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A a. - U3. - firs..." ,..._ .5... ~..". ’4‘ c. .. I‘m-4, 7 _ 3L)". 9' ' .,.. :v...,....,z> u Ivy-r .1." n -J . ..-. .. ' n .1‘ ~t -. n).,,.,,,. J'l."\"‘f We: .. .. I 1.: .u “a q-K .. r "1 , .,.' ........-- , a... -22». 93h- w- .. n l " o2 :- .-4 .- fur" 1.121", rrrr . .,. .04--. ”m...” .,... . ..., .. . . « .-:.-.~ - ,.--.-- J .. - ...- . . ".9 mm... . MA w, .- m9 . .. -- .. _..,...:,:, .., - ., 1?: -’~" , ,- . .r-r , ,. .. l"!"1-PL ""' .1” h—w . “4...... .. (4" " .. fHEb'lb 8T Illllllllllllllll ‘ This is to certify that the dissertation entitled Responses of Soil Aggregation to Root Activity and Root Extracts of Alfalfa and Ryegrass presented by Stephanie Lynn Schroeder Murphy has been accepted towards fulfillment of the requirements for Ph.D. degreein Crop and Soil Sciences QL-W Ma#r professor Mafia; IO: [94/ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before due due. ll DATE DUE DATE DUE DATE DUE MSU Ie An Affirmative ActiorVEquel Opportunity Institution omens-9.1 RESPONSES OP SOIL AGGREGATION TO ROOT ACTIVITY AND ROOT EXTRACTS OP ALPRLPA AND PERENNIAL RYEGRASS BY Stephenie Lynn Schroeder Murphy A DISSERTATION Bunnitted to nichigen state University in partial tuliillnent or the requirements for the degree of DOCTOR OF PHILOSOPHY Depertnent of Crop and soil Science: 1991 ABSTRACT RESPONSES OP SOIL AGGREGATION TO ROOT ACTIVITY AND ROOT EXTRACTS OP ALPALFA AND RYEGRASS BY Stephanie Lynn'Schroeder Murphy Plant roots have been shown to affect soil aggregation, but the mechanisms of their influence are not well established. The objectives of this study were to investigate the effects of root extracts, simulating root exudates, and root growth of two species, alfalfa (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.), on the aggregation of a disturbed soil (loamy, mixed, mesic, Glossoboric Hapludalf). A laboratory experiment was conducted in which the disturbed soil was repeatedly wet with either water or the root extracts of alfalfa or ryegrass and dried to 0.3 MPa. Soil treatment cells (STC) were designed and constructed for this purpose. After eight cycles of wetting and drying, the root extracts and soil leachate were analyzed for carbohydrate contents, and soil aggregation was measured by several methods. In the greenhouse, alfalfa or ryegrass was grown in soil for 1 mo and 4 mo periods, and unplanted soil served as the control. For the long-term experiment, top and bottom sections of soil were analyzed separately to consider differences in evaporative water loss and root distribution. For the short-term experiment, two irrigation schedules were applied to evaluate the effect of soil drying by plant roots on soil aggregation. At harvest, subsamples of the soil were analyzed for soil aggregation, and roots were extracted from the soil and quantified by length and/or dry weight. No significant differences in soil aggregation were found in the laboratory experiment; root extracts appeared to have little effect on aggregation of this soil under the drying and time conditions of the experiment. The STCs were operated successfully and will be valuable tools for soil research in many sub-disciplines. In the greenhouse experiments, plant roots were responsible for aggregation differences according to some tests. Ryegrass aggregates had greater crushing resistance than either alfalfa or unplanted soil. Alfalfa was generally associated with greater stability of aggregates in water. No aggregation differences were statistically related to drying treatment, and few were related to the soil position factor. Organic matter content was greater in planted soil. Quantity of roots was sometimes related to soil aggregation indices, but plant species was important as well. to Jim- thanks for your love and support and the joy you bring me iv ACKNOILEDGENENTS I would like to thank the members of my graduate committe, Dr. Alvin Smucker (chair), Dr. Jim Crum, Dr. Steve Boyd, Dr. Alan Putnam, and Dr. Ron Perry, for their advice, guidance, and interest. Many others have contributed to this research through their advice, technical assistance, actual labor, and moral support. The many abilities of John C. Ferguson have been particularly appreciated. Jeff Lounds constructed the soil treatment cells described in Chapter 3. Rosemond Soanes contributed in the collection of "unpublished data" described in Chapter 5. I also would like to thank friends and other graduate students for their encouragement and fellowship. Finally, I would like to express appreciation to family members, whose support and love has been important to me throughout my academic career. TABLE OF CONTENTS List Of TableSOOOOOOO0.0...OOOOOOOOOOOOOOOOOO...OOOOOOOOVii List Of FigureSOOOOOOOOOO0.0.0....OOOOOOOOOOOOOOOOOOOO0.0ix Chapter 1. IntrOductionOIOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.1 List Of ReferenceSOOOOOOOOOOO0.0...0.0.0.0000000000004 Chapter 2. Literature Review..............................5 Soil structure.......................................5 Cropping effect.....................................18 Root exudates and soil carbohydrates................24 Summary.............................................31 List of references..................................33 Chapter 3. Soil stability analyses following repeated application of root extract solutions and drying cycles in the laboratory.................4O Abstract............................................40 Introduction........................................42 Materials and methods...............................43 Results and discussion..............................50 Conclusions.........................................61 List of references..................................63 Chapter 4. Influence of alfalfa and ryegrass root growth on soil aggregation in greenhouse studies....65 Abstract............................................65 Introduction........................................67 Materials and methods...............................68 Results and discussion..............................77 Conclusions.........................................91 List of references..................................94 Chapter 5. A comparison of alfalfa and ryegrass root quantification......................................97 Abstract............................................97 Introduction........................................98 Materials and methods...............................98 Results and discussion..............................99 Conclusions........................................107 List of references.................................108 Chapter 6. conCIuSionSOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.000109 vi LIST OP TABLES PAGE Concentration of identified compounds in root extracts and soil leachates.........................52 Mean gravimetric water content of soil after each STC dry-down cycle.............................56 Size distribution of soil aggregates after 8 cycles of treatments..................... ......... 57 Mean wet aggregate stability (WAS) of soil aggregates after 8 cycles of treatments.............59 Mean transmission of soil suspensions following 2 min (T2) or 20 min (T20) of end-over-end shaking of 1 to 2 mm aggregates................ ..... 59 Organic matter (OM) content of soil aggregates (1 to 2 mm) after 8 cycles of treatments............60 Mean dry weight of plant shoots and roots for the significant factors in GHl......................77 Proportion of soil (by weight) found in the size range <1 mm by dry sieving for GHl........ ..... 79 Mean crushing resistance (CR) of 4 to 6 mm soil aggregates for GHlOOOOOOOOOOOO0.0.0.0....0.0... ..... 