud break, 15N was still not detected in trees growing in the 8 degrees C soil treatment, although total 15N uptake and uptake rate continued to increase with increasing soil temperatures between 12 and 20 degrees C. Twenty-one days after bud break, trees in all temperature treatments were able to acquire 15N from the soil, although the amount of uptake increased with increasing soil temperature. Distribution of 15N in trees changed as plants grew. Most of the 15N absorbed by trees before bud break (approximately 5% of 15N supplied per tree) remained in the roots. Forty-six days after bud break, approximately one-third of the 15N absorbed by the trees in the 12-20 degrees C soil temperature treatments remained in the roots, whereas the shank, stem and new growth contained about two-thirds of the 15N taken up by the roots. Total amino acid concentration and distribution of amino acids in trees changed with plant growth stage, but only the amino acid concentration in new growth and roots was affected by soil temperature. We conclude that a combination of low soil temperature and plant developmental stage influences the ability of apple trees to take up and use N from the soil in the spring. Thus, early fertilizer application in the spring when soil temperatures are low or when the aboveground portion of the tree is not actively growing may be ineffective in promoting N uptake.

Morphotype community structure of ectomycorrhizas on Douglas fir (Pseudotsuga menziesii Mirb. Franco) seedlings grown under elevated atmospheric CO2 and temperature.

Mycorrhizas alter the acquisition of carbon and nutrients, thereby affecting numerous plant and ecosystem processes. It is important, therefore, to determine how mycorrhizal populations will change under possible future climate conditions. Individual and interactive effects of elevated atmospheric CO2 concentration and atmospheric temperature were assessed in a 2×2 factorial design [ambient and elevated (200 ppm above ambient) CO2 concentrations, and ambient and elevated (4°C above ambient) temperatures]. In June 1993, 2-year-old Douglas fir (Pseudotsuga menziesii Mirb. Franco) seedlings were planted in 12 environment-tracking chambers (n=3) containing reconstructed, low-nitrogen, native forest soil. Climate treatments were imposed shortly thereafter, and the seedlings grew until June 1997. Soil cores were taken twice per year during the exposure period. We present findings on changes in the community structure of ectomycorrhizal (ECM) root tips, categorized into morphotypes using gross morphological traits. A diverse and stable community of morphotypes (a total of 40) was encountered; no more than 30 of which were seen at any sampling time. In the first sample, there were only 15 morphotypes found in the 12 chambers. Morphotype numbers increased during the first half of the experiment, remaining fairly constant thereafter. Near the end of the exposure, elevated-temperature treatments maintained more morphotypes than ambient treatments. However, overall, absolute measures (number of ECM tips) were affected primarily by CO2 treatment, whereas proportional measures (e.g., Simpson's index) were affected primarily by temperature. While some morphotypes were negatively affected seasonally by higher temperatures (putative Rhizopogon group), others (Cenococcum) seemed to thrive. Underlying the dominant patterns of change in diversity, driven by the Rhizopogon group, subdominant populations responded slightly differently. Community diversity through time tended to increase at a greater rate for all subdominant populations compared with the rate when dominant populations were included.

Understanding plant-soil relationships using controlled environment facilities.

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.

90Sr uptake by Pinus ponderosa and Pinus radiata seedlings inoculated with ectomycorrhizal fungi.

Strontium-90 ((90)Sr) is a radionuclide characteristic of fallout from nuclear reactor accidents and nuclear weapons testing. Prior studies have shown that Pinus ponderosa and P. radiata seedlings can remove appreciable quantities of (90)Sr from soil and store it in plant tissue. In this study, we inoculated P. ponderosa and P. radiata seedlings with one of five isolates of ectomycorrhizal fungi. Inoculated and noninoculated (control) seedlings were compared for their ability to remove (90)Sr from an organic growth medium. Seedlings were grown in a growth chamber in glass tubes containing 165 cm(3) of sphagnum peat moss and perlite (1 : 1 (v/v)) and, except in the controls, the fungal inoculum. After 3 months, 5978 Bq of (90)Sr in 1 ml of sterile, distilled, deionized water was added. Seedlings were grown for an additional month and then harvested. P. ponderosa seedlings with ectomycorrhizae accumulated 3.0-6.0% of the (90)Sr; bioconcentration ratios (Bq (90)Sr cm(-3) plant tissue/Bq (90)Sr cm(-3) growth medium) ranged from 98-162. Ectomycorrhizal P. radiata seedlings accumulated 6.0-6.9% of the (90)Sr; bioconcentration ratios ranged from 88-133. Nonmycorrhizal P. ponderosa and P. radiata seedlings accumulated only 0.6 and 0.7% of the (90)Sr and had bioconcentration ratios of 28 and 27, respectively. Ectomycorrhizal P. ponderosa and P. radiata seedlings are able to remove 3-5 times more (90)Sr from contaminated soil than seedlings without ectomycorrhizae.

Stress interactions and mycorrhizal plant response: understanding carbon allocation priorities.

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.

Temporal allocation of (14)C to extramatrical hyphae of ectomycorrhizal ponderosa pine seedlings.

