Automated image analyses for separating plant roots from soil debris elutrated from soil cores

Historically, destructive root sampling has been labor intensive and requires manual separation of extraneous organic debris recovered along with the hydropneumatic elutriation method of separating plant roots from soils. Quantification of root system demographics by public domain National Institute of Health (NIH-Image) and Root Image Processing Laboratory (RIPL) image processing algorithms has eliminated much of the labor-intensive manual separation. This was accomplished by determining the best length to diameter ratio for each object during image analyses. Objects with a length to diameter ratio less than a given threshold are considered non-root materials and are rejected automatically by computer algorithms. Iterative analyses of length to diameter ratios showed that a 15:1 ratio was best for separating images of maize (Zea mays L.) roots from associated organic debris. Using this threshold ratio for a set of 24 soil cores, a highly significant correlation (r² = 0.89) was obtained between computer image processed total root length per core and actual root length. A linear relationship (r² = 0.80) was observed between root lengths determined by NIH-Image analyses and lengths determined independently by the RIPL imaging system, using the same maize root + debris samples. This correlation demonstrates that computer image processing provides opportunities for comparing root length parameters between different laboratories for samples containing debris.     

A method to separate plant roots from soil and analyze root surface area

Analysis of the effects of soil management practices on crop production requires knowledge of these effects on plant roots. Much time is required to wash plant roots from soil and separate the living plant roots from organic debris and previous years’ roots. We developed a root washer that can accommodate relatively large soil samples for washing. The root washer has a rotary design and will accommodate up to 24 samples (100 mm diam. by 240 mm long) at one time. We used a flat-bed scanner to digitize an image of the roots from each sample and used a grid system with commercially-available image analysis software to analyze each sample for root surface area. Sensitivity analysis and subsequent comparisons of ‘dirty’ samples containing the roots and all the organic debris contained in the sample and ‘clean’ samples where the organic debris was manually removed from each sample showed that up to 15% of the projected image could be coveredwith debris without affecting accuracy and precision of root surface area measurements. Samples containing a large amount of debris may need to be partitioned into more than one scanning tray to allow accurate measurements of the root surface area. Sample processing time was reduced from 20 h, when hand separation of roots from debris was used, to about 0.5 h, when analyzing the image from an uncleaned sample. The method minimizes the need for preprocessing steps such as dying the roots to get better image contrast for image analysis. Some information, such as root length, root diameter classes and root weights, is not obtained when using this technique. Root length measurements, if needed, could be made by hand on the digital images. Root weight measurement would require sample cleaning and the advantage of less processing time per sample with this method would be lost. The significance of the tradeoff between information not obtained using this technique and the ability to process a greater number of samples with the time and personnel resources available must be determined by the individual researcher and research objectives.     

Challenges and opportunities for quantifying roots and rhizosphere interactions through imaging and image analysis

The morphology of roots and root systems influences the efficiency by which plants acquire nutrients and water, anchor themselves and provide stability to the surrounding soil. Plant genotype and the biotic and abiotic environment significantly influence root morphology, growth and ultimately crop yield. The challenge for researchers interested in phenotyping root systems is, therefore, not just to measure roots and link their phenotype to the plant genotype, but also to understand how the growth of roots is influenced by their environment. This review discusses progress in quantifying root system parameters (e.g. in terms of size, shape and dynamics) using imaging and image analysis technologies and also discusses their potential for providing a better understanding of root:soil interactions. Significant progress has been made in image acquisition techniques, however trade‐offs exist between sample throughput, sample size, image resolution and information gained. All of these factors impact on downstream image analysis processes. While there have been significant advances in computation power, limitations still exist in statistical processes involved in image analysis. Utilizing and combining different imaging systems, integrating measurements and image analysis where possible, and amalgamating data will allow researchers to gain a better understanding of root:soil interactions.     