80 Mean wet aggregate stability (WAS) for 1 to ZMSOil aggregates Of GHlOOOOI0.00.00.00.00000000081 Mean transmission of soil suspensions following 2 (T ) and 20 minutes (T20) of end-over-end shaking of 1 to 2 mm soil aggregates for GHl........82 Soil organic matter (OM) content of soil after GHl treatments, as determined by low-temperature combustionto.0.0000000000000000000000.0.0.0.0...00.083 Correlation coefficients for comparisons between root parameters and soil aggregate analyses for GHl.......................... ..... .....84 vii Correlation coefficients for comparisons among methods of soil aggregate analysis for GHl..........85 Mean dry weights of plant shoots and roots for the significant factors in GH2......................86 Proportions of soil (by weight) found in various aggregate size ranges by dry sieving for GHZ........86 Mean crushing resistance (CR) of 4 to 6 mm sail aggregates for GHZOOOOOOOOOOOOOOOOOOOOO0.00.0008? Mean wet aggregate stability (WAS) of 1 to 2 mm soil aggregates for GHZ........................88 Mean transmission of soil suspensions following 2 (T2) and 20 minutes (T20) of end- over-end shaking of 1 to 2 mm soil aggregates for GHZ.............................................89 Correlation coefficients for comparisons between root dry weight and soil aggregate analyses for GHZOOOOOOOOOOOO0.000......COO...0.0.0.089 Correlation coefficients for comparisons among methods of soil aggregate analysis for GHZOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.0...0......00.0.90 Mean length of washed roots of alfalfa and ryegrass before and after applying algorithms to exclude debris..................................103 viii LIST OF FIGURES FIGURE PAGE 2.1 Diagram of major variables and interactions in the formation and maintenance of soil structure.....17 3.1 Cross section and plan view of a Soil Treatment cell (STC)OOOO0....OI0.00.00000000000000000000000.0047 3.2 Water desorption curve for the disturbed, <2 mm loam soil obtained by Soil Treatment Cells (STCS).0.00.0000000000000000000000000.00.0000...0.0.53‘ 3.3 Mean cumulative outflow from Soil Treatment Cells (STCs) during dry-down periods................55 3.4 Mean crushing resistance (CR) for 4 to 6 mm soil aggregates after 8 cycles of treatments........58 4.1 Water desorption curve for the disturbed Marlette 8011....OOOOOOOOOOOOIOOOOO00.0.0.0...0.0.0.72 4.2 Equipment setup for determining the crushing resistance (CR) of soil aggregates..................75 5.1 The relationship between the line intersect method and image analysis (IA) to determine rOOt length.OOOOOOOOOOOOOOOOOOOO00.0.0.0....0.0.0.0102 5.2 The relationship between root dry weight and root length determined by the line intersect methOdOIOO0.0...OOOOOOOOOOOOOOOOO0.0.0.0....0.0.0.0104 5.3 The relationship between root dry weight and root length (RL) determined by image analysis......106 INTRODUCTION Soil structure is defined as the arrangement of primary soil particles into secondary particles, or peds (Soil Sci. Soc. Am., 1984). In a general sense, soil structure controls and indirectly describes the arrangement of the pore space in soil. Soil structure is an important variable determining other soil characteristics such as water retention, gaseous diffusion, and hydraulic conductivity. Besides the arrangement, or form itself, an important aspect of structure is its stability, i.e. the ability to retain its original form over time when exposed to various forces (Ray et al., 1988). The major factors of soil aggregation and structural stability have been identified as organic matter, clay content and mineralogy, soil wetness, cations, drying history, stresses imposed, freezing/thawing, and biological factors (including microbial). The individual factors have not been completely separated to determine their relative importance. Because of the significance of soil structure, particularly in agricultural production, it is beneficial to l manage soil in such a way as to maintain or improve aggregation. Man's most common effort at managing soil structure is through tillage with various implements, stressing and breaking bonds between soil particles by shear or packing and remolding by compression. Although there are good reasons for tillage (e.g. to improve seed-soil contact, decrease mechanical impedance of root growth, and increase soil drainage and aeration), tillage may have detrimental effects as well, such as destruction of aggregation, compaction, and increased organic matter decomposition. Control of plant growth is another means of soil management that affects soil structure. Crop rotations and use of cover or green manure crops are common demonstrations of soil management beneficial to soil structure and fertility. It is increasingly evident that different plants can have distinct effects on soil. Plant influences on soil structure have been observed for some time and for many species. Researchers have investigated the mechanisms of aggregate stabilization, but the possible effects have not been fully separated to quantify their relative effects, and the potential for improving damaged soils has not been realized or developed to any great degree. Isolating and modeling several of the root effects to quantify their short term contributions to soil structure will not only help to understand the physical system but also have important applications in soil management. The objective of the current research was to investigate the short-term root effects on the formation and stabilization of soil aggregates and to determine the direct and indirect contributions of plant roots to soil structure. It was hypothesized that biochemical exudates of plant roots contribute directly (binding soil particles) and indirectly (supporting beneficial microbial populations) to the structural development of soil aggregates, that drying of soil by root extraction at rates and to degrees characteristic of different species is a variable affecting aggregate formation/stabilization, and that the presence of roots in the soil is a structure-forming factor whose significance can be related to root system morphology (length, diameter, distribution). LIST 0! “133.1038 List of References Kay, B.D., D.A. Angers, P.H. Groenevelt, and J.A. Baldock. 1988. Quantifying the influence of cropping history on soil structure. Can. J. Soil Sci. 68:359-368. Soil Science Society of America. 1984. Glossary of Soil Science Terms. Soil Sci. Soc. Am., Madison, WI. LITERATURE REVIII Boil Structure Aggregate size distribution affects mechanical, physical, and agronomic properties of the soil. Processeso::f7f‘ such as compaction, erosion, crusting, evaporation, aeration, and heat transfer are affected by soil aggregate size (Braunack and Dexter, 1989a). The importance of soil aggregation on seed germination and plant growth has been reviewed, especially with regard to size of soil aggregates (Braunack and Dexter, 1989b). Soil structure also influences the incidence and/or severity of root diseases (Carter and Johnston, 1989). Theories of structural development have been proposed. Russell (1971) described the major mechanisms creating structure in soils: shrinkage with drying, plant roots forming or altering channels, soil faunal activity, and cultivation. Stabilizing agents include clay, iron oxide, polysaccaride and polyuronide gums, and fungal hyphae. 