Root sampling methods

[1] Varying root sizes that are observed via root sampling

Overview

Interest in root sampling was first stimulated on an ecological scale in 1960 by an ecologist testing soil water availability in plants[5]. Methods have been developed since that are able to produce both rough estimations and almost exact measurements of root biomass. Rhizodeposition is a key factor in Plant establishment and these sampling methods become useful when gathering information on plant nutrient allocation and development. Plant roots are highly variable in growth. Therefore, results from any root sampling method can be challenging to interpret.[6] It’s been estimated that in order to have a 90% confidence interval using any technique, 40 or more samples must be taken, which is unfeasible for the majority of research purposes.[13]

Root Length Equation

[2]

Where R = total length of the root, N = # of intersections between the root and straight lines, A = area of the sampled rectangle, and H = total length of the straight lines. The line intersect method (including the root length equation) was created by E.I. Newman after he recognized that absorption of nutrients and water from the soil depends on root length and surface area rather than overall biomass.[9] Newman used various mathematical and ecological equations to create this method specifically for complicated root systems to avoid direct counting and measurement under a microscope. Through this calculation, ecologists were able to precisely measure the root lengths contained in a system in one-third of the time it took prior.[14] This was one of the first methods offering a quicker, more accurate approach to counting roots and fine root hairs.

Uses for Root Sampling

Root samples are useful for many agricultural, ecological, and educational purposes. Depending on the situation and ecosystem, different methods may be preferred over others. Generally, root data is collected to analyze the overall health and development of a tree or plant.

With the increasing occurrence of habitat restoration projects and wildlife rehabilitation, root sampling is a vital step to see the extent to which introduced plants have assimilated into new territory. Plant establishment will be checked at constant intervals after a site is designed, until the restoration efforts can be confirmed as successful. These experimental techniques allow the rhizosphere of the modified ecosystems to be checked, and aid in detecting potential Ectomycorrhizal Fungi and Arbuscular Mycorrhizal Fungi connections.[13]

Destructive Sampling Methods

The Harvest Method

[3] Monolith collection at the University of Idaho

The harvest method is performed by extracting an undisturbed, vertical sample of ground soil and keeping it preserved in situ to examine the characteristics of the different Soil Horizons. First, the size of the desired sample must be determined and the auger to extract the soil must be chosen. These auger devices may come in small, hand-held sizes or larger sizes which are mechanical and sometimes mounted on trucks.

The soil is either kept intact and preserved as a monolith or the roots in the sample are rinsed free of the soil particulates. Monoliths are created by cutting the cylindrical soil core in half and transferring one of the profiles to a solid surface, like a plyboard, using an acrylic bonding agent for mounting.[6][15] The other half not used for display purposes is used for lab sampling or classification purposes. Monoliths can be kept for decades if done correctly. When root samples are desired, water and patience are required. Roots are generally pre-soaked to minimize water usage, and in some cases dispersing chemicals are applied. [10] Separated root samples can be stored for up to 10 weeks which allows for ample time to study the root systems.

Despite this being considered a destructive sampling technique, the harvest method minimizes site disturbance while allowing a lot of valuable information to be gathered.[6]

Isotope-Dilution Method

The isotope-dilution method is commonly used to determine below-ground biomass turnover in grasslands. It utilizes the 14C-dilution technique to sample roots. 12C is also incorporated into the structural tissues of root systems after the 14C. This is typically done a few weeks to months after 14C is added to root samples. A ratio of 14C/12C is later measured in the structural tissues of root samples to measure tissue loss. [3]

Root-Ingrowth

[4] 'A dug up ingrowth core with visible newly grown herbaceous roots (picture by Dr. Emily Solly)

The root ingrowth method is beneficial for measuring the rate of growth for fine root hyphae (diameter <2 mm). It is very labor-intensive and one of the more controversial root sampling procedures.[10] This is because (I) natural growth patterns can easily be altered chemically and physically, (II) current roots are injured, (III) growth starts after a period of delay, (IV) decomposition rates are not considered, and (V) artificial and low densities are recorded in the cores for the majority of the experiment.[17]

First, the chosen ground area in the root zone of plants is cored wide and deep enough to fit the parameters of the experiment. This coring is what cuts off the living roots of present systems. Mesh, nylon bags are filled with sieved soil free of any root hairs or nodules, brought to the site, and inserted into the cored space. Women’s stockings can be used for a tight-budget project. The mesh soil bags are left either long or short-term but must be kept buried long enough to allow for roots to transect and occupy the bag. This usually takes at least 2 months.[11] After the bags are collected, the roots are separated from the adhered soil using methods such as pre-soaking or dispersing chemicals which are also used in the Harvest Method (See above). Primary and secondary roots are left out to air dry while the fine root hyphae are oven dried at 50°C to constant weights.[17]

Non-destructive Sampling Methods

Rhizotrons

[5] The Northern Research Station's rhizotron located in Houghton, Michigan

Rhizotrons are underground walkways with glass walls on either one or both sides that expose the Rhizosphere including the Organisms and Soil surrounding the structure. These structures are unique because they allow scientists to go inside and study the root systems that are still living and developing. Individual roots are easy to keep track of and measure which is great for succession and development research. A big limitation of this type of research is that large rhizotrons can be very costly to construct and operate.[12]

More advanced structures are designed to change the temperature, pH, and other elements of the surrounding soil, altering the observed Soil processes and root behaviors. Cameras are often mounted and set on time lapse in the observatory facing the roots to account for any changes such as diurnal swelling and shrinking[1] that scientists may miss unless they were constantly monitoring the roots.