Portable rhizotron and color scanner system for monitoring root development

Rhizotrons allow the examination of spatial and temporal in situ root development. Permanent rhizotron installations provide 2-D images of whole root profiles, but their immobility limits the number of soil-plant systems that can be studied. Our objectives were to develop a portable rhizotron and color scanning system for studying the development of whole root systems. Potato root development was monitored in an irrigated experiment at Othello, WA. Covered, rectangular hollow boxes with a transparent glass face were installed perpendicular to planted potato rows, and a seed piece was planted in the soil adjacent to the glass. Rooting in the hill furrow topography was measured at 2 to 4 week intervals. Images of roots growing along the glass face are captured with five scans with a portable, color scanner and a portable computer. Image thresholding discriminated roots from soil using primary color values, color intensity differences, color proportions, or overall intensity. Seasonal patterns of computed root lengths by image analysis were comparable to manual tracing. Primary roots extended to 15 cm from the seed piece prior to shoot emergence, 21 days after planting. Lateral roots began to develop shortly thereafter. Potato roots extended to depths of 60 cm by 4 to 6 weeks after planting, and maximum root density in the hill and furrow was observed by tuber initiation to early tuber bulking. Temporal and spatial trends were similar to previous results using destructive sampling. The method has promise for studying the root growth and development of field-grown plants.     

Visualization of Toyoura sand-grown plant roots by X-ray computer tomography

Plants establish their root system as a three-dimensional structure, which is then used to explore the soil to absorb resources and provide mechanical anchorage. Simplified two-dimensional growth systems, such as agar plates, have been used to study various aspects of plant root biology. However, it remains challenging to study the more realistic three-dimensional structure and function of roots hidden in opaque soil. Here, we optimized X-ray computer tomography (CT)-based visualization of an intact root system by using Toyoura sand, a standard silica sand used in geotechnology research, as a growth substrate. Distinct X-ray attenuation densities of root tissue and Toyoura sand enabled clear image segmentation of the CT data. Sorghum grew especially vigorously in Toyoura sand and it could be used as a model for analyzing root structure optimization in response to mechanical obstacles. The use of Toyoura sand has the potential to link plant root biology and geotechnology applications.     

Root-soil contact of maize,as measured by a thin-section technique

In models of oxygen, water and nutrient uptake by plant roots, the degree of root-soil contact is an important parameter. An observation technique is required to evaluate to what extent root-soil contact depends on plant species, soil texture and structure. Thin sections for studying soil structure may be used for this purpose, provided that roots do not shrink during section preparation, and that all root cross sections are recognized.Maize was grown in pots with soil aggregates obtained by sieving and compacting to three bulk densities. Thin sections were made by freeze-drying samples before impregnating the soil with resin. Two checks were made on the validity of the method. Firstly, visual appearance of roots with intact epidermis, cortex and other tissues did not show signs of shrinkage. Secondly, the agreement was checked between root lengths obtained by washing duplicate soil samples and the number of root cross sections counted on horizonal and vertical thin sections. For the latter, the angle at which roots intersected the thin-section plane was determined from the shape of the cross sections. The frequency distribution of calculated angles was in agreement with the frequency distribution expected for a randomly oriented set of cylinders when an error term was included in the simulated measurements.Some results are presented for a field test of the thin-section method with barley on a calcareous marine sandy loam. Root hairs, apparently undamaged by sample preparation, are important for bridging the gap between roots and soil in this situation. According to the experience presented, the thin-section technique is suitable to derive the degree of root-soil contact, as influenced by species, soil texture and structure, in samples obtained from pot or field experiments.Communication No. 43 of the Dutch Programme on Soil Ecology of Arable Farming Systems.Communication No. 43 of the Dutch Programme on Soil Ecology of Arable Farming Systems.     

Root effects on soil water and hydraulic properties

Plants can affect soil moisture and the soil hydraulic properties both directly by root water uptake and indirectly by modifying the soil structure. Furthermore, water in plant roots is mostly neglected when studying soil hydraulic properties. In this contribution, we analyze effects of the moisture content inside roots as compared to bulk soil moisture contents and speculate on implications of non-capillary-bound root water for determination of soil moisture and calibration of soil hydraulic properties. In a field crop of maize (Zea mays) of 75 cm row spacing, we sampled the total soil volumes of 0.7 m × 0.4 m and 0.3 m deep plots at the time of tasseling. For each of the 84 soil cubes of 10 cm edge length, root mass and length as well as moisture content and soil bulk density were determined. Roots were separated in 3 size classes for which a mean root porosity of 0.82 was obtained from the relation between root dry mass density and root bulk density using pycnometers. The spatially distributed fractions of root water contents were compared with those of the water in capillary pores of the soil matrix. Water inside roots was mostly below 2–5% of total soil water content; however, locally near the plant rows it was up to 20%. The results suggest that soil moisture in roots should be separately considered. Upon drying, the relation between the soil and root water may change towards water remaining in roots. Relations depend especially on soil water retention properties, growth stages, and root distributions. Gravimetric soil water content measurement could be misleading and TDR probes providing an integrated signal are difficult to interpret. Root effects should be more intensively studied for improved field soil water balance calculations. Presented at the International Conference on Bioclimatology and Natural Hazards, Pol’ana nad Detvou, Slovakia, 17–20 September 2007.     