6 Soil structure is greatly affected by clay content and mineralogy because of cohesive and adhesive properties associated with clays. The proportion of clay and its mineralogy determine a soil's relative reactivity. Iron and aluminum oxides may be particularly important in interactions with clays and organic matter because of their positive charge. Ions in soil solution can have a significant effect on soil structure. They affect the thickness of the diffuse double layer and potentially the spacing of clay laminae (Abu-Sharar et al., 1987). Cations are also involved in various mechanisms of organic material adsorption to clay (Mortland, 1986). Abu-Sharar et al. (1987) helped to clarify the effect of cations on aggregate stability. Increasingly greater electrolyte (CaClz) concentrations caused lesser degrees of clay dispersion and slaking. At 3 at sodium the higher concentrations (e.g. >3.2 mol m' adsorption ratio=0), slaking into subaggregates larger than 5 um diameter occurred with very little clay dispersion. Expansion of the diffuse double layer with lower electrolyte concentration caused shearing stresses which were resolved by slaking and at some lower concentration caused mutual repulsion of clay particles and therefore dispersion. Climate is a widely variable and independent factor. The temperature aspect of climate has influences on soil structure. Freeze/thaw cycles of soils have been shown to have structural implications in certain latitudes (Pawluk, 1988; Formanek et al., 1984). Temperature also has an important effect on the rates of chemical reactions, including those of bond formation and of mineral weathering, and on rates of biological metabolism, e.g. microbial decomposition. Precipitation is a major determinant of vegetation type and total biomass and therefore of organic matter input to soil. Total precipitation also affects soil processes such as leaching of ions, reduction/oxidation of soil minerals, and translocation of clay. Precipitation intensity and distribution determine rate and degree of soil wetting. The I rate of wetting is important because pneumatic pressures can develop in rapidly wetted aggregates from entrapped air that may explode the aggregates. The amount of water affects the concentration of ions and the water film thickness between clay laminae and other soil particles. Intensity, number, and time of swelling and drying events have been suggested as the major factors determining aggregate strength (Horn, 1990). Drying of soil allows particles to come in contact or closer proximity, and the strength of a single aggregate depends on the number of contact points or the forces that can be transmitted at each contact point (Horn, 1990). Utomo and Dexter (1982) investigated the effects of wetting and drying on sterile and nonsterile tilled, nontilled, and remolded soil. Remolded soil formed more water-stable aggregates when subjected to wetting and drying cycles, especially at the wetter end of the matric potential scale 8 (-1 to -100 kPa), and this effect was greater for nonsterile soil than for sterile soil. When nonsterile tilled soil was subjected to wetting/drying cycles, water-stable aggregation increased to a maximum and then decreased, but wetting/drying cycles always decreased the water-stable aggregation of sterile tilled soil. This demonstrated that variation in soil structural stability was also affected by microbial activity. Cycles of wetting and drying had been known to stimulate microbial activity, which promoted aggregation of disturbed soil in this study. Untilled soil decreased in the proportion of water-stable aggregates with wetting/drying cycles regardless of microbiological population. A theory of equilibrium states of soil aggregation was introduced to explain the increasing or decreasing proportions of water-stable aggregates resulting from wetting and drying: each soil has an equilibrium value or range of aggregate water stability which will be approached as it is wetted and dried and which the untilled soil is initially above and the tilled soil is initially below (Utomo and Dexter, 1982). Hagen et al. (1988) investigated abrasion of soil aggregates by aeolian particles and found a strong interaction of soil water content and dry aggregate stability on determining abrasive loss. A sandy loam soil (1.07% soil organic matter, s.o.m.) and a loam (1.90% s.o.m.) had reduced abrasive losses as soil water content increased. Abrasive losses of a silt loam (2.33% s.o.m.) were little affected by water content, 9 and increased losses with increasing water content distinguished a silty clay loam (1.93% s.o.m.). Coote et al. (1988) also demonstrated the importance of soil water content at the time of structural measurement for a range of soil textures. According to Greacen (1960), unsaturated aggregates under strain experience an increase in pore water tension, which acts as an equivalent applied load on the water-filled fraction of the soil and increases its potential resistance to strain. Organic matter content probably has the most drastic “‘==:;’ effect on soil structure (Tisdall and Oades, 1982). Organic I matter accumulates over the long term to a steady state level, which is determined by the amount of biological contributions over time, soil water content and temperature (regulating decomposition), and other factors such as texture. Fire and other natural processes can interrupt the attained steady-state level of organic matter. Other disturbances to the ecosystem can also alter organic matter content in the short run, e.g. soil tillage, removal of plants or plant parts, or control of species populations. All of the latter "disturbances" are activities of crop production. Other agricultural activities may tend to counteract the reduction of organic matter, such as spreading manure, adding (imported) plant residue, and rotating cultivation seasons with periods of pasture or "green manure" crop, but usually the level of organic matter cannot be maintained at its equilibrium level. This is a 10 primary reason of soil aggregate stability decline after the initiation of intensive row crop production agriculture (e.g., McKeague et al., 1987; Weill et al., 1989). Sharma and Aggarwal (1984) found that organic carbon content was correlated with variation of structural indices. Gregorich et al. (1987) investigated the nature of organic matter within microaggregates. Results suggested that physical protection of carbon within microaggregates had prevented decomposition of easily-decomposable organic substances. The nature of the organic matter is a factor in its effect on soil structure. A trend was found for decreased fixation of humic substances by montmorillonite (smectite) with increasing acidity of the humic samples under low moisture conditions (Nayak et a1, 1990). The cause was thought to be the lowering of interparticle repulsive forces and reduction in hydration energy with decreasing content of acidic groups. Nonionic forces seemed to be important in clay-humus complexes that had been dried. Pojasok and Kay (1990) found that longer chained C compounds of bromegrass exudates extracted by acetone sorbed less readily to soil and decomposed more slowly than water- extracted C compounds. McBride (1987) found that clay and organic matter contents of soil affected results of compression tests but proposed that organic matter effects on water potential and soil strength be better defined with regard to amount, type and degree of organic matter humification. 