Miniature versions of rhizotrons, not to be confused with minirhizotrons, are more common as they are simple to build and cost less.

[6] Minirhizotron diagram

Minirhizotrons

Minirhizotrons consist of a transparent tube that is sometimes designed with a reflective surface mounted to the inside of it. The tube is inserted in the root zone of the soil and a high-resolution, thin camera is drawn through the tube. Once inside, the camera provides clear, in situ root images which can then be further used for quantitative data analysis by converting two-dimensional image data into three-dimensional root biomass data.[8] Minirhizotrons are similar to rhizotrons in that they allow for a close-up study of root systems growing without human interaction or destruction. The obtained images are used to examine root biomass growth and are greatly beneficial for analyzing restoration efforts. Minirhizotrons can monitor soil moisture, temperature, and water potential using tensiometers, time domain reflectometer probes, and matrix water potential sensors.[2] Monitoring the Water Behavior in Soils is important along with root development because the two are so closely related.

References

1. “Science.” Science, 4434th ed., vol. 207, American Association for the Advancement of Science, 1980, p. 975. [7]

2. Cai, GC, et al. “Construction of Minirhizotron Facilities for Investigating Root Zone Processes.” VADOSE ZONE JOURNAL, vol. 15, no. 9, Sept. 2016, doi:10.2136/vzj2016.05.0043. [8]

3. Coleman, D. C., Callahan Jr, M. A., & Crossley Jr., D. A. (2018). Fundamentals of Soil Ecology (3rd ed.). Academic Press.

4. Solly, E.F., Brunner, I., Helmisaari, HS. et al. Unravelling the age of fine roots of temperate and boreal forests. Nat Commun 9, 3006 (2018). https://doi.org/10.1038/s41467-018-05460-6

5. Gardener, W. R. “DYNAMIC ASPECTS OF WATER AVAILABILITY TO PLANTS.” SOIL SCIENCE, vol. 89, no. 2, Feb. 1960, pp. 63–73., journals.lww.com/soilsci/Citation/1960/02000/DYNAMIC_ASPECTS_OF_WATER_AVAILABILITY_TO_PLANTS.1.aspx. [9]

6. Haddad, N.i., et al. “Improved Method of Making Soil Monoliths Using an Acrylic Bonding Agent and Proline Auger.” Geoderma, vol. 151, no. 3-4, 9 June 2009, pp. 395–400., doi:10.1016/j.geoderma.2009.05.012. [10]

7. Johnson, Jane M.F., Morgan, Jack. “Sampling Protocols.” Plant Sampling Guidelines. IN Sampling Protocols, Ch. 2. R.F. Follett, editor. 2010, pp. 2-10. www.ars.usda.gov/research/GRACEnet [11]

8. Lee, Chol Gyu, et al. “Estimation of Fine Root Biomass Using a Minirhizotron Technique among Three Vegetation Types in a Cool-Temperate Brackish Marsh.” Soil Science and Plant Nutrition, vol. 62, no. 5-6, 2016, pp. 465–470., doi:10.1080/00380768.2016.1205957. [12]

9. Newman, E. I. “A Method of Estimating the Total Length of Root in a Sample.” Journal of Applied Ecology, vol. 3, no. 1, 1966, pp. 139–145. JSTOR, www.jstor.org/stable/2401670. [13]

10. “Pedology Laboratory.” uidaho.edu, n.d. [14]

11. Soil Survey Staff. 2009. Soil Survey Field and Laboratory Methods Manual. Soil Survey Investigations Report No. 51, Version 1.0. R. Burt (ed.). U.S. Department of Agriculture, Natural Resources Conservation Service. [15]

12. Steingrobe, Bernd, et al. “The Use of the Ingrowth Core Method for Measuring Root Production of Arable Crops – Influence of Soil and Root Disturbance during Installation of the Bags on Root Ingrowth into the Cores.” European Journal of Agronomy, vol. 15, no. 2, 5 Oct. 2001, pp. 143–151., doi:10.1016/s1161-0301(01)00100-9. [16]

13. Taylor, H. M., et al. “Applications and Limitations of Rhizotrons and Minirhizotrons for Root Studies.” Plant and Soil, vol. 129, no. 1, 1990, p. 29. [17]

14. Taylor, H.M. 1986. Methods of studying root systems in the field. Hortscience 21:952-956. [18]

15. Tennant, D. “A Test of a Modified Line Intersect Method of Estimating Root Length.” Journal of Ecology, vol. 63, no. 3, 1975, pp. 995–1001. JSTOR, www.jstor.org/stable/2258617. [19]

16. United States, Congress, Kiniry, Lauren N., and Conrad L. Neitsch. “Monolith Collection and Preparation For Soils without Restrictive Layers*.” Monolith Collection and Preparation For Soils without Restrictive Layers*, 1994. [20]

17. Vanderford, C. F. "The soils of Tennessee. Univ. Tennessee Agr. Experiment Station." Bulletin 10.3 (1897): 1-139. [21]

18. Xuefeng Li, Jiang Zhu, Holger Lange, Shijie Han, A modified ingrowth core method for measuring fine root production, mortality and decomposition in forests, Tree Physiology, Volume 33, Issue 1, January 2013, Pages 18–25, https://doi.org/10.1093/treephys/tps124 [22]