Soil Anti-Scouribility Enhanced by Plant Roots

The magnitude of soil anti-scouribility depends on the physical condition of the soil. Plant roots can greatly enhance soil stability and anti-erodibility. A scouring experiment of undisturbed soil was conducted to investigate the effects of roots on soil anti-scouribility and its distribution in the soil profile. At the end of each erosion test, plant roots were collected from soil samples and root surface area was calculated by means of a computer image analysis system (CIAS). Root surface area density (RSAD), the surface area of the roots per unit of soil volume, was related to soil anti-scouribility. More than 83% of root surface area was concentrated in the 0 - 30 cm soil layer. Soil anti-scouribility increased with an increase in RSAD and the value of intensified soil anti-scouribility (△AS) can be expressed by exponential equations, depending on the plant species. These equations were △AS = 9.578 6 RSAD0.8321 (R2 = 0.951) for afforested Pinus tabulaeformis Cart., △AS = 7.808 7 RSAD0.7894 (R2 = 0.974) for afforested Robinia pseudoacacia L., and △AS = 9.256 6 RSAD0.8707 (R2 = 0.899) for Bothriochloa ischemum L.     

Non-invasive imaging of roots with high resolution X-ray micro-tomography

X-ray micro-tomography is a well-established technique for non-invasive imaging and evaluation of heterogeneous materials. An inexpensive X-ray micro-tomography system has been designed and built for the specific purposes of examining root growth and root/soil interactions. The system uses a silver target X-ray source with a focal spot diameter of 80 m, an X-ray image intensifier with a sampling aperture of about 100 m, and a sample with a diameter of 25 mm. Pre-germinated wheat and rape seeds were grown for up to 8–10 days in plastic containers in a sandy loam soil sieved to < 250 m, and imaged with the X-ray system at regular intervals. The quality of 3 D image obtained was good allowing the development and growth of both root axes and some first-order laterals to be observed. The satisfactory discrimination between soil and roots enabled measurements of root diameter (wheat values were 0.48–1.22 mm) in individual tomographic slices and, by tracking from slice to slice, root lengths were also measured. The measurements obtained were generally within 10% of those obtained from destructive samples measured manually and with a flat-bed scanner. Further developments of the system will allow more detailed examination of the root:soil interface.     

An approach to minirhizotron root image analysis

Minirhizotrons speed up research on root demography, but image quality often hampers standardization of the image processing method. A simple procedure working on the blue band of colour images was tested on fibrous roots of sugarbeet (Beta vulgaris var. saccharifera). With respect to green and red, the blue band allows better detection of roots when their luminance is very similar to that of the background. The method makes use of an exponential algorithm of contrast stretching, which takes luminance frequency distribution into account. Based on a single threshold level, the procedure includes skeletonization. A minimum segment length was adopted to discriminate roots from extraneous objects. Although a specific minimum root length (MRL) value was calculated for each soil type, results show that a single value can be applied, indicating that this method can be profitably used for processing large samples of images. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of soil conditions in fine root ecomorphology in Norway spruce (Picea abies (L.) Karst.)