11 Another aspect of crop production affecting soil structure is applied external force. Animal trafficking may be a natural external force affecting structure (forming platy structure). Another example of larger magnitude is tillage. Cultivation implements are generally designed to overcome the bonds stabilizing structure, breaking aggregates into smaller ones for increased water holding capacity and maximum soil-seed contact. However, compression by modern machinery often destroys soil structure in a subsurface layer of soil. Horn (1988) discussed soil structural resistance to mechanical loading. Internal soil parameters considered important in determining soils' mechanical compressibility included water content and suction, organic content, and structural units and their strength (as induced by swell/shrinking and stabilization by humic matter), as well as particle size distribution, mineralogy, cations, bulk density, pore size distribution, and pore continuity. These strength factors were seen as equilibrated with external forces when soil is "stable". Some biological effects on soil structure are related to the other factors already mentioned. Soil drying through extraction by plant roots increases the rate and average degree of drying that would occur by mere drainage and evaporation. Biological factors obviously determine organic matter contribution to soil. External force is applied by plant roots as they grow; the pressure may approach 0.9 MPa, causing a zone of compaction around the root and reorienting 12 particles tangential to the root (Foster, 1986). Plants and animals also affect soil structure as they create channels by root growth or burrowing; soil peds may be completely destroyed, have a new face defined, or be transected in the process. Earthworms also produce casts as they burrow, and these casts may be the initial form of new aggregates (Marinissen and Dexter, 1990). The distribution of nutrient ions in soil is significantly affected by root uptake (Jungk and Claassen, 1986), however several studies have indicated that cation gradients in the rhizosphere did not affect a rhizosphere aggregate stability significantly (Reid et al., ’J 1982; Reid and Goss, 1981). Other very important biological effects must be noted as well. Microbial populations in the soil are responsible for degradation of dead tissue to produce humified organic matter. Microorganisms and plant roots exude organic compounds into the soil which may directly bind soil particles together or be incorporated into humified organic matter. Root exudates also serve as a rich carbon and energy source for many microorganisms. Reid and Goss (1982b) suggested that microorganisms prefer fresh exudates to dead tissue, therefore affecting the rate of decomposition of residue, but Cheng and Coleman (1990) found that the increased microbial activity associated with living roots resulted in greater decomposition of soil organic matter. Fine roots and microbial hyphae also have been reported to physically bind soil particles together. 4 i 13 Networks of dead and especially living roots resist compactive loads (Soane, 1990) and shear stress (Waldron, 1977; Terwilleger and Waldron, 1990); fungal hyphae work similarly within soil aggregates (Soane, 1990). Lynch and Bragg (1985) reviewed the influences of microorganisms on soil aggregate stability. Time may affect all other factors in the long run, but may also have a significant short term effect. Bond formation and/or strengthening may occur in a matter of days; achievement of a thermodynamically stable state may occur in a period of weeks or months (Dexter et al., 1988). Dexter et al. (1988) discussed two mechanisms of "age- hardening", or strength regain of disturbed soils after some time period. Rearrangement toward a minimum free energy orientation with new inter-particle bonds was postulated as the mechanism operating when the increase in strength over time is independent of compaction pressure but the relative change in strength (S/So) is inversely related to compaction pressure. Cementation (increase of bond strength) was occurring in cases (soils) where the increase in strength over time was dependent on compaction pressure but 8/80 was not. The optimum soil water content for cementation (also called thixotropic hardening) was found to be approximately equal to the lower plastic limit (LPL). Organic matter seemed to inhibit cementation. Many techniques and modifications have been used as indices of soil structure, but no single measurement can 14 completely characterize structure. Researchers must determine which techniques are most applicable to their investigation. Some of the more popular and useful soil structural indices are the proportion of solids and porosity in a volume of soil (bulk density/porosity), the size distribution of peds, the size distribution of pores, soil permeability to air or water, and the stability of peds upon immersion and shaking in water (wet aggregate stability). Sharma and Aggarwal (1984) found that several indices of structural quality were necessary to compare different soil types under different land uses. Percentage of water-stable aggregates, mean weight diameter, bulk density, and total porosity were used to evaluate some soils of India's humid tropics. In most measurements of soil structure, the initial condition of the soil aggregates must be standardized especially with regard to the water content of the sample. Reid and Goss (1981) determined that aggregate stability must be measured with soil samples at an appropriate water content. It may be reasonable to use dry aggregates when soil surface conditions are of interest. The wet aggregate stability method itself reasonably simulates the wetting conditions and erosion processes at the soil surface (Grieve, 1980). Skidmore et al. (1975) compared wet aggregate stability using two methods of aggregate wetting: rapid or flash wetting and gradual wetting under vacuum. Pneumatic pressures of entrapped air under flash-wetting 15 apparently accelerated breakdown, causing greater treatment differences in stability. Other investigators (Stone and Buttery, 1987; Monroe and Kladivko, 1987) have supported this observation. Turbidimetry is a method which measures the light transmission of soil suspensions after end-over-end shaking and a period of settling (Williams et al., 1966) Reid and Goss (1981) used turbidimetry to measure differences in soil cropped to different species, and Chen et al. (1987) used this method to determine flocculation of clays by organic materials. Pojasok and Kay (1990a) measured both wet aggregate stability and dispersible clay (turbid transmission) of the same sample of soil aggregates. Field soil scientists have always recognized the roughness of soil fracture surfaces as important, and a recently proposed measure of soil microstructure attempts to quantify this feature (Grant et al., 1990). The fracture surfaces were produced by tensile stress imposed by hand or by parallel plates. The index of fracture surface roughness was defined as the standard deviation of the differences between measured elevations of soil fracture surface and their corresponding running mean values. Direct laser scanning (multiple scanning) or bisection and image analysis of an epoxy-embedded sample (single transect) were used to determine relative elevations. Tensile strength of individual aggregates, which measures almost directly the force required to break the 16 inter-particle bonds per unit area Of fracture surface (Dexter et al., 1988), can be quantified by crushing tests. Mechanical devices are used to determine the strain that individual aggregates experience before rupturing (Dexter, 1975; Boyd et al., 1983). Nearing et al. (1988) designed a different method to test tensile strength of low density (disturbed) soil at controlled water contents. Conditions or treatments tested included water tension, time, and density. Applied suction had a positive linear relationship with tensile strength over the range 0 to 6 kPa. Tensile strength was also greater with greater density. The confined condition of the sample preventing volume change may have prevented strength increase as a result of pre-stress history or time. A diagram of the major factors and interactions controlling the dynamics of soil structure is presented in Figure 2.1. It was based on the conceptual model of soil structure change presented by Gibbs and Reid (1988) but is more detailed or expanded in terms of the root effects. The model of Gibbs and Reid (1988) emphasized macropore (> 100 um) dynamics. The direct root effects on macroporosity included in the model were creation and blockage of macropores, and roots' decomposition to organic matter. Activities of living roots were cited as indirect influences on organic matter humification, macropore stability, shrinkage (due to water uptake), and planar micropore expansion by mechanical pressure. 