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.     

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.     

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.     

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.     

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.     

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.     

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.     

Root growth dynamics of Brussels sprouts (Brassica olearacea var.gemmifera) and leeks (Allium porrum L.) as reflected by root length,root colour and UV fluorescence

Minirhizotron observations of roots of leeks and Brussels sprouts grown in the Wageningen Rhizolab were used to study the dynamics of root length. Day of appearance and the time of decay were assessed for individual root segments visible on the minirhizotron surface.A Brussels sprouts crop produced much more root length than leeks, but the average longevity of these roots was about half that of leek roots.To investigate whether root colour or UV fluorescence could be used as a quantitative index of root functionality or root age, changes in root colour (on a scale of greys) over time were measured with interactive image analysis. In both crops a gradual change towards black was found with ageing. Measurements of the intensity of the UV fluorescence showed that leek roots fluoresced more than Brussels sprouts roots. Over time, UV fluorescence decreased in Brussels sprouts roots but increased in leek roots. It is concluded that UV fluorescence cannot be used as a universal indicator of root age or root functionality, but in some plant species it may be used to separate (transparent) roots from the background with image analysis techniques.     

用Minirhizotrons观测柠条根系生长动态

 Minirhizotrons是一种非破坏性、定点直接观察和研究植物根系的新方法。该文介绍了用Minirhizotrons测定植物根系的方法,并同根钻取原状土样法进行了比较;探讨了根系生长动态同土壤含水量间的关系。试验于2004年植物生长季在沙坡头沙漠试验研究站的水分平衡观测场的人工柠条(Caragana korshinskii)林进行,结果表明：Minirhizotrons 管埋入土壤后需要10个月时间允许柠条根系在其周围定居,其观测图片中的根系代表了管子周围2.6 mm土层的根系。柠条根系生长动态和土壤水分变化相关,含水量的升高导致根系的大量繁殖,而根系吸水及蒸发散又导致含水量的减少;在2004年植物生长季,土壤水分和根系的这种相互作用出现了两次,但根系生长高峰比土壤含水量高峰滞后20 d左右。     

A comparison of soil core sampling and minirhizotrons to quantify root development of field-grown potatoes

Root growth of potato (Solanum tuberosum L.) is sensitive to soil conditions. A reduced root system size can result in reduced uptake of water and/or nutrients, leading to impaired crop growth. To understand the mechanisms by which soil conditions affect crop growth, study of temporal and spatial development of roots is required.In field experiments, effects of soil temperature, soil compaction and potato cyst nematodes (Globodera pallida) on root growth of potato cultivars were studied using two methods: core sampling and vertically oriented minirhizotrons.Minirhizotrons showed relatively more roots in deeper soil layers than core sampling, probably because of preferential root growth along the tube. Spatial distribution of roots should therefore be analysed by core sampling.To eliminate differences in spatial distribution, total root systems as measured by both methods were compared. Nematodes, cultivars and time did not affect the relationship between both methods. Soil compaction, however, affected it because of a strong response of root length in bulk soil and small differences in root number against the minirhizotron, suggesting that soil coring has to be used to study effects of different bulk densities.With both methods, sequential measurements of roots give the net effect of root growth and decay. Data on root turnover can only be obtained with minirhizotrons by comparing video recordings of different dates. Other information obtained with minirhizotrons is the average orientation of roots. Moreover, the minirhizotron method has the advantage of demanding less labour.     

Rates of rhizodeposition and ammonium depletion in the rhizosphere of axenic oat roots

Summary A multiple split root chamber and artificial soil were developed which allowed for maintenance of axenic conditions and for the isolation of soil from specific regions of single roots. A sterile minirhizotron was used to measure patterns and rates of root extension under sterile conditions. Carbon and nitrogen distributions in the rhizosphere of sterile oat roots were measured in combination with rates of root elongation to calculate specific rates of rhizodeposition and ammonium nitrogen uptake. The highest rates of rhizodeposition C production and N depletion occurred at the root tip (first day segment). Rhizodeposited soluble and insoluble C compounds represented up to 50% of the standing root biomass C. Within 48 hours after root entry, levels of rhizosphere ammonium-N decreased by 40–50%. The results were summarized in a simple model of root growth, rhizodeposition, and NH ₄ ⁺ −H uptake. From a dissertation by the senior author submitted to the Academic Faculty of Colorado State University in partial fulfillment of the requirements for the Ph.D. degree.     