Ponderosa pine seedlings were inoculated with Hebeloma crustuliniforme either in growth pouches before they were transplanted to root-mycocosms (P seedlings), or at the time of transfer to root-mycocosms (V seedlings). Uninoculated seedlings served as controls (U seedlings). The use of root-mycocosms allowed examination of portions of hyphae separate from roots and rooting substrate but still in symbiosis with the host. The results thus provided a quantitative basis for estimating hyphal mass and carbon allocation to extramatrical hyphae. The amount of (14)CO(2) fixed after a 2-h exposure was greatest for P seedlings and least for uninoculated seedlings. Four and nine days after exposure, (14)C content was greatest in uninoculated seedlings and least in inoculated seedlings. In isotope distribution and dry mass accumulation, V seedlings were more similar to U than to P seedlings. Calculated on a dry weight basis, the allocation of isotope to mycelium suggested that extramatrical hyphae of P seedlings were a stronger sink for carbon than extramatrical hyphae of V seedlings. Differences in inoculation methods resulted in differences in carbon allocation and physiology of extramatrical hyphae that could affect seedling establishment and survival. Seedlings inoculated by one method cannot serve as surrogates for mycorrhizal seedlings produced by other inoculation techniques.

Effects of pretreatment conditions on ammonium and nitrate uptake by Douglas-fir seedlings.

Effects of pretreatment solutions containing varying concentrations of calcium, potassium, ammonium and nitrate were evaluated by measuring fluxes of the same ions during a subsequent 4-hour uptake by Douglas-fir seedlings. Maximal rate of ammonium uptake (11 microeq g(-1) root dry weight h(-1)) was about 5 times faster than that of nitrate (2 microeq g(-1) root dry weight h(-1)). Ammonium uptake was most rapid after pretreatment with low potassium levels and was unaffected by ammonium pretreatment. Nitrate uptake was most rapid after pretreatment with high levels of nitrate and low levels of potassium. Calcium uptake was greater when nitrate replaced ammonium as the N source. High calcium pretreatment levels depressed subsequent calcium uptake or resulted in calcium release in both ammonium and nitrate experiments. Potassium efflux occurred with both N sources, but the release was less during nitrate uptake than during ammonium uptake. Efflux of potassium is probably associated with the high potassium status of the seedlings and the exchange between potassium and other cations. High levels of potassium in the pretreatment solutions enhanced potassium efflux and caused a reduction in the subsequent rates of uptake of both ammonium and nitrate. Length of pretreatment and seedling size generally did not affect uptake rates.

A comparison of methods for determining compartmental analysis parameters.

The traditional method for determining compartmental analysis parameters relies on a visual selection of data points to be used for regression of data from each cellular compartment. This method is appropriate when the compartments are kinetically discrete and are easily discernible. However, where treatment effects on compartment parameters are being evaluated, a more objective method for determining initial parameters is desirable.Three methods were examined for determining initial isotopic contents and half-times of (86)Rb elution from cellular compartments using theoretical data with known parameters. Experimental data from roots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) and barley (Hordeum vulgare L.) intact seedlings were also used. The three methods were a visually assisted, linear regression on data of semilog plot of isotope elution versus time, a microcomputer-assisted, linear regression on semilog plot where maximization of the square of the correlation coefficient (r(2)) was the criterion to determine data points needed for each regression and a mainframe computer-assisted, direct nonlinear regression on elution data using a model of the sum of three exponential decay functions. The visual method resulted in the least accurate estimates of compartmental analysis parameters. The microcomputer-assisted and nonlinear regression methods calculated the parameters equally well.

Mycorrhizal effects on potassium fluxes by northwest coniferous seedlings.

In ectomycorrhizae, the relative abilities of mycobiont and host plant to take up and store inorganic nutrients are not easily determined due to the intimate physical relationship of the two components forming the association. Since compartmental analysis of solute elution can estimate cellular compartment pool sizes and unidirectional fluxes across membranes, we have used this method to study ectomycorrhizal coniferous roots. Rubidium-86, used as a tracer for potassium, was loaded into and eluted from intact roots of nonmycorrhizal and mycorrhizal (with the fungus Hebeloma crustuliniformme [Bull.: St. Amans Quél] Douglas fir (Pseudotsuga menziesii [Mirb.] Franco), western hemlock (Tsuga heterophylla [Raf.] Sarg.) and Sitka spruce (Picea sitchensis [Bong.] Carr.) seedlings.Mycorrhizas significantly increased (86)Rb uptake rates while decreasing the amount of (86)Rb released to the external solution. Using compartmental analysis, the flux data suggest that the primary mycorrhizal effects were to increase inward potassium fluxes across the fungal tonoplast and to decrease potassium efflux across the fungal tonoplast, as compared with nonmycorrhizal seedling roots. The result was greater potassium storage, presumably in the fungal vacuole. The three coniferous species responded differently to fungal infection with respect to potassium fluxes. Both cytoplasmic and vacuolar fluxes for mycorrhizal hemlock were 2-fold greater than for spruce and 3-fold greater than for Douglas fir. These results demonstrate the usefulness of compartmental analysis for study of ion fluxes in intact mycorrhizal root systems and suggest that the fungal tonoplast may be the site for regulation of potassium fluxes in these coniferous roots.