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.     

Soil core and minirhizotron comparison for the determination of root length density

Detailed knowledge of the distribution of roots in the soil is important in understanding the extraction of water and nutrients from soil. Various techniques have been developed to monitor root-length density under field conditions. Excavation techniques, including soil cores, have long been considered to give reliable estimates of root-length density, but these techniques are laborious in sample collection and tedious in determination of root lengths. An attractive alternative for monitoring root-length density has been the minirhizotron whereby a periscope is inserted into a clear tube permanently installed in the soil for repeated and rapid measures of root development. The objective of this study was to compare the ability of the minirhizotron technique to measure root-length density as compared to the root-core technique.As in previous studies, substantial disagreement existed between the two techniques in the top 30-cm of the soil. The results from the minirhizotron consistently indicated a much lower root population than the root-core technique in the surface layer of soil. This is especially worrisome because more than 45% of the root-length density was found in this layer with the root-core technique. At deeper soil layers, the minirhizotron data proved to be no less variable than the root-core technique making the determination of statistically significant results difficult. Finally, the relationship between the minirhizotron and soil-core results varied with time even when the observations from the soil surface layer were ignored. Attempts to directly translate minirhizotron observations into a root-length density using a correlation approach would be suspect based on the results of this experiment.Mention of company names or commercial products does not imply recommendation or endorsement by the United States Department of Agriculture over others not mentioned.     

Automatic discrimination of fine roots in minirhizotron images

Minirhizotrons provide detailed information on the production, life history and mortality of fine roots. However, manual processing of minirhizotron images is time-consuming, limiting the number and size of experiments that can reasonably be analysed. Previously, an algorithm was developed to automatically detect and measure individual roots in minirhizotron images. Here, species-specific root classifiers were developed to discriminate detected roots from bright background artifacts. Classifiers were developed from training images of peach (Prunus persica), freeman maple (Acer x freemanii) and sweetbay magnolia (Magnolia virginiana) using the Adaboost algorithm. True- and false-positive rates for classifiers were estimated using receiver operating characteristic curves. Classifiers gave true positive rates of 89-94% and false positive rates of 3-7% when applied to nontraining images of the species for which they were developed. The application of a classifier trained on one species to images from another species resulted in little or no reduction in accuracy. These results suggest that a single root classifier can be used to distinguish roots from background objects across multiple minirhizotron experiments. By incorporating root detection and discrimination algorithms into an open-source minirhizotron image analysis application, many analysis tasks that are currently performed by hand can be automated.     

A color composite technique for detecting root dynamics of barley (Hordeum vulgare L.) from minirhizotron images

Quantification of root dynamics by destructive methods is confounded by high coefficients of variation and loss of fine roots. The minirhizotron technique is non-destructive and allows for sequential root observations to be made at the same depth in situ. Observations can be stored on video tape which facilitates data handling and computer-aided image processing. A color composite technique using digital image analyses was adapted in this study to detect barley root dynamics from sequential minirhizotron images. Plants were grown in the greenhouse in boxes (80 × 80 × 75 cm) containing soil from a surface horizon of a Typic Cryoboroll. A minirhizotron was installed at a 45°C angle in each box. Roots intersecting the minirhizotron were observed and video-recorded at tillering, stem extension, heading, dough and ripening growth stages. The images from a particular depth were digitized from the analog video then registered to each other. Discrimination of roots from the soil matrix gave quantitative estimates of root appearance and disappearance. Changes in root appearance and disappearance were detected by assigning a separate primary color (red, green, blue) to selected growth stages, then overlaying the images to create red-green and red-green-blue color composites. The resulting composites allowed for a visual interpretation and quantification of barley root dynamics in situ.     

An inflatable minirhizotron system for root observations with improved soil/tube contact

Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images.     

应用微根管法测定细根指标方法评述

树木细根(直径<2mm)在森林生态系统能量流动和物质循环中起着重要的作用。原有的细根生产周转研究中常采用的土钻法、内生长法、挖掘法、根室法和土柱法等,均不能直接观察到细根的动态变化。微根管法是一种非破坏性、可定点直接观察和研究植物根系的方法,为研究细根的生长、衰老、死亡、分解和再生长的过程提供了有效的工具,尤其适用于细根周转、寿命和分解等方面的研究。但该技术不能直接测定单位面积的细根生物量、细根化学组成及细根周转对土壤碳和养分循环的影响,需要与土钻法结合。本文就运用微根管法对细根生物量、生产、周转和寿命等指标的研究方法进行了评述。     

Root sampling methods - applications and limitations of the minirhizotron technique

