Over the past few years there has been a growing realization that our knowledge about roots, which has been applied globally to managing plant systems, has been based on false premises of simplicity (Wells & Eissenstat, 2001; King et al., 2002; Pregitzer, 2002; Pregitzer et al., 2002; Zobel, 2003; Hodge, 2004). In a discussion paper about the fine roots of trees Pregitzer (2002) stressed the fact that our traditional views of fine tree roots – our knowledge and understanding of their length and diameter, structural and functional diversity/complexity and life history/turnover – are probably deeply flawed. More recently, Zobel (2003) revisited and reaffirmed Pregitzer's original points based on examples which demonstrate that we still lack clear anatomical, dimensional, functional and physiological definitions of what a fine root is, not only for trees, but for all plant species.
It is now certain that in both annual and perennial plants, roots <1 mm in diameter form a structurally and functionally complex population which is the dominant component of the root system. While it is widely recognized that fine roots amount to most of the root length in many plant species, it is also acknowledged that this is often underestimated because of their small size and near transparency (Costa et al., 2001). We need to improve our understanding of fine roots to support better prediction and management of biogeochemical cycles from the scale of single plants to the globe (Jackson et al., 1997; McCully, 1999; Norby et al., 2004; Thomas et al., 2004). To do this, it is necessary to come to terms with measuring the quantity and functions of fine roots under a variety of conditions. It is striking that there is no firmly established procedure for the accurate measurement of fine root length and biomass (Vogt et al., 1998).
In trees, the importance of fine roots is well supported by field evidence. For example, King et al. (2002) showed that in loblolly pine, 96% of the mycorrhizal and 77% of the nonmycorrhizal root length occurred in roots with diameters ranging from 0.2–0.6 and 0.4–1 mm, respectively. In a study which demonstrated that overwinter survivorship of apple roots is positively correlated to root diameter, Wells & Eissenstat (2001) showed that the majority of roots (>64.5%) were 0.1–0.3 mm in diameter. Pregitzer et al. (2002) confirmed for eight north American tree species that lateral roots <0.5 mm in diameter appear to account for >75% of the root length. Norby et al. (2004) found that under both ambient and CO2-enriched conditions, ≈80% of the root length of 10–15 yr old sweetgums (Liquidambar styraciflua L.) was in roots <0.5 mm in diameter.
Quantified evidence that fine roots are the principal contributors to root length in annual plants has also been consistently reported. For example, Pavlychenko (1937) found that, depending on the degree of competition between plants, the root length of oats, wheat and spring rye was formed of 45–93% of second-order laterals <0.1 mm in diameter. In a detailed quantitative study of winter rye roots, Dittmer (1937) reported that the length of second- and third-order laterals averaging 0.13 and 0.12 mm in diameter, respectively, made up >99% of the root system length, with over two-thirds of the length consisting of the finest third-order laterals. Kooistra et al. (1992) observed that, depending on soil bulk density, roots <0.2 and <0.3 mm in diameter could encompass up to 40 and 80%, respectively, of the root length developed by maize plants. Pallant et al. (1993) showed that in maize, roots <0.24 mm in diameter made the largest contribution to total root length. Moran et al. (2000) found that ≈80% of the root length of wheat was in roots <0.3 mm in diameter.
The main methods used to measure root length/biomass can be classified as (1) extraction methods (generically known as root washing); (2) mapping techniques; (3) in situ imaging techniques; and (4) other (often sophisticated) imaging techniques.
