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      Traceable Calibration, Performance Metrics, and Uncertainty Estimates of Minirhizotron Digital Imagery for Fine-Root Measurements

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          Abstract

          Even though fine-root turnover is a highly studied topic, it is often poorly understood as a result of uncertainties inherent in its sampling, e.g., quantifying spatial and temporal variability. While many methods exist to quantify fine-root turnover, use of minirhizotrons has increased over the last two decades, making sensor errors another source of uncertainty. Currently, no standardized methodology exists to test and compare minirhizotron camera capability, imagery, and performance. This paper presents a reproducible, laboratory-based method by which minirhizotron cameras can be tested and validated in a traceable manner. The performance of camera characteristics was identified and test criteria were developed: we quantified the precision of camera location for successive images, estimated the trueness and precision of each camera's ability to quantify root diameter and root color, and also assessed the influence of heat dissipation introduced by the minirhizotron cameras and electrical components. We report detailed and defensible metrology analyses that examine the performance of two commercially available minirhizotron cameras. These cameras performed differently with regard to the various test criteria and uncertainty analyses. We recommend a defensible metrology approach to quantify the performance of minirhizotron camera characteristics and determine sensor-related measurement uncertainties prior to field use. This approach is also extensible to other digital imagery technologies. In turn, these approaches facilitate a greater understanding of measurement uncertainties (signal-to-noise ratio) inherent in the camera performance and allow such uncertainties to be quantified and mitigated so that estimates of fine-root turnover can be more confidently quantified.

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          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.
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            Environmental controls and the influence of vegetation type, fine roots and rhizomorphs on diel and seasonal variation in soil respiration.

            Characterization of spatial and temporal variation of soil respiration coupled with fine root and rhizomorph dynamics is necessary to understand the mechanisms that regulate soil respiration. A dense wireless network array of soil CO2 sensors in combination with minirhizotron tubes was used to continuously measure soil respiration over 1 yr in a mixed conifer forest in California, USA, in two adjacent areas with different vegetation types: an area with woody vegetation (Wv) and an area with scattered herbaceous vegetation (Hv). Annual soil respiration rates and the lengths of fine roots and rhizomorphs were greater at Wv than at Hv. Soil respiration was positively correlated with fine roots and rhizomorphs at Wv but only with fine roots at Hv. Diel and seasonal soil respiration patterns were decoupled with soil temperature at Wv but not at Hv. When decoupled, higher soil respiration rates were observed at increasing temperatures, demonstrating a hysteresis effect. The diel hysteresis at Wv was explained by including the temperature-dependent component of soil respiration and the variation dependent on photosynthetically active radiation. The results show that vegetation type and fine root and rhizomorph dynamics influence soil respiration in addition to changes in light, temperature and moisture.
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              Conventional detection methodology is limiting our ability to understand the roles and functions of fine roots.

              We lack a thorough conceptual and functional understanding of fine roots. Studies that have focused on estimating the quantity of fine roots provide evidence that they dominate overall plant root length. We need a standard procedure to quantify root length/biomass that takes proper account of fine roots. Here we investigated the extent to which root length/biomass may be underestimated using conventional methodology, and examined the technical reasons that could explain such underestimation. Our discussion is based on original X-ray-based measurements and on a literature review spanning more than six decades. We present evidence that root-length recovery depends strongly on the observation scale/spatial resolution at which measurements are carried out; and that observation scales/resolutions adequate for fine root detection have an adverse impact on the processing times required to obtain precise estimates. We conclude that fine roots are the major component of root systems of most (if not all) annual and perennial plants. Hence plant root systems could be much longer, and probably include more biomass, than is widely accepted.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                12 November 2014
                : 9
                : 11
                : e112362
                Affiliations
                [1 ]National Ecological Observatory Network (NEON), Boulder, Colorado, United States of America
                [2 ]Institute of Arctic and Alpine Research (INSTAAR), University of Colorado, Boulder, Colorado, United States of America
                DOE Pacific Northwest National Laboratory, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JAR MDS HWL EA. Performed the experiments: JAR MDS. Analyzed the data: JAR MDS HWL EA. Contributed reagents/materials/analysis tools: JAR MDS HWL EA. Wrote the paper: JAR MDS HWL EA.

                Article
                PONE-D-14-34438
                10.1371/journal.pone.0112362
                4229195
                25391023
                d8b7954a-8b73-4885-a30c-532b677a81de
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 31 July 2014
                : 10 October 2014
                Page count
                Pages: 16
                Funding
                Funding provided by National Science Foundation ( http://www.nsf.gov/); Grant #: DBI-0752017. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Plant Science
                Plant Anatomy
                Plant Roots
                Lateral Roots
                Root Hairs
                Root Structure
                Engineering and Technology
                Equipment
                Optical Equipment
                Cameras
                Digital Cameras
                Instrumentation
                Instrument Calibration
                Research and Analysis Methods
                Equipment Preparation
                Imaging Techniques
                Image Analysis
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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                Uncategorized

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