Applications and limitations of the minirhizotron technique (non-destructive) in relation to two frequently used destructive methods (soil coreing and ingrowth cores) is discussed. Sequential coreing provides data on standing crop but it is difficult to obtain data on root biomass production. Ingrowth cores can provide a quick estimate of relative fine-root growth when root growth is rapid. One limitation of the ingrowth core is that no information on the time of ingrowth and mortality is obtained.The minirhizotron method, in contrast to the destructive methods permits simultaneous calculation of fine-root length production and mortality and turnover. The same fine-root segment in the same soil space can be monitored for its life time, and stored in a database for processing. The methodological difficulties of separating excavated fine roots into living and dead vitality classes are avoided, since it is possible to judge directly the successive ageing of individual roots from the images. It is concluded that the minirhizotron technique is capable of quantifying root dynamics (root-length production, mortality and longevity) and fine-root decomposition. Additionally, by combining soil core data (biomass, root length and nutrient content) and minirhizotron data (length production and mortality), biomass production and nutrient input into the soil via root mortality and decomposition can be estimated.     

Applications of minirhizotrons to understand root function in forests and other natural ecosystems

Minirhizotrons have proved useful to understand the dynamics and function of fine roots. However, they have been used comparatively infrequently in forests and other natural plant communities. Several factors have contributed to this situation, including anomalous root distributions along the minirhizotron surface and the difficulty of extracting data from minirhizotron images. Technical and methodological advances have ameliorated some of these difficulties, and minirhizotrons have considerable potential to address some questions of long standing interest. These questions include more fully understanding the role of roots in carbon and nutrient cycling, rates of root decomposition, responses to resource availability and the functional significance of interactions between plant roots and soil organisms. Maximizing the potential for minirhizotrons to help us better understand the functional importance of fine roots in natural plant communities depends upon using them to answer only those questions appropriate to both their inherent strengths and limitations.     

A comparison between minirhizotron and monolith sampling methods for measuring root growth of maize (Zea mays L.)

Transparent plastic minirhizotron tubes have been used to evaluate spatial and temporal growth activities of plant root systems. Root number was estimated from video recordings of roots intersecting minirhizotron tubes and of washed roots extracted from monoliths of the same soil profiles at the physiological maturity stage of a maize (Zea mays L.) crop. Root length was measured by the line intercept (LI) and computer image processing (CIP) methods from the monolith samples.There was a slight significant correlation (r=0.28, p<0.005) between the number of roots measured by minirhizotron and root lengths measured by the LI method, however, no correlation was found with the CIP method. Using a single regression line, root number was underestimated by the minirhizotron method at depths between 0–7.6 cm. A correlation was found between root length estimated by LI and CIP. The slope of estimated RLD was significant with depth for these two methods. Root length density (RLD) measured by CIP showed a more erratic decline with distance from the plant row and soil surface than the LI method.     

Measuring Fine Root Turnover in Forest Ecosystems

Development of direct and indirect methods for measuring root turnover and the status of knowledge on fine root turnover in forest ecosystems are discussed. While soil and ingrowth cores give estimates of standing root biomass and relative growth, respectively, minirhizotrons provide estimates of median root longevity (turnover time) i.e., the time by which 50% of the roots are dead. Advanced minirhizotron and carbon tracer studies combined with demographic statistical methods and new models hold the promise of improving our fundamental understanding of the factors controlling root turnover. Using minirhizotron data, fine root turnover (y⁻¹) can be estimated in two ways: as the ratio of annual root length production to average live root length observed and as the inverse of median root longevity. Fine root production and mortality can be estimated by combining data from minirhizotrons and soil cores, provided that these data are based on roots of the same diameter class (e.g., < 1 mm in diameter) and changes in the same time steps. Fluxes of carbon and nutrients via fine root mortality can then be estimated by multiplying the amount of carbon and nutrients in fine root biomass by fine root turnover. It is suggested that the minirhizotron method is suitable for estimating median fine root longevity. In comparison to the minirhizotron method, the radio carbon technique favor larger fine roots that are less dynamics. We need to reconcile and improve both methods to develop a more complete understanding of root turnover.     

Fine root dynamics of pedunculate oak and narrow-leaved ash in a mixed-hardwood plantation in clay soils

Plant and Soil - The minirhizotron technique was used to study the temporal dynamics of fine roots (diameter ≤2&;nbsp;mm) over a twelve-month period in pedunculate oak (Quercus robur L.)...     

Advancing the use of minirhizotrons in wetlands

Background

Wetlands store a substantial amount of carbon (C) in deep soil organic matter deposits, and play an important role in global fluxes of carbon dioxide and methane. Fine roots (i.e., ephemeral roots that are active in water and nutrient uptake) are recognized as important components of biogeochemical cycles in nutrient-limited wetland ecosystems. However, quantification of fine-root dynamics in wetlands has generally been limited to destructive approaches, possibly because of methodological difficulties associated with the unique environmental, soil, and plant community characteristics of these systems. Non-destructive minirhizotron technology has rarely been used in wetland ecosystems.