Extraction methods are based on collecting soil samples of known volume (core or monolith) from which roots are physically separated by carefully washing the soil away, and finally measuring the length of the separated roots using stereological or image analysis techniques (do Rosario et al., 2000). The principle of mapping methods is to record the occurrence of root contacts on a destructively exposed soil surface (van Noordwijk et al., 2000). Root contacts, whether enumerated on a pit face or a core surface with the naked eye, or on soil thin/polished sections using a microscope, are subsequently converted to length measurements according to a calibration procedure. With in situ imaging methods (chiefly involving reflection of visible light on observed objects), roots are observed at transparent interfaces with soil, such as the walls of transparent plastic tubes inserted into the soil for several months (minirhizotrons) (Smit et al., 2000). This allows dynamic monitoring of root growth and measurement of root length either directly, or based on calibration procedures. Finally, other imaging techniques involve probing (using electromagnetic radiation such as light, X-rays or γ-rays, particle beams or variable magnetic fields) of either field specimens or whole root systems confined within the delimited volume of specifically designed containers (the size of which is a function of the probing technique). The result is the reconstruction of either 2D (e.g. X-radiography) or 3D (X-ray CAT scanning, NMRI) images from which a range of root measurements can be derived by means of image analysis (Moran et al., 2000; Pierret et al., 2003).
It is well documented that all these techniques yield highly variable results (e.g. CV > 100% for minirhizotron and washing techniques), and that results obtained using two different techniques are, more often than not, difficult to compare. For example, Kucke et al. (1995) compared the core-break, trench-profile, core and monolith methods. They found good agreement between core and monolith methods, but obtained variable results with the core-break and trench-profile methods, the results from the two latter being poorly correlated with results from the two former. Unlike Heeraman & Juma (1993), they found more consistently lower CVs with monoliths (4500 cm3) than with cores (754 cm3). These authors interpreted the differences between mapping and destructive techniques as the result of (1) preferential orientations of roots, and (2) differences in root visibility depending on contrast with soil matrix. Tierney & Fahey (2002) noted differences between minirhizotrons and a radiocarbon method, but were able to analyse their results making sense of both data sets.
It would be somewhat hasty to dismiss the possibility that the variability of results gained through one technique may reflect real processes such as root proliferation in response to heterogeneous supplies of nutrient (Caldwell et al., 1992; Hodge, 2004). However, the regularity with which root-length measurements appear to vary depending on the technique used (see Literature review below) highlights the methodological, conceptual and scientific issues with which experimenters estimating root quantities are faced. One of the most startling of these issues is that the definition of the object itself – roots in general, and fine roots in particular – is still the subject of debate (Pregitzer, 2002; Zobel, 2003). As described by Zobel (2003), because we basically ignore much of how roots are organized and operate within a root system, we are still studying roots according to arbitrary size (diameter) classes and not according to more logical, function-related parameters. Hence issues as basic as the size distribution of root diameters down to the finest roots remain very unclear. Likewise, precise knowledge about root ontogeny and its morphological expressions, as well as root longevity/turnover, is lacking.
This paper provides a critical appraisal of methods used to measure the quantity (length and biomass) of fine roots. The critique is based on original measurements of fine roots obtained by means of a high-resolution X-ray imaging technique (Moran et al., 2000) and a review of literature. The high-resolution X-ray imaging technique was chosen because it permits the codetection of fine plant roots (down to ≈50 µm in diameter) and calibrated soil structure, and facilitates the quantification of the root/soil couple as a mutually interacting system. We investigated the extent to which root length may be underestimated, using a range of techniques, as well as the technical reasons that could explain such an underestimation. We present evidence showing that, because of the dominance of fine roots in most plant root systems, root-length recovery is strongly dependent on the observation scale at which the length measurement is carried out. Further, observation scales adequate for the quantification of fine root length require long processing times to obtain precise quantity estimates. As a consequence, plants could actually grow much longer (and larger biomass) root systems than is widely accepted. Our ability to understand fine roots will continue to be limited as long as these systematic errors are not taken into account in interpretations of plant functions.