The present study is an attempt to investigate the pattern of morphological variability of the short roots of Norway spruce (Picea abies (L.) Karst.) growing in different soils. Five root parameters – diameter, length and dry weight of the root tip, root density (dry weight per water-saturated volume) and specific root area (absorbing area of dry weight unit) were studied with respect to 11 soil characteristics using CANOCO RDA analysis. The investigation was conducted in seven study areas in Estonia differing in site quality class and soil type. Ten root samples per study area were collected randomly from the forest floor and from the 20 cm soil surface layer. Eleven soil parameters were included in the study: humus content, specific soil surface area, field capacity, soil bulk density, pH (KCl and H₂O dilution's), N and Ca concentrations, Ca/Al and C/N ratios, and the decomposition rate of fine roots (<2 mm dia.). Root morphological characteristics most strongly related to the measured soil characteristics in the different sites were specific root area, root density and diameter of the short roots, the means varying from 29 to 42 m² kg⁻¹, from 310 to 540 kg m⁻³ and from 0.26 to 0.32 mm, respectively; root density being most sensitive. The most favourable site and soil types resulting in fine roots with morphological characteristics for optimizing nutrient uptake (e.g. low short root density and high specific root area) were Umbric Luvisol (Oxalis), Dystric Gleysol (Oxalis) and Gleyic Luvisol (Hepatica). These soil types correspond to highly productive natural forest stands of Norway spruce in Estonia. All measured soil variables explained 28% of total variance of the root characteristics. The most important variables related to root morphology were the humus content, field capacity and specific soil surface area. This revised version was published online in June 2006 with corrections to the Cover Date.     

Root distribution at various distances from clove trees growing in Indonesia

Efficient fertilizer application requires an understanding of the distribution of roots and soil nutrients in the soil profile. Cultural practices for clove trees in Indonesia has resulted in phosphorus (P) fertilizer being applied at the canopy edge; however, in these high P fixing soils efficient P fertilizer application should occur with the highest root densities. The objective of this study, therefore, was to determine the rooting distribution at various distances from the tree and soil depths for clove (Eugenia aromatica OK; variety Zanzibar) trees growing on an Andosol soil at Modoinding, Indonesia. Root distributions were determined to a 100-cm soil depth using soil cores at 0.5, 1.0 and 1.5 times the canopy radius for five 10-year-old clove trees grown on either level terrain or 23% slopes. Clove root length and weight densities decreased with soil depth and distance from the tree base. Fine clove roots (1 mm dia) comprised 72% of the total root length and was three to five times higher underneath the canopy than that outside the canopy. Roots were concentrated in the upper soil horizons; however, up to 36% of the total root length was found at a depth of 50–100 cm. Clove roots for trees growing at the level landscape position had the highest root length densities. Intercropped species root length densities were higher than clove root length densities at 1.5 times the canopy radius whereas intercropped root weight densities were higher than that for clove roots at both 1.5 and 1 times the canopy radius. Results suggest that fertilizer applications should be placed closer to the tree trunk rather than at the canopy edge to maximize P uptake by clove roots.     

Root:soil adhesion in the maize rhizosphere: the rheological approach

This study was designed to investigate the strength of attachment of plant seedling roots to the soil in which they were grown. The study also assessed the effects of differing soil textures and differing soil matric potentials upon the strength of the root:soil attachment. A device for growing roots upon a soil surface was designed, and was used to produce roots which were attached to the soil. In order to quantify root:soil adhesion, roots of maize seedlings, grown on the soil surface, were subsequently peeled off using a universal test machine, in conjunction with simultaneous time-lapse video observation. To clarify the partitioning of energy in the root:soil peeling test, separate mechanical tests on roots, and on two adherent remoulded topsoil balls were also carried out. The seedling root was characterised by a low bending stiffness. The energy stored in bending was negligible, compared to the root:soil adhesion energy. The mechanical properties of two adherent remoulded topsoil balls were a decrease of the soil:soil adhesion energy as the soil:soil plastic energy increased. These two parameters were therefore interdependent. Using a video-camera system, it was possible to separate the different processes occurring during the root:soil peeling test, in particular, the seed:soil adhesion and the root:soil soil adhesion. An interpretation of the complex and variable force:displacement curves was thus possible, enabling calculation of the root:soil interfacial rupture energy. At a given suction (10 kPa), the results of the peeling test showed a clear soil texture effect on the value of the root:soil interfacial rupture energy. In contrast, for the same silty topsoil, the effect of the soil water suction on the value of the interfacial rupture energy was very moderate. The root:soil interfacial rupture energy was controlled mainly by a product of microscopic soil specific surface area and the macroscopic contact surface area between the root and the soil. Biological and physical interactions contributing to root:soil adhesion such as root:soil interlocking mechanics were also analysed and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Soil Anti-Scouribility Enhanced by Plant Roots

The magnitude of soil anti-scouribility depends on the physical condition of the soil. Plant roots can greatly enhance soil stability and anti-erodibility. A scouring experiment of undisturbed soil was conducted to investigate the effects of roots on soil anti-scouribility and its distribution in the soil profile. At the end of each erosion test, plant roots were collected from soil samples and root surface area was calculated by means of a computer image analysis system (CIAS). Root surface area density (RSAD), the surface area of the roots per unit of soil volume, was related to soil anti-scouribility. More than 83% of root surface area was concentrated in the 0-30 cm soil layer. Soil anti-scouribility increased with an increase in RSAD and the value of intensified soil anti-scouribility (ΔAS) can be expressed by exponential equations, depending on the plant species. These equations were ΔAS=9.578 6 RSAD^0.8321 (R^2=0.951) for afforested Pinus tabulaeformis Carr.ΔAS=7.8087 RSAD^0.7894(R^2=0.974) for afforested Robinia pseudoacacia L., and ΔAS=9.256 6 RSAD^0.8707(R^2=0.899) for Bothriochloa ischemum L.     

Contribution of root cap mucilage and presence of an intact root cap in maize (Zea mays) to the reduction of soil mechanical impedance

BACKGROUND AND AIMS: The impedance to root growth imposed by soil can be decreased by both mucilage secretion and the sloughing of border cells from the root cap. The aim of this study is to quantify the contribution of these two factors for maize root growth in compact soil. METHODS: These effects were evaluated by assessing growth after removing both mucilage (treatment I -- intact) and the root cap (treatment D -- decapped) from the root tip, and then by adding back 2 micro L of mucilage to both intact (treatment IM -- intact plus mucilage) and decapped (treatment DM -- decapped plus mucilage) roots. Roots were grown in either loose (0.9 Mg m(-3)) or compact (1.5 Mg m(-3)) loamy sand soils. Also examined were the effects of decapping on root penetration resistance at three soil bulk densities (1.3, 1.4 and 1.5 Mg m(-3)). KEY RESULTS: In treatment I, mucilage was visible 12 h after transplanting to the compact soil. The decapping and mucilage treatments affected neither the root elongation nor the root widening rates when the plants were grown in loose soil for 12 h. Root growth pressures of seminal axes in D, DM, I and IM treatments were 0.328, 0.288, 0.272 and 0.222 MPa, respectively, when the roots were grown in compact soil (1.5 Mg m(-3) density; 1.59 MPa penetrometer resistance). CONCLUSIONS: The contributions of mucilage and presence of the intact root cap without mucilage to the lubricating effect of root cap (percentage decrease in root penetration resistance caused by decapping) were 43 % and 58 %, respectively. The lubricating effect of the root cap was about 30 % and unaffected by the degree of soil compaction (for penetrometer resistances of 0.52, 1.20 and 1.59 MPa).     

Root Reinforcement by Hawthorn and Oak Roots on a Highway Cut-Slope in Southern England

Highway embankments and cutting slopes in the United Kingdom, particularly in the South East of England, are often constructed of or within stiff over-consolidated clays. These clays are prone to softening with time leading to shallow slope failures and costly repairs. Reinforcement by natural vegetation is potentially a cost-effective method of stabilising these types of slopes over the medium–long term. However, there is a lack of information on how natural vegetation reinforces and stabilises clay slopes. To investigate this problem, the potential reinforcement of selected oak (Quercus robur L.) and hawthorn (Crataegus monogyna Jacq.) roots was assessed by conducting in situ root pull-out experiments on a London Clay cutting in south-east England. Pull-out tests were carried out using specifically designed clamps and either a hand pull system with a spring balance and manual recording of force for oak roots or a jacking system with electronic data logging of applied force and displacement for hawthorn roots. Oak roots had a mean pull-out resistance of 7 MPa and that of hawthorn roots was 8 MPa. The electronic data logging of applied force (pull-out resistance) and displacement of the hawthorn roots provided additional data on the failure of branched roots which could be correlated with variations in root morphology. The failure of the roots can be categorised into three modes: Type A: single root failure with rapid rise in pull-out resistance until failure occurs; Type B: double peak failure of a forked or branched root and Type C: stepped failure with multiple branches failing successively. The different types of root–soil bonds are described in relation to root anchorage and soil stability.     