Scope

Our goal was to develop a consensus on, and a methodological framework for, the appropriate installation and use of minirhizotron technology in wetland ecosystems. Here, we discuss a number of potential solutions for the challenges associated with the deployment of minirhizotron technology in wetlands, including minirhizotron installation and anchorage, capture and analysis of minirhizotron images, and upscaling of minirhizotron data for analysis of biogeochemical pools and parameterization of land surface models.

Conclusions

The appropriate use of minirhizotron technology to examine relatively understudied fine-root dynamics in wetlands will advance our knowledge of ecosystem C and nutrient cycling in these globally important ecosystems.     

Soil temperature effects from minirhizotron lighting systems

Observing root dynamics or soil fauna with minirhizotrons requires the use of incandescent or ultraviolet (UV) lighting systems. These light sources can generate heat which would be transferred to the surrounding soil adjacent to the minirhizotron observation tubes and thus may influence root growth and development or fauna activity. The objective of this study was to determine the effect of incandescent and UV light from a minirhizotron camera system on soil temperatures next to minirhizotron tubes. Temperature probes were attached next to and at 0.5 cm from the tube surface and the tubes were then placed in boxes with either a fine sand or a loamy clay soil. Incandescent light was operated stationary for 5 min or moved at 1 cm increments every 10 s down the tube for both dry and wet soils. The UV light was used in a stationary position for 10 minutes in both dry soils. Maximum temperature increases were 3.41–3.52 °C and 1.69–2.14 °C next to the tube for the dry and wet soils, respectively with 5 min of stationary incandescent light. Ultraviolet lights increased soil temperatures to a maximum of approximately 2.5 °C in the dry soil. Probes placed 0.5 cm from the tube surface also showed temperature increases up to 2.15 °C. Moving the light source every 10 s, however, resulted in lower temperature increases (<0.8 °C). Therefore short durations of light resulted in small temperature increases suggesting minimal impact on root development. Increased soil temperatures from longer durations of light, however, may alter root growth and development as well as soil fauna activity and warrants further study.     

Root architecture and wind-firmness of mature Pinus pinaster

This study aims to link three-dimensional coarse root architecture to tree stability in mature timber trees with an average of 1-m rooting depth. Undamaged and uprooted trees were sampled in a stand damaged by a storm. Root architecture was measured by three-dimensional (3-D) digitizing. The distribution of root volume by root type and in wind-oriented sectors was analysed. Mature Pinus pinaster root systems were organized in a rigid 'cage' composed of a taproot, the zone of rapid taper of horizontal surface roots and numerous sinkers and deep roots, imprisoning a large mass of soil and guyed by long horizontal surface roots. Key compartments for stability exhibited strong selective leeward or windward reinforcement. Uprooted trees showed a lower cage volume, a larger proportion of oblique and intermediate depth horizontal roots and less wind-oriented root reinforcement. Pinus pinaster stability on moderately deep soils is optimized through a typical rooting pattern and a considerable structural adaptation to the prevailing wind and soil profile.     

森林生态系统根系生物量研究进展

在森林生态系统功能过程研究中,特别是在生产力和生物地化学循环方面,根系的作用不容忽视,但是,由于研究方法和研究者的观念等方面的限制,对于根系的研究还远不及地上部分受到重视。而关于细根生物量,周转率和生产力等方面的是很少有人问津。为推动我国根系生物学研究的发展,本一面地介绍了９种典型的测度细根生物量的方法。包括：收获法、钻土芯法、内生长土芯法、平衡法、根观测实验室法、土壤碳平衡法、挖土块法、间接法和     

A comparison of minirhizotron techniques for estimating root length density in soils of different bulk densities

Flexible- and rigid-walled minirhizotron techniques were compared for estimating root length density of 14- to 28-day-old Pinto bean (Phaseolus vulgaris L.) and spring whet (Triticum aestivum L.) plants in soil boxes under controlled environment conditions at three soil bulk densities (1.3, 1.5 and 1.7 g cm^–3). The flexible-tube system consisted of bicycle inner tubes inflated inside augered access holes and removed only when measurements were taken. Rigid tubes were constructed of extruded polybutyrate plastic. In both cases tubes were oriented horizontally. Despite similar root densities for wheat and beans based on measurements obtained from soil cores, root densities estimated from both types of minirhizotron were higher in bean than in wheat in uncompacted soil. Estimates of root density by the flexible tube minirhizotron were more closely correlated with soil core image analysis estimates than were those by the rigid minirhizotron system. At high soil bulk density, rigid tube measurements consistently overestimated actual rooting density of both wheat and bean. The relationship between estimated and actual rooting densities in the case of flexible tube measurements was not significantly influenced by soil bulk density. These findings were consistent with the theory that preferential root growth is induced by gaps at the soil-observation tube interface, inherent in the rigid tube technique, and was accentuated under conditions of high soil strength.     

Estimating Root Production: Comparison of 11 Methods in Shortgrass Steppe and Review of Biases

Estimating root production has been difficult due to multiple potential biases associated with both old and new methods. This shortgrass steppe site is the only place we are aware of that can compare most methods including sequential coring, ingrowth cores, and ingrowth donuts, ¹⁴C pulse-isotope dilution, ¹⁴C pulse-isotope turnover, rhizotron windows, and minirhizotron, and indirect methods including nitrogen budget, carbon flux, simulation carbon flow model, and regression model. We used the studies at this site, other comparisons, a summary of potential directional biases, and different ways of calculating estimates in a logical, comparative approach of evaluating methods. Much of the literature for root production is based on sequential biomass coring, a method resulting in erroneous estimates. Root ingrowth estimates of production are generally conservative compared to minirhizotron and isotope turnover methods. The size of the ingrowth area may be the most important determinant of the underestimation. Estimates based on pulse-isotope dilution are also erroneous due to non-uniform labeling of tissues. Uniform labeling is not an assumption of the pulse-isotope turnover method, and this method has the least severe potential biases. Root production estimates from pulse-isotope turnover were lower than those using minirhizotron when the most common method of calculation was used. This agrees with literature concerning bomb ¹⁴C continuous-isotope labeling comparisons with minirhizotron, although some potential biases between isotope methods are different. However, good agreement between pulse-isotope turnover and minirhizotron were obtained when minirhizotron estimates were calculated from regression of decomposition versus production to equilibrium and when pulse-isotope turnover estimates were calculated from two-phase life-span regressions. This minirhizotron method bypasses biases associated with the artificial surface similar to root-cohort methods that may be practical only in mesic systems, and takes into account both short- and long-lived roots and corrects for soil-isotope contamination that the continuous-isotope labeling bomb ¹⁴C method is not able to account for. Comparisons of these direct methods are also made with four indirect methods.     

A plane intersect method for estimating fine root productivity of trees from minirhizotron images

The advent of minirhizotrons more than a decade ago has made the careful and widespread study of fine root dynamics of trees possible. However, to this day, the estimation of fine root productivity in terms of mass production per unit of ground surface from the minirhizotron data remains hampered by the difficulty in transforming images of roots captured along a two-dimensional plane into estimates of root volume or mass within a soil volume. In this work, we propose that the date of fine root appearance and the diameter of fine roots are the most robust variables that can be obtained from minirhizotron measurements of tree roots and that these two variables should be the basis of productivity estimates. The method proposed for estimating fine root productivity expands the line intersect method of Van Wagner (1968) into a plane intersect method that permits, with the appropriate volumetric transformations and corrections for tube and slope angles, the estimation of fine root productivity per unit ground area for specific periods. Examples of calculations are presented for two datasets obtained within two different forested sites, as well as a comparison with a methodology based on camera depth-of-view. The main weakness of the plane intersect method is the assumption that all fine root segments are independent. The correction for the fraction of coarse particles also creates uncertainty in the final estimate.     

Improved scaling of minirhizotron data using an empirically-derived depth of field and correcting for the underestimation of root diameters

Background and aims

Accurate data on the standing crop, production, and turnover of fine roots is essential to our understanding of major terrestrial ecological processes. Minirhizotrons offer a unique opportunity to study the dynamic processes of root systems, but are susceptible to several measurement biases.

Methods

We use roots extracted from minirhizotron tube surfaces to calculate the depth of field of a minirhizotron image and present a model to correct for the underestimation of root diameters obscured by soil in minirhizotron images.

Results

Non-linear regression analysis resulted in an estimated depth of field of 0.78 mm for minirhizotron images. Unadjusted minirhizotron data underestimated root net primary production and fine root standing crop by 61 % when compared to adjusted data using our depth of field and root diameter corrections. Changes in depth of field accounted for >99 % of standing crop adjustments with root diameter corrections accounting for <1 %.

Conclusions

Our results represent the first effort to empirically derive depth of field for minirhizotron images. This work may explain the commonly reported underestimation of fine roots using minirhizotrons, and stands to improve the ability of researchers to accurately scale minirhizotron data to large soil volumes.     

A new dimension to observations in minirhizotrons: A stereoscopic view on root photographs

Summary With a stereoscope, as used for the inspection of aerial photographs, sequential photographs of roots obtained by the endoscope method from minirhizotrons can yield much more information than hitherto. A series of photographs shows that most of the roots seen in a minirhizotron in grassland grew on the surface of the lexan tube, while there was a gap between the roots and the soil. Decay of the extensive root hair zones around the roots may make new root growth in the gap between rhizotron wall and soil invisible. Some consequences of these observations for the endoscope method are discussed.