Acoustic classification of dolphins in the California Current using whistles,echolocation clicks,and burst pulses

Passive acoustic monitoring of dolphins is limited by our ability to classify calls to species. Significant overlap in call characteristics among many species, combined with a wide range of call types and acoustic behavior, makes classification of calls to species challenging. Here, we introduce BANTER, a compound acoustic classification method for dolphins that utilizes information from all call types produced by dolphins rather than a single call type, as has been typical for acoustic classifiers. Output from the passive acoustic monitoring software, PAMGuard, was used to create independent classifiers for whistles, echolocation clicks, and burst pulses, which were then merged into a final, compound classifier for each species. Classifiers for five species found in the California Current ecosystem were trained and tested using 153 single‐species acoustic events recorded during a 4.5 mo combined visual and acoustic shipboard cetacean survey off the west coast of the United States. Correct classification scores for individual species ranged from 71% to 92%, with an overall correct classification score of 84% for all five species. The conceptual framework of this approach easily lends itself to other species and study areas as well as to noncetacean taxa.     

水曲柳和兴安落叶松人工林细根分解研究

  细根分解是陆地生态系统C和养分循环的重要环节。以往的细根分解研究以埋袋法的应用为主。然而, 由于埋袋法对分解材料的干扰以及对分解环境的改变使其很难揭示原位环境下根系的自然分解过程。该研究应用微根管(Minirhizotron)技术连续3年对水曲柳(Fraxinus mandshurica)和兴安落叶松(Larix gmelinii)细根的分解过程进行原位监测, 运用Kaplan–Meier方法估算细根分解的保存率及分解期中位值(即50%细根完全分解的时间, Median root decomposition time), 做分解曲线, 用对数秩检验(Log-rank test)方法分析不同树种、直径、根序及土层对细根保存率的影响。结果表明, 伴随时间延长, 细根的保存率逐渐下降, 兴安落叶松细根保存率的下降显著快于水曲柳(p<0.001), 两树种分解期中位值分别为(82±7) d 和(317±28) d; 不同直径等级(≤0.3、0.3～0.6、＞0.6 mm)细根的分解速率不同, 两树种最长分解期中位值均出现在最细直径(≤0.3 mm)根中; 高级根分解速率显著低于一级根(p<0.05); 土壤上层分解速度快, 随着土壤深度增加细根分解速率减小。微根管技术为了解细根自然分解过程提供了有效途径。     

Modelling minirhizotron observations to test experimental procedures

In order to help design experiments with minirhizotrons or interpret data from such experiments, a modelling approach is a valuable tool to complement empirical approaches. The general principle of this modelling approach is to calculate and to study the part of a theoretical root system that is intersected by passes through a virtual minirhizotron tube (modelled here as a cylinder). Various outputs can be calculated from this part of the root system, and related to the surrounding root system which is perfectly known, since it has been simulated and stored in a data structure. Therefore, the method involves two levels of modelling that are presented and discussed: the root system architecture of a crop, and the observations that can be achieved with minirhizotron tubes. Illustrations of the method are presented to study the effect of several factors on the rooting depth curves, and to show how images may be calculated to mimic what can actually be viewed from inside the tube. These first results show that the maximum rooting depth curves, as virtually observed in the minirhizotron tube, present large variations and strongly underestimate the maximum rooting depth of the modelled root system (up to 60 cm in average). The underestimation is still more critical when the radius of the tube is lower than 3 cm, and when the tube is close to the vertical (angle lower than 0.2 rad). The use of the 0.9 quantile instead of the average value, for each of the observation dates, leads to a better estimation of the maximum rooting depth.     

Fine root biomass estimates from minirhizotron imagery in a shrub ecosystem exposed to elevated CO2