Materials and Methods
Original X-ray root-length measurements
Samples analysed in this paper were taken under a canola crop (Brassica napus L. var Rainbow) grown on a farm located 40 km north-west from Wagga Wagga, NSW, Australia (34°52′ S, 147°05′ E). Mean annual rainfall and temperature are ≈560 mm and ≈15°C, respectively, with winter-dominant rainfall. The soil, a red Kandosol (Soil Survey Staff, 1998), was sampled at depths ranging from 5–115 cm on three occasions during the 1998 growing season: in June (just after sowing); at the end of August/early September (early grain filling); and at the end of October (before harvest). The canola crop measured in this study followed crops of lucerne (Lucerneago sativa L.) in 1996 and 1997. Before sowing, the soil was cultivated to a depth of ≈100 mm. Because of the underlying principles of the X-ray imaging technique (see below), the observations and measurements reported here not only correspond to the roots of the canola crop grown during the 1998 season, but also include information about decaying roots from previous seasons (Moran et al., 2000). More detail about sampling and cropping history is given by Pankhurst et al. (2002).
Soil samples were obtained by manually excavating monoliths ≈200 × 200 × 200 mm. For each sampling date, up to six such monoliths were extracted from the surface down a vertical sequence. In general, two replicates were obtained from each 20 cm depth increment. In the most favourable cases (when the monoliths did not crumble down while being excavated), the two replicates could be X-rayed effectively. However, a certain number of samples had to be discarded because their structure was not preserved intact. Undisturbed soil blocks (≈200 × 200 × 200 mm cubes) were air-dried in the laboratory for ≈12 wk and impregnated with polyester resin using a vacuum drip method (Moran et al., 1989). Subsamples (≈100 × 100 × 100 mm cubes) were extracted after completion of impregnation. From these subsamples, horizontal, 70 × 70 mm sections, 1 mm thick, were produced using a high-precision flatbed diamond-head grinder. Twenty such horizontal sections (six corresponding to the June sampling and seven for both September and October samplings) were imaged using a microfocus X-ray imaging system, the setup of which is described by Moran et al. (2000). To optimize image resolution, a geometric magnification of ×2.86 was applied by placing the sample at some distance from the image detector (Fuji BAS, imaging plates).
Once digitized, X-ray images were calibrated in terms of density using the method of Bresson & Moran (1998). Root traces were clearly visible on the images and were selected using a semi-automatic (computer-aided) procedure. Digital X-ray images were edited using adobe photoshop software and visualized at full resolution on a Wacom PL-400 LCD integrated tablet which allowed direct draw-on-screen interaction with the edited image. Detection of roots and root-related porosity (channels created by roots decomposing or decomposed at the time of exposure) relies on the recognition of a combination of densitometric and geometrical properties. This includes morphometric features of individual objects, but also contextual visual information related to the overall spatial arrangement of the root network imaged in a radiograph. In a previous study (Moran et al., 2000), we recognized the risk of mistaking small cracks for roots, and defined simple morphological criteria to discriminate them. When parallel enough to the section plane, roots and root-related pores appear as fibrous objects of increasing density from the centre to the edges (directly related to their cylindrical geometry). The more perpendicular to the section plane they become, the more roots appear as circular areas of low density. As opposed to these characteristics, small cracks are visible only when they are nearly perpendicular to the section plane, and show sharp edges with no gradual change in intensity from the edges to the centre as observed for roots. Based on these criteria, roots were traced using photoshop's inbuilt pixel-selection tools. The tracing, even though manual, was quite accurate because the operator could adjust the width of drawn lines down to 1 pixel (equivalent to 17.5 µm in the case of the images dealt with here) and could zoom in and out while editing the image.
Once the root images had been obtained, they were processed to derive root diameter distributions, root-length density estimates, and quantified indicators of the interplay between roots and soil structure such as the density of soil adjacent to roots, or the density of soil at increasing distances from roots of different diameter classes (see Moran et al., 2000; Pierret et al., 2000 for detail of procedures). Each X-ray image represents a snapshot, at a given point in time and soil depth, of the long-term co-evolution between successions of roots from crops locally. Objects designated as ‘roots’ can be live roots either recolonizing root channels established by previous crops or creating new pores (thus participating in the progressive refilling of ancient/abandoned root channels in the soil nearby), or they can be decaying and dead roots. The comparison of images corresponding to samples taken at close enough time intervals and similar soil depths can give some indication of the root dynamics corresponding to the growth of a given crop.
Literature-based comparison of techniques used to measure root length
We reviewed a number of papers dealing (whether chiefly or not) with root length measurements to test the hypothesis that there exists a correlation between measuring times and observation scales on the one hand, and root-length recovery on the other. Most references were found using the cab abstracts search engine facility (CABI Publishing North America, http://www.cabi-publishing.org) with various combinations of the words ‘root’, ‘length’ and ‘density’. Based on this initial search – which yielded >600 references for the expression ‘root-length density’– we retained studies dealing with both annual and perennial species, in which length density was expressed on a volumetric basis (as opposed to surface area) or, alternatively, in which the volume of soil from which root length was recovered was clearly reported. Minimum detail was also sought of (1) soil depth at which measurements had been made and (2) diameters of roots measured. A total of 29 such studies were found. These requisites were set to circumvent the need for excessive interpretation of published data and to provide consistency between the figures compared. As a direct consequence, studies in which root densities were expressed as root biomass per unit soil volume or surface area were not included in our comparison.
The studies selected encompass the five broad families of techniques available to measure root length: minirhizotron tubes (7); root washing (11); micromorphology (8); X-ray computed tomography (1); and projection X-ray imaging (2). A sixth category includes three references corresponding to extremely detailed measurements carried out in the 1930s, and based on a combination of very careful root washing and optical microscopy (Dittmer, 1937). For all but one of these categories of techniques (X-ray CT, because of the lack of references), we calculated the average, minimum and maximum root-length density. In addition, we used the results of a review study of 92 references by van Noordwijk & Brouwer (1991) to compare the root density values most commonly reported for 20 plant species as derived from root washing and minirhizotron observations.
A review of the literature has demonstrated that root-length recovery varies by as much as one order of magnitude depending on the technique used. As a general principle, there is a clear correlation between fine root detection and/or processing times on the one hand, and root-length recovery on the other. There exists ample evidence that profuse fine roots can easily be overlooked when using root washing and minirhizotron, the most convenient, affordable and rapid, and hence most widely used, techniques to assess root length and biomass.
Based on original root measurements and a review of an array of literature spanning more than six decades, this paper confirms that the fine roots of an annual crop (canola) contribute significantly to its overall root length. In our study, roots <0.2 mm in diameter represent up to >50% of root length, a result that is in agreement with the findings of Pallant et al. (1993), who showed that in maize, roots <0.175 mm in diameter can account for more than 56% of root length.
Statistical analysis of the X-ray-based root-length and diameter information leads us to two potential interpretations. First, the lack of statistical difference between root distributions indicates that two different crops, and one crop at two different times, have the same relationship between root length and root diameter. This is consistent for coarse and fine roots. Such a result, if true, warrants further investigation. Second, if the lack of significant differences is caused by local spatial variation, the canola crop has a finer overall root distribution than lucerne, and this becomes more emphasized throughout the growing season. The latter interpretation is consistent with visual inspection of the distribution functions.
We conclude that plant root systems are likely to be much longer and include more biomass than is widely accepted. Despite sustained technological progress leading to improved understanding of root function and biogeochemical cycles, we believe there is an urgent need for careful studies aimed at accurately quantifying root length and biomass under a range of biophysical conditions and for a wide array of plant species.
This research was financially supported by CSIRO Land & Water, the Australian Grains Research and Development Corporation and INRA. We thank Mr Colin McLachlan, CSIRO Land & Water, Canberra, for invaluable technical assistance with the production of most of the original measurements presented in this work. We are grateful to Dr John Passioura, CSIRO Plant Industry, for support and guidance during the several laborious years of work required to gain confidence that the early results from X-ray imaging were not an aberration of the technique. We thank Dr Yves Dudal, INRA Climat, Sol & Environnement, Avignon, for the helpful comments on the manuscript.