Marjorie J. Storm

Advancing fine root research with minirhizotrons

Minirhizotrons provide a nondestructive, in situ method for directly viewing and studying fine roots. Although many insights into fine roots have been gained using minirhizotrons, a review of the literature indicates a wide variation in how minirhizotrons and minirhizotron data are used. Tube installation is critical, and steps must be taken to insure good soil/tube contact without compacting the soil. Ideally, soil adjacent to minirhizotrons will mimic bulk soil. Tube installation causes some degree of soil disturbance and has the potential to create artifacts in subsequent root data and analysis. We therefore recommend a waiting period between tube installation and image collection of 6-12 months to allow roots to recolonize the space around the tubes and to permit nutrients to return to pre-disturbance levels. To make repeated observations of individual roots for the purposes of quantifying their dynamic properties (e.g. root production, turnover or lifespan), tubes should be secured to prevent movement. The frequency of image collection depends upon the root parameters being measured or calculated and the time and resources available for collecting images and extracting data. However, long sampling intervals of 8 weeks or more can result in large underestimates of root dynamic properties because more fine roots will be born and die unobserved between sampling events. A sampling interval of 2 weeks or less reduces these underestimates to acceptable levels. While short sample intervals are desirable, they can lead to a potential trade-off between the number of minirhizotron tubes used and the number of frames analyzed per tube. Analyzing fewer frames per minirhizotron tube is one way to reduce costs with only minor effects on data variation. The quality of minirhizotron data should be assessed and reported; procedures for quantifying the quality of minirhizotron data are presented here. Root length is a more sensitive metric for dynamic root properties than the root number. To make minirhizotron data from separate experiments more easily comparable, idiosyncratic units should be avoided. Volumetric units compatible with aboveground plant measures make minirhizotron-based estimates of root standing crop, production and turnover more useful. Methods for calculating the volumetric root data are discussed and an example presented. Procedures for estimating fine root lifespan are discussed.

Lifetime and temporal occurrence of ectomycorrhizae on ponderosa pine (Pinus ponderosa Laws.) seedlings grown under varied atmospheric CO2 and nitrogen levels

Climate change (elevated atmospheric CO2, and altered air temperatures, precipitation amounts and seasonal patterns) may affect ecosystem processes by altering carbon allocation in plants, and carbon flux from plants to soil. Mycorrhizal fungi, as carbon sinks, are among the first soil biota to receive carbon from plants, and thereby influence carbon release from plants to soil. One step in this carbon release is via fine root and mycorrhizal turnover. It is necessary to know the lifetime and temporal occurrence of roots and mycorrhizae to determine the capacity of the soil ecosystem to sequester carbon assimilated aboveground. In this study, ponderosa pine (Pinus ponderosa Laws) seedlings were grown under three levels of atmospheric CO2 (ambient, 525 and 700 μmol CO2 mol-1) and three levels of annual nitrogen additions (0,100 and 200 kg N ha-1) in open-top chambers. At a two-month frequency during 18 months, we observed ectomycorrhizal root tips observed using minirhizotron tubes and camera. The numbers of new mycorrhizal root tips, the numbers of tips that disappeared between two consecutive recording events, and the standing crop of tips at each event were determined. There were more mycorrhizal tips of all three types seen during the summer compared with other times of the year. When only the standing crop of mycorrhizal tips was considered, effects of the CO2 and N addition treatments on carbon allocation to mycorrhizal tips was weakly evident. However, when the three types of tips were considered collectively, tips numbers flux of carbon through mycorrhizae was greatest in the: (1) high CO2 treatment compared with the other CO2 treatments, and (2) intermediate N addition treatment compared with the other N addition treatments. A survival analysis on the entire 18 month cohort of tips was done to calculate the median lifetime of the mycorrhizal root tips. Average median lifetime of the mycorrhizal tips was 139 days and was not affected by nitrogen and CO2 treatments.

Effects of elevated CO2 and N fertilization on fine root dynamics and fungal growth in seedling Pinus ponderosa

Abstract The effects of elevated CO 2 and N fertilization on fine root growth of Pinus ponderosa Dougl. ex P. Laws. C. Laws., grown in native soil in open-top field-exposure chambers at Placerville, CA, were monitored for a 2-year period using minirhizotrons. The experimental design was a replicated 3 × 3 factorial with a treatment missing; plants were exposed to ambient (≈ 365 μmol mol −1 ) air or ambient air plus either 175 or 350 μmol mol −1 CO 2 and three levels of N addition (0, 100 and 200 kg ha −1 year −1 ). By the second year, elevated CO 2 increased fine root occurrence and root length while N fertilization had no effect. The CO 2 × N interactions were not significant. Neither elevated CO 2 nor N fertilization altered fine root diameter. Fine root mortality was increased by increasing soil N but was reduced in elevated CO 2 . Highest fine root mortality occurred during summer and was lowest during winter. Elevated CO 2 increased mycorrhizal and fungal occurrence earlier than N fertilization.

Germination and early plant development of ten plant species exposed to TiO2 and CeO2 nanoparticles

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.

Germination and early plant development of ten plant species exposed to titanium dioxide and cerium oxide nanoparticles.

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.

Evaluating the Role of Habitat Quality on Establishment of GM Agrostis stolonifera Plants in Non-agronomic Settings

We compared soil chemistry and plant community data at non agronomic mesic locations that either did or did not contain genetically modified (GM) Agrostis stolonifera. The best two-variable logistic regression model included soil Mn content and A. stolonifera cover and explained 90% of the variance in the probability of a site having GM A. stolonifera. Inclusion of NH4 as a third predictor variable increased the variance explained by the logistic model to 100%. Soils at GM locations were characterized by significantly lower (P < 0.05) Mn, A. stolonifera cover, and NH4. Pairwise comparisons indicated that sites in which the GM plants became established had a significantly higher % of bare ground and significantly lower A. stolonifera cover, Mn, organic matter, and carbon (P < 0.05). The pH of soil at GM plant locations varied from 5.9 to 9.5. Our results suggest potential roles of soil disturbance and nutrient status in the establishment of Agrostis in mesic habitats. Additional research is needed to evaluate the ecological consequences of gene flow of GM Agrostis to non-agronomic plant communities.

Elevated CO2 and O3 Effects on Fine-Root Life Span in Ponderosa Pine

Atmospheric carbon dioxide (CO2) and ozone (O3) concentrations are rising, which may have opposing effects on tree C balance and allocation to fine roots. More information is needed on interactive CO2 and O3 effects on roots, particularly fine-root life span, a critical demographic parameter and determinant of soil C and N pools and cycling rates. We conducted a study in which ponderosa pine (Pinus ponderosa) seedlings were exposed to two levels of CO2 and O3 in sun-lit controlledenvironment terracosms for three years. Minirhizotrons were used to monitor individual fine roots in three soil horizons every 28 days. Proportional hazards regression was used to analyze effects of CO2, O3, diameter, depth, and season of root initiation on fine-root survivorship. More fine roots were produced in the elevated CO2 treatment than in ambient CO2. Median life spans varied from 140-448 days depending on the season of root initiation. Elevated CO2, increasing root diameter, and increasing root depth all significantly increased fine-root survivorship and median life span. Life span was slightly, but not significantly, lower in elevated O3, and increased O3 did not reduce the effect of elevated CO2. These results indicate the potential for elevated CO2 to increase the number of fine roots and their residence time in the soil, which is also affected by root diameter, root depth, and phenology. Elevated CO2 and O3 Effects on Fine-Root Life Span in Ponderosa Pine Donald L. Phillips1, Mark G. Johnson1, David T. Tingey1, Marjorie J. Storm2 1 US Environmental Protection Agency, National Health & Environmental Effects Laboratory, Corvallis, OR; 2 Dynamac International, Inc., c/o US EPA, Corvallis, OR Conclusions Elevated CO2 led to longer-lived fine roots in growing ponderosa pine seedlings, as did increasing depth & diameter No compensatory effects of elevated CO2 and elevated O3 on root life span were observed Fine root C turnover at end of study was lower for EC (290 g/m2/yr) than for AC (374 g/m2/yr) Thus, elevated CO2 may lead to increased root C residence time in the soil (at least for tree seedlings) Introduction CO2 and O3 are generally increasing worldwide Leaves are sites of uptake and direct action, but CO2 and O3 can affect C assimilation and allocation with consequences for whole plant Fine roots are especially important because of: role in water and nutrient uptake significant contribution to NPP and its responses to CO2 and O3 CO2 and O3 have potentially offsetting effects of on fine roots (allocation, growth, mortality) Fine root life span is an important determinant of soil C pools and cycling rates Multi-year studies are needed on CO2 & O3 effects on fine root longevity Methods 3 year experiment on ponderosa pine seedlings Sun-lit controlled environment mesocosms at EPA lab in Corvallis, OR 2-way factorial (2 CO2 x 2 O3 with 3 replicates) CO2: ambient (AC), elevated (EC; AC+270 ppm) O3: low (LO; 0 ppm-h seasonal SUM06) high (HO; 10-26 ppm-h seasonal SUM06) Minirhizotron tubes at 4 depths Each fine root (<2 mm diameter) was measured in images recorded every 28 days Proportional hazards regression was used to analyze the effects on fineroot life span of: CO2 and O3 treatments season of root formation, horizon, diameter