A method for extracting plant roots from soil which facilitates rapid sample processing without compromising measurement accuracy

This study evaluates a novel method for extracting roots from soil samples and applies it to estimate standing crop root mass (+/- confidence intervals) in an eastern Amazon rainforest. Roots were manually extracted from soil cores over a period of 40 min, which was split into 10 min time intervals. The pattern of cumulative extraction over time was used to predict root extraction beyond 40 min. A maximum-likelihood approach was used to calculate confidence intervals. The temporal prediction method added 21-32% to initial estimates of standing crop root mass. According to predictions, complete manual root extraction from 18 samples would have taken c. 239 h, compared with 12 h using the prediction method. Uncertainties (percentage difference between mean, and 10th and 90th percentiles) introduced by the prediction method were small (12-15%), compared with uncertainties caused by spatial variation in root mass (72-191%, for nine samples per plot surveyed). This method provides a way of increasing the number of root samples processed per unit time, without compromising measurement accuracy.     

Structure,ecology and physiology of root clusters – a review

Hairy rootlets, aggregated in longitudinal rows to form distinct clusters, are a major part of the root system in some species. These root clusters are almost universal (1600 species) in the family Proteaceae (proteoid roots), with fewer species in another seven families. There may be 10–1000 rootlets per cm length of parent root in 2–7 rows. Proteoid roots may increase the surface area by over 140× and soil volume explored by 300× that per length of an equivalent non-proteoid root. This greatly enhances exudation of carboxylates, phenolics and water, solubilisation of mineral and organic nutrients and uptake of inorganic nutrients, amino acids and water per unit root mass. Root cluster production peaks at soil nutrient levels (P, N, Fe) suboptimal for growth of the rest of the root system, and may cease when shoot mass peaks. As with other root types, root cluster production is controlled by the interplay between external and internal nutrient levels, and mediated by auxin and other hormones to which the process is particularly sensitive. Proteoid roots are concentrated in the humus-rich surface soil horizons, by 800× in Banksia scrub-heath. Compared with an equal mass of the B horizon, the A₁ horizon has much higher levels of N, P, K and Ca in soils where species with proteoid root clusters are prominent, and the concentration of root clusters in that region ensures that uptake is optimal where supply is maximal. Both proteoid and non-proteoid root growth are promoted wherever the humus-rich layer is located in the soil profile, with 4× more proteoid roots per root length in Hakea laurina. Proteoid root production near the soil surface is favoured among hakeas, even in uniform soil, but to a lesser extent, while addition of dilute N or P solutions in split-root system studies promotes non-proteoid, but inhibits proteoid, root production. Local or seasonal applications of water to hakeas initiate non-proteoid, then proteoid, root production, while waterlogging inhibits non-proteoid, but promotes proteoid, root production near the soil surface. A chemical stimulus, probably of bacterial origin, may be associated with root cluster initiation, but most experiments have alternative interpretations. It is possible that the bacterial component of soil pockets rich in organic matter, rather than their nutrient component, could be responsible for the proliferation of proteoid roots there, but much more research on root cluster microbiology is needed.     

The distribution and abundance of wheat roots in a dense,structured subsoil – implications for water uptake

We analysed the abundance, spatial distribution and soil contact of wheat roots in dense, structured subsoil to determine whether incomplete extraction of subsoil water was due to root system limitations. Intact soil cores were collected to 1.6 m below wheat crops at maturity on a red Kandosol in southern Australia. Wheat roots, remnant roots, soil pores and root–soil contact were quantified at fresh breaks in the soil cores. In surface soil layers (<0.6 m) 30–40% of roots were clumped within pores and cracks in the soil, increasing to 85–100% in the subsoil (>0.6 m), where 44% of roots were in pores with at least three other roots. Most pores contained no roots, with occupancy declining from 20% in surface layers to 5% in subsoil. Wheat roots clumped into pores contacted the surrounding soil via numerous root hairs, whereas roots in cracks were appressed to the soil surface and had very few root hairs. Calculations assuming good root–soil contact indicated that root density was sufficient to extract available subsoil water, suggesting that uptake is constrained at the root–soil interface. To increase extraction of subsoil water, genetic targets could include increasing root–soil contact with denser root hairs, and increasing root proliferation to utilize existing soil pores.