Fine root biomass and C content are critical components in ecosystem C models, but they cannot be directly determined by minirhizotron techniques, and indirect methods involve estimating 3-dimensional values (biomass/ soil volume) from 2-dimensional measurements. To estimate biomass from minirhizotron data, a conversion factor for length to biomass must be developed, and assumptions regarding depth of view must be made. In a scrub-oak ecosystem in central Florida, USA, root length density (RLD) was monitored for 10 years in a CO₂ manipulation experiment using minirhizotron tubes. In the seventh year of the study, soil cores were removed from both ambient and elevated CO₂ chambers. Roots from those cores were used to determine specific root length values (m/g) that were applied to the long-term RLD data for an estimation of root biomass over 10 years of CO₂ manipulation. Root length and biomass estimated from minirhizotron data were comparable to determinations from soil cores, suggesting that the minirhizotron biomass model is valid. Biomass estimates from minirhizotrons indicate the <0.25 mm diameter roots accounted for nearly 95% of the total root length in 2002. The long-term trends for this smallest size class (<0.25 mm diameter) mirrored the RLD trends closely, particularly in relation to suspected root closure in this system. Elevated CO₂ did not significantly affect specific root length as determined by the soil cores. A significant treatment effect indicated smallest diameter fine roots (<0.25 mm) were greater under elevated CO₂ during the early years of the study and the largest (2–10 mm) had greater biomass under elevated CO₂ during the later years of the study. Overall, this method permits long-term analysis of the effects of elevated CO₂ on fine root biomass accumulation and provides essential information for carbon models.     

Environment-induced Modifications to Root Longevity in Lolium perenne and Trifolium repens

Understanding how environmental factors affect the longevityof roots is essential if root mortality linked nutrient cyclingprocesses within ecosystems are to be understood, and the impactof natural and anthropogenic climate change properly evaluated.In this study the longevity of roots at two geographically andclimatically distinct sites were compared to identify the scaleof change that can occur due to environmental differences. Minirhizotrontubes were inserted into swards sown with the same variety ofLolium perenne and Trifolium repens at sites in the UK and Italy.Roots were viewed using a video camera and digitized imagesof roots generated at intervals. From these images the lifehistory of individual roots was determined and compared. Therewere few differences in patterns of longevity between differentspecies at the same site. Major differences, however, were observedbetween roots of the same species at different sites. For example,73% of Trifolium repens roots failed to survive for 21 d inItaly compared to only 29% at the UK site. Similarly, over 84%of Lolium perenne roots failed to survive for more than 21 din Italy compared to 38% in the UK. These data suggest thatenvironmental factors can have a major impact on root longevity.Copyright 2000 Annals of Botany Company Climate change, Lolium perenne L., minirhizotron, perennial ryegrass, root longevity, Trifolium repens L., white clover     

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.     

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.     

Estimating Fine Root Turnover in Tropical Forests along an Elevational Transect using Minirhizotrons

Growth and death of fine roots represent an important carbon sink in forests. Our understanding of the patterns of fine root turnover is limited, in particular in tropical forests, despite its acknowledged importance in the global carbon cycle. We used the minirhizotron technique for studying the changes in fine root longevity and turnover along a 2000-m-elevational transect in the tropical mountain forests of South Ecuador. Fine root growth and loss rates were monitored during a 5-mo period at intervals of four weeks with each 10 minirhizotron tubes in three stands at 1050, 1890, and 3060 m asl. Average root loss rate decreased from 1.07 to 0.72 g/g/yr from 1050 to 1890 m, indicating an increase in mean root longevity with increasing elevation. However average root loss rate increased again toward the uppermost stand at 3060 m (1.30 g/g/yr). Thus, root longevity increased from lower montane to mid-montane elevation as would be expected from an effect of low temperature on root turnover, but it decreased further upslope despite colder temperatures. We suggest that adverse soil conditions may reduce root longevity at high elevations in South Ecuador, and are thus additional factors besides temperature that control root dynamics in tropical mountain forests.     

Apical diameter and branching density affect lateral root elongation rates in banana

Thick roots elongate faster than thinner ones. However, within one species, the growth achieved by roots of a given diameter can be very variable, and root diameter can only be considered as a determinant of root potential elongation rate. As root elongation is highly correlated to carbon availability, it can be hypothesized that local competition for resources, expressed as the number of lateral roots per unit length (i.e. the branching density), modulates root elongation. Using novel methods in field conditions, we have estimated apical diameters, elongation rates and growth durations of nearly 3500 banana lateral roots, in a field experiment with high radiations and a shaded glasshouse experiment with low radiations. Apical diameters and branching densities were lower in the experiment with low radiation, but elongation rates were higher. In both experiments, mean elongation rates of first-order laterals and thick second-order laterals were negatively correlated with bearing root branching densities. It is hypothesized that, even though apical diameters were lower, low branching densities in the shaded glasshouse allowed enhanced lateral root elongation. In both experiments, second-order laterals elongated more slowly than first-order laterals of similar diameter. A specific effect of root order, independent of branching density and apical diameter, contributed to explain these slow second-order lateral elongation rates. Most lateral roots elongated between 9 and 21 days and growth duration was mainly correlated with root diameter.