17 CLAY AMOWT. MRALOOY IONS IN SOLUTION sneezme/ l\‘ SOIL STRUCTURE THAWING _ 7 FORM AND STABILITY EXTERNAL WETTINGI DRYING MICROORGANISMS ORGANIC MATTER \ (HUMIFIED) , PLANT SOIL WETNESS Figure 2.1. Diagram of major variables and interactions in the formation and maintenance of soil structure. 18 Kay et al. (1988) presented a model to quantify effects of cropping on soil structure. The model was based upon the relative levels of stabilizing materials, organic and inorganic, as well as biological factors and external forces which also influence the rate of structural form change. Losses and additions of stabilizing materials over time were theorized to largely determine the relative stability parameters, which in turn are a factor of structural form change. Cropping Effect Plant species identified as beneficial to soil aggregation include bromegrass (Barber, 1959; Baldock and Kay, 1987), alfalfa (Barber, 1959; Reid and Goss, 1981) and ryegrass (Tisdall and Oades, 1979; Reid and Goss, 1981). Pasture in general has been found to increase aggregation of previously tilled soil (Tisdall and Oades, 1980a; Elustondo et al., 1990; Haynes and Swift, 1990). Stone and Buttery (1987) found that nine forages differed in their ability to improve structure. Sharma and Aggarwal (1984) found that soils with forests or plantings of tea exhibited "better" structure than grassland, and arable soils had the "poorest" structure. The land uses associated with continuous soil cover and lack of soil manipulation provided greater opportunity for development of stable soil structure. Species reported to be detrimental to soil structure were corn (Reid et al., 1982), soybeans (Bathke and Blake, 1984; 19 Fahad et al., 1982) and cereal species in general (Grieve, 1980). Angers et al. (1987) compared soil cropped to corn and to bromegrass with respect to compaction characteristics. Increasing duration of bromegrass culture increased tensile strength of aggregates, which was positively correlated with both aggregate density and interaggregate porosity. Although the compression index was also increased with time under bromegrass, calculations suggest that normal loads (<500 kPa) encountered in the field would not reduce the interaggregate porosity. The observed cropping effects occurred within a few seasons. Skidmore et al. (1975) compared native pasture soil to adjacent soil cropped continuously for more than 60 years with numerous methods and concluded that structure of sod soil was more stable than that of tilled soil. Electron micrographs of low magnification showed that sod soils had more roots and cavities in aggregates than those from tilled soil. The mechanisms of soil aggregate stabilization by plants have been studied. Total organic carbon often has been correlated with aggregate stabilization (Tisdall and Oades, 1980a, Elustondo et al. 1990); qualitative differences in organic matter and the mechanisms or efficiency involved may account for unexplained differences (Tisdall and Oades, 1980a, Reid and Goss, 1981). Often much of the increase in organic carbon has been found in the 20 sand-size fraction, which includes fragments of plant tissue and roots (Elustondo et al., 1990; Cheshire and Mundie, 1990). However, periodate-sensitive materials (polysaccharide and/or polyuronides) have been shown to be stabilizing agents for aggregates in many cases (Angers and Mehuys, 1989; Baldock and Kay, 1987; Reid and Goss, 1981; Tisdall and Oades, 1979). Pyrophosphate-sensitive materials, probably bound to minerals by polyvalent cations, were partially responsible in some cases (Baldock and Kay, 1987, Tisdall and Oades, 1979, 1980a). It was postulated that the polysaccharide compounds which stabilize aggregates are components of root exudate and that qualitative differences in root exudates might be responsible for species' effects on soil aggregation (Reid and Goss, 1981). Extracted exudates of bromegrass increased wet aggregate stability and decreased dispersible clay content of aggregates compared to corn exudates (Pojasok and Kay, 1990b). Carbon content of the corn exudates was greater than that of bromegrass exudates, but bromegrass had greater exudate C per unit length of root. Also, exudates of corn were more decomposable than those of bromegrass. Cheshire and Mundie (1990) found less than 1% of 14C fixed by corn in the soil as soluble substances and suggested that higher estimates of soluble exudates were due to lysis caused by separation of roots from soil and/or removal of the shoot. Shoot harvest has been shown to affect the amount of root exudation; alfalfa harvest increased root 21 release of nitrogenous compounds (Brophy and Heichel, 1989). Tisdall and Oades, (1980b) found that monthly clipping enhanced the effect of ryegrass on soil aggregate stabilization and suggested root turnover processes which liberate organic compounds to the soil as a possible explanation. The detrimental effect of corn on soil aggregates was believed to be directly related to the corn root mucilage (Reid et al., 1982), although that theory has been disputed (Pojasok and Kay, 1990b). Root and associated mycorrhizal binding of soil particles have been shown to stabilize soil aggregates (Tisdall and Oades, 1979, 1980a, 1980b, Miller and Jastrow, 1990). Micrographs have shown fungal hyphae attached to clay particles with amorphous mucilage (Tisdall and Oades, 1979) containing polysaccharide. Rooting patterns (i.e., total root length, distribution, root length density, branching frequency, and root hairs) were suggested to be important in explaining differences in soil stabilization by different species (Reid and Goss, 1981). Miller and Jastrow (1990) found that the length of fine roots (0.2 to 1 mm) and especially the length of extra-radical VAM hyphae were very important biological factors for soil structural development in restored tallgrass prairie. The amount of fine roots that the C4 grass and perennial Composite lifeforms produce and their dependence on VAM colonization make them more important than other forbs and C3 grasses in producing stable aggregates. Thomas et al. (1986) measured water 22 stable aggregation differences resulting from onion roots with and without mycorrhizal infection. VAM-colonized onion roots were associated with greater water-stable aggregation as opposed to the disaggregation and consolidation which occurred with uninfected onion roots. There was a positive correlation of aggregation with root mass and with vesicular-arbuscular mycorrhizae density. However, it was determined that root effects were primarily responsible for the aggregation and that VAM effects were related to the symbiosis-stimulated growth of the host plant (including roots). Stone and Buttery (1987) also found that frequency of VA mycorrhizae was not as much a factor in affecting soil structure as root growth. Warm season, C4 prairie graminoids seemed to promote aggregation better than cool season C3 grasses (Jastrow, 1987), perhaps due to total production, length and timing of growth, physiological differences affecting root exudates, root morphology, and/or microbial population differences (esp. mycorrhizal infection). Soil drying by root extraction of water has been indicated as a stabilizing force in several studies (Monroe and Kladivko, 1987; Reid and Goss, 1982a). Water extraction by corn roots partially diminshed the detrimental effect of corn root on soil structure, but drying to -1.5 MPa matric potential did not completely counteract the structure degradation under this crop (Reid and Goss, 1982a). Reid and Goss (1982a) speculated that drying allowed greater 23 adsorption of organic materials onto mineral surfaces. In the absence of plants, Utomo and Dexter (1982) increased wet aggregate stability of remolded soil by wetting and drying in the -1 to -100 KPa matric potential range. However, the aggregate stability of untilled soil and sterilized, tilled soil decreased in response to wetting and drying. Aggregates of unsterilized, tilled soil initially increased in stability with cycles of wetting and drying but then steadily decreased, and it was concluded that short-term changes in water-aggregate stability in the field were related to microbial activity (Utomo and Dexter, 1982). In another study involving remolded soil, stability was significantly increased by a single drying/wetting cycle, which seemed unrelated to organic binding materials and separate from effects of fungal growth (Marinissen and Dexter, 1990). Remolded soil responded differently than intact soil aggregates, however, and therefore the results apply only to similar situations, such as with earthworm casts (Marinissen and Dexter, 1990). Despite evidence that drying may contribute to soil aggregation, Tisdall and Oades (1980b) found that maintaining adequate water content in the soil to favor ryegrass root growth was more influential on soil structure than a greater degree of drying. Time is an important factor in the stabilization of soil aggregates by plants. Only 8 weeks were necessary before increases in the aggregate stability with ryegrass could be measured (Tisdall and Oades, 1979), but the effect 24 leveled off at 16 weeks. In another study, alfalfa and ryegrass caused linear increases in soil aggregation from one to four years (Barber, 1959), but a degraded soil, such as has been under continuous row cropping, did not recover aggregation levels equal to a virgin site even 30 years after conversion to meadow. Jastrow (1987) studied the dynamics of soil aggregation upon restoring cultivated soil to tallgrass prairie. It was suggested that biologically and physically significant recovery of aggregation may occur after 5 - 10 growing seasons at this site in eastern Illinois, which is relatively rapid compared to estimates of other investigators. Rapid recovery may be aided by high degrees of initial aggregation, very high production of biomass (1 to 1.5 kg m72), lack of soil manipulation, and vegetation type with associated microorganisms. Separating the effect of time without disturbance from the effect of vegetation type was not entirely possible, but such effort indicated that the time factor may predominate. Root Exudatea and Soil Carbohydrates Roots of many species have been shown to exude organic compounds of varying composition and complexity. It is not determined conclusively whether root exudation is an evolutionary advantage or a failure of the plant. Supporting the theory that root exudation has a definite role in plant physiology, Robinson et al. (1989) suggested that the availability of inorganic N to plants is increased 25 when microbial activity is stimulated by carbon release of the roots. Also, proteoid roots of lupin grown in P- deficient soil released citric acid, which is very effective in dissolving phosphate minerals in soil (Dinkelaker et al., 1989). On the other hand, increases in nitrogen release from alfalfa roots under specific treatments (nonsterile, water deficit, and shoot harvest) was attributed to passive loss (Brophy and Heichel, 1989). Nevertheless, it is clear that both genetic and environmental factors influence root exudation. Rovira (1969) compiled a list of these factors affecting root exudation: plant species, plant age, temperature, light, plant nutrition, soil moisture, root damage, and foliar applications. Age of plant affected root exudate composition or amount (Martinez-Toledo et al., 1988). Barber and Martin (1976) found that significantly greater exudation is associated with presence of microorganisms when compared to sterile conditions. Barber and Gunn (1974) showed that mechanical stress increased root exudation of amino acids and carbohydrates. However, Cheshire and Mundie (1990) claimed that the release of large amounts of water-soluble carbohydrate from corn roots was due to cell lysis upon removal of the shoot and separation of roots from soil. Rapid extraction of the soil indicated that as little as 0.5% of 14C fixed during 36 d was released as water-soluble substances. Root lysate solution contained mainly glucose but also galactose and mannose. After 1 yr of incubation, labelled soluble materials remaining were 26 negligible in quantity but still dominated by glucose, perhaps having been adsorbed or being typical of cell contents released by microorganisms. Corn mucigel - distinct from root exudate - was considered water insoluble and likely to remain associated with mineral surfaces after dispersion. Four sugars were measured in maize seedling exudate: glucose, fructose, sucrose, and maltose (Schonwitz and Ziegler, 1989). Glucose was dominant; 617 to 1472 ug plant-1. Rovira (1969) estimated that 0.1 to 0.4% of carbon assimilated was released as exudate to the soil. Barber and Gunn’s (1974) minimum estimate of exudates released was 9% of the dry matter of the root increment grown. Meharg and Killham (1989) demonstrated the effect of temperature on the amount of 14C released from roots of perennial ryegrass. Much of the literature on soil exudation is based on research with corn because of its profuse production of exudates and mucilage. An SEM study of corn roots (Fyson et al., 1988) indicated a mucilage layer nearly covering the entire length. The mucilage was most abundant at the root cap. Soil aggregates were seen embedded in the mucilage at the root cap and on root hairs. The root surface below the root hairs, the elongation zone, and the zone below the kernel were all relatively bare and free of adhering soil or microorganisms. The "soil sheath" (the mantle of adhering soil) in the root hair zone is persistant and is evidence of the adhesive binding of soil by mucilage and root hairs. Schonwitz and Ziegler (1989) found that the percentage of 27 corn root surface covered by microflora (assuming a monolayer) was 4% in the region where root hairs emerge, 7% in the root hair zone, and 20% on the oldest part of roots. Short rods and some filaments dominated the microbial population of these roots. Guinel and McCully (1986) also studied the root mucilage of corn. It appeared to be extruded from root cap secretory cells as one or two small molecular weight components which polymerize extramurally. At water potentials greater than -7.3 kPa, the mucilage hydrated to a water content of 99.9% (w/w). Martinez-Toledo et al. (1988) found that corn root exudates stimulated growth of Azotobacter chroococcum by serving as a carbon and energy source, and production of auxins, gibberellins, and cytokinins by A. chroococcum was doubled. Rovira (1973) labelled carbon in plants to investigate the release of carbonaceous compounds to the "rhizosphere", in this case, a sandwich of chromatography paper. The majority of exudation was seen to occur in the zone of elongation, probably due to leaking of photosynthate intended for the apical meristem. The materials measured on the chromatography paper were largely non-diffusible in water, therefore root cap cells and polysaccharide exuded at the apex seemed to be a major component of root contributions to the rhizosphere. Differences in exudation patterns of wheat and lupin were seen. Wheat exudation was 28 largely confined to the root tip, whereas lupin released soluble exudates from the older region of roots. Moody et al. (1988) determined that the compositions of wheat and cowpea root slimes were similar to that of cell wall preparations of the respective species. They also found that water soluble components of root slime were mostly (> 95%, w/w) carbohydrate. There were species differences in uronic acid content and neutral monosaccharide composition of the root slimes. Dormaar (1988) studied the chemical and biochemical properties of the rhizosphere of two grasses. Eight monosaccharides of soil hydrolysates were identified: rhamnose, fucose, ribose, arabinose, xylose, mannose, galactose, and glucose. Total monosaccharides decreased in the non-rooted soil, but generally increased in the root zone. The relationship was very complex, with soil, crop, distance from root, and all interactions thereof significant at P < 0.001. Polysaccharides formed under the direct influence of the root and rhizosphere were theorized to be predominantly of plant origin, while polysaccharides at a small distance from the root (0.4 to 0.8 cm) would be from microorganisms. Baldock et al. (1987) took samples of soil under bromegrass, corn, and a rotation of both and analyzed the soil hydrolysates for monosaccharides to evaluate their relation to structural stability. Only minor variations were found in monomeric sugar content due to cropping 29 history, and no significant differences in total carbohydrates were found. Eight sugars were found in all soils in the approximate quantitative order glucose > mannose, galactose > arabinose > xylose > rhamnose > fucose, ribose. Only four sugar monomers were found in root tissue of corn and bromegrass: glucose, xylose, arabinose, galactose. Bromegrass roots had larger proportions of arabinose and xylose and smaller proportions of galactose and glucose than corn roots. There is evidence that a higher percentage of soil carbohydrates are plant-derived after 15 yr of bromegrass, as compared to higher proportions- with microbial origins after 15 yr of corn. Only very small aggregates (<0.50 mm diameter) were found to have significant differences in total carbohydrate content with cropping treatment: carbohydrate content in those aggregates was greatest after 15 yr of bromegrass, intermediate after 15 yr of corn, and least after 13 yr of corn followed by 2 yr of bromegrass. The total carbohydrate content increased (on a mg/kg soil basis) as aggregate size decreased below 0.50 mm. Substantial quantities of arabinose and xylose are plant-derived whereas microorganisms synthesize galactose and mannose, therefore the ratio (G+M):(A+X) has been used to indicate the dominant source of soil carbohydrates (Oades, 1984). However, galactose was found in substantial quantities in corn and bromegrass plants, and so the ratio M:(A+X) has been recommended instead (Baldock et al., 1987). 30 Dalal and Henry (1988) determined that carbohydrates constitute 8 to 16% of the soil organic matter in virgin soils, and the amount generally increased with clay content. Monosaccharides released upon hydrolysis of carbohydrates were dominated by glucose and galactose. Polysaccharides were of both microbial and plant origin. Carbohydrates were found in larger quantities in soil cropped to barley and alfalfa for 2 yr compared to fallow soil especially in the sand fraction, which had 46 to 83% more carbohydrate than the fallow (Angers and Mehuys, 1990). However, composition and origin of soil carbohydrate from the soil and its size fractions did not differ greatly between treatments. In a similar study, (Angers and Mehuys, 1989) carbohydrate content of soil was increased up to 25% by cropping to barley and alfalfa for 2 yr compared to corn or fallow. There was a strong correlation between carbohydrate content and mean weight diameter of soil aggregates. On the other hand, cropping to continuous corn for 2 yr decreased carbohydrate content by 30% and organic matter by 9% compared to an adjacent hayfield (Benzing- Purdie and Nikiforuk, 1989). Levels of carbohydrates, arabinose, galactose, glucose, mannose, and xylose mostly of plant origin, were related to qualitatively-assessed soil structure of the well-structured 2-year corn soil compared to a compacted 15-yr corn soil. When combined over soils and cropping histories, the data of Haynes and Swift (1990) indicated that hot-water extractable carbohydrate content 31 was more closely correlated with aggregate stability than either organic C or hydrolysable carbohydrate content. Foster (1986) reviewed various aspects of the root-soil interface and proposed terminology to address it. "Rhizoplane" was defined as the ca. 10 um region surrounding roots, based on the presence of a dense gel of carbohydrate material heavily populated with microorganisms. The "inner rhizosphere" was the 10 to 400 um zone with a microbial population density ten times less than that of the rhizoplane. In this zone, electron microscopy has shown a less dense gel permeating the soil fabric to a distance of 100 um and a nutrient concentration discontinuity at 400 um from the root wall. The zone 400 to 3000 um from the root surface was called the "outer rhizosphere", which contained the diffusion limit of water soluble exudates (1000 um) and extends to the limit of root detection by pathogens (3000 um). The boundaries of the zones were only meant to be estimates since differences in root exudation rates and compounds occur between species and with position relative to the root apex, in addition to many other factors. Summary Soil structure is a very important factor for the biological and physical processes. Development of soil structure involves the aggregation of primary soil particles and stabilization of the aggregates, or peds. There are many varied determinants in the development of soil 32 structure, and the one of interest here is biological, specifically plant roots. Many researchers have shown that plants have various effects on soil structure. There are hypotheses about the mechanisms of plant root effects on soil structure, and evidence indicates that the strongest influences are the exudates of roots, the physical binding of soil aggregates by roots, soil drying by water extraction, and enhanced microbial activity of the rhizosphere. While specific exudates of certain plant roots have been shown to have definite purpose, the most common components of root exudates are not known to have a special reason. Plants produce different exudate components in different amounts, but carbohydrates seem to be the major component of most exudates. It has been shown that soil carbohydrate content and composition varies with cropping treatments and that they can affect the stability of soil structure. The need for further research into the topic of plant root effects on soil structure is evident. LIST OF REFERENCES List of Referenoee Abu-Sharar, T.M., F.T. Bingham, and J.D. Rhoades. 1987. Stability of soil aggregates as affected by electrolyte concentration and composition. Soil Sci. Soc. Am. J. 51:309-314. Angers, D.A., B.D. Kay, and P.H. Groenevelt. 1987. Compaction characteristics of a soil cropped to corn and bromegrass. Soil Sci. Soc. Am. J. 51:779-783. Angers, D. A., and G. R. Mehuys. 1990. 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Effect of living roots of different plant species on the aggregate stability of two arable soils. J. Soil Sci. 32:521-541. Reid, J.B., M.J. Goss, and P.D. Robertson. 1982. Relationship between the decreases in soil stability effected by the growth of maize roots and changes in ‘ organically bound iron and aluminum. J. Soil Sci. 33:397- 410. Robinson, D., B. Griffiths, K. Ritz, and R. Wheatley. 1989. Root-induced nitrogen mineralisation: A theoretical analysis. Plant and Soil 117:185-193. Rovira, A.D. 1973. Zones of exudation along plant roots and spatial distribution of microorganisms in the rhizosphere. Pestic. Sci. 4:361-366. Rovira, A.D. 1969. Plant root exudates. Adv. Agron. 35- 57. Russell, E.W. 1971. Soil structure: Its maintenance and‘ improvement. J. Soil Sci. 22:137-151. Schonwitz, R., and H. Ziegler. 1989. Interaction of maize roots and rhizosphere microorganisms. Z. Pflanzerernahr. Bodenk. 152:217-222. Sharma, P.K., and G.C. Aggarwal. 1984. Soil structure under different land uses. Catena 11:197-200. Skidmore, E.L., W.A. Carstenson, and E.E. Banbury. 1975. Soil changes resulting from cropping. Soil Sci. Soc. Am. PrOC. 39:964-967. Soane, B.D. 1990. The role of organic matter in soil compactibility: A review of some practical aspects. Soil & Tillage Res. 16:179-201. 39 Stone, J.A., and B.R. Buttery. 1987. Nine forages and the structure of a clay loam soil. Agron. Abstr. 79:247. Terwilleger, V.J., and L.J. Waldron. 1990. Assessing the contribution of roots to the strength of undisturbed, slip prone soils. Catena 17:151-162. Thomas, R.S., S. Dakessian, R.N. Ames, M.S. Brown, and G.J. Bethlenfalvay. 1986. Aggregation of a silty clay loam soil by mycorrhizal onion roots. Soil Sci. Soc. Am. J. 50:1494- 1499. Tisdall, J.M., and J.M. Oades. 1982. Organic matter and water stable aggregates in soil. J. Soil Sci. 33:141-164. Tisdall, J.M., and J.M. Oades. 1980a. The effect of crop rotation in a Red-Brown Earth. Aust. J. Soil Res. 18:423- 433. Tisdall, J.M., and J.M. Oades. 1980b. The management of ryegrass to stabilize aggregates of a Red-Brown Earth. Aust. J. Soil Res. 18:415-422. Tisdall, J.M., and J.M. Oades. 1979. Stabilization of soil aggregates by the root systems of ryegrass. Aust. J. Soil Res. 17:429-441. Utomo, W.H., and A.R. Dexter. 1982. Changes in soil aggregate water stability induced by wetting and drying cycles in non-saturated soil. J. Soil Sci. 33:623-637. Waldron, L.J. 1977. The shear resistance of root-permeated homogenous and stratified soil. Soil Sci. Soc. Am. J. 41:843-849. Weill, A. N., C.R. De Kimpe, and E. McKyes. 1989. Effect of tillage reduction and fertilizer on soil macro- and microaggregation. Can. J. Soil Sci. 69:489-500. Williams, E.G., D.J. Greenland, G.R. Lindstrom, and J.P. Quirk. 1966. Techniques for the determination of the stability of soil aggregates. Soil Sci. 101:157-163. CHAPTER 3 SOIL STABILITY ANALYSES POLLO'ING REPEATEE APPLICATION OR ROOT EXTRACT SOLUTIONS AND DRYING CYCLES IN THE LABORATORY Abstract Soil drying by water extraction and the contribution cf exuded compounds to soil are processes of plant roots that may influence soil structure. A laboratory experiment was conducted to simulate these processes without the presence of roots to avoid other effects that plant roots have on soil structure. The root exudates were simulated by water soluble extracts of roots obtained from seedlings grown in sterile sand culture. Treatments included the application of water (control), root extracts of alfalfa (Medicago sativa, L.), and root extracts of perennial ryegrass (Lolium perenne, L) to wet the soil. Eight cycles of wetting and drying to 0.3 MPa were imposed on the samples. Individual pressure cells (soil treatment cells, STC) were developed for extraction of soil solution from each soil sample. Soil (aggregation analyses included dry aggregate distribution, crushing resistance, wet aggregate stability, and turbidity tests. Results indicated that root exudate treatments did 40 41 not affect soil aggregation to a significant degree. Further research in this area might be improved by collection and application of actual root exudates, increased degree of drying, more cycles of wetting and drying, sequential sampling to determine temporal changes, improved methods of soil aggregate characterization, and more complete analysis of root exudates. Introduction Vegetation has been observed to have an influence on soil aggregate stability. Ryegrass (Tisdall and Oades, 1979; Reid and Goss, 1981), bromegrass (Barber, 1959; Baldock and Kay, 1987), and alfalfa (Barber, 1959; Reid and Goss, 1981) have been reported to stabilize soil aggregation, while corn (Reid and Goss 1981; Reid et al., 1982), soybean (Fahad et al., 1982; Bathke and Blake, 1984), and cereal cropping, (Grieve, 1980) were reported to be detrimental to soil structure. Soil structural improvements were sometimes related to periodate-sensitive materials (Reid and Goss, 1981; Reid et al., 1982; Tisdall and Oades, 1979, 1980a, 1980b; Baldock and Kay, 1987) or pyrophosphate- sensitive materials (Tisdall and Oades, 1979, 1980a,; Baldock and Kay, 1987), presumably polysaccharides and polyuronides. Total C content was correlated in some cases (Grieve, 1980; Tisdall and Oades, 1980a,b) to cropping effects and aggregate stability. Physical binding of soil particles by roots and by associated mycorrhizal fungi has been shown by micrograph and otherwise implicated in the aggregation process (Tisdall and Oades, 1979, 1980a, 1980b). Chelating agents of Fe3+land Al3+ in the mucilage of corn roots may account for the detrimental effects of corn on soil structure (Reid et al., 1982), but drying of soil by ,corn roots was seen to moderate their detrimental effect (Reid and Goss, 1982a). Other suggested reasons for structural degradation due to vegetation have been: reduced 42 43 particle bonding, greater root decomposition rate, physical forces from roots, and rhizosphere microbial activity (Fahad et al., 1982). To better understand the contribution of roots to soil structural development, a laboratory study was initiated to investigate the effects of soluble root extracts on soil aggregation in the absence of roots. The stability of soil aggregates after treatment with root extract solutions from alfalfa (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.) was compared to that of control aggregates (water-treated). The hypothesis was that plant roots contribute to the stability of soil aggregates through the exudation of water-soluble carbohydrates into the rhizosphere. The specific objectives of the study were: 1) to grow alfalfa and perennial ryegrass in sterile sand culture and harvest the roots, 2) to extract and quantify the water soluble compounds of the roots, 3) to apply the two root solutions and water (control) to individual soil samples and impose drying cycles, and 4) to determine the soil aggregate stability by four methods for assessing the effects of the root solutions. Materials and Methods Bog;_£x§;ggtg. The application of unaltered root compounds to the soil was the immediate goal in this study, although microbial alteration after application was not 44 prevented. To obtain the root compounds, seedlings were grown in a sterile environment and harvested using sterile technique. Glass jars (240 ml) were filled with sand to within 1 cm of the rim. Beakers (400 ml) were inverted over the jars and closely fitted the jars, preventing contamination due to air currents and yet allowing gas exchange. These glass assemblies were autoclaved three times for 20 min at 121 C with a 15 min dry cycle at 24 hr intervals. In the environment of a laminar flow hood, seeds of alfalfa (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.) were surface-sterilized with 30% bleach solution (1.6% hypochlorite) for 5 min and rinsed with sterile water five times. The seeds were immediately planted in the sterile sand jars, and sterile water (30 ml) was added to maintain the plants until harvest. Sterile plastic bags (Whirl-Pak, 540 ml) were placed over the jars and loosely secured so as to maintain sterility but allow gaseous exchange. The seeds were germinated in the dark for 2 d, and the seedlings were grown for 16 to 19 d under fluorescent growth lights at constant temperature (30 C). Eighteen to twenty- one days after planting, the seedlings were harvested in the laminar flow hood. Shoots were cut near the sand surface. After cutting through the sand media below the seed depth and discarding the seed zone, the remaining sand was spread onto a sterile surface, and roots were combed out of the sand with sterile forceps. The roots were dipped in sterile 45 water to rinse away loose sand; some sand remained adhered to root surfaces, however. The rinsed roots were placed in a sterile plastic bag (Whirl-Pak, 180 ml) and frozen in liquid nitrogen. This served to lyse cells, releasing their contents, and to arrest any microbial activity in case of contamination during the harvest process. The root samples were stored in a -20 C freezