- Top of page
- Materials and Methods
- Results and Discussion
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.
- Top of page
- Materials and Methods
- Results and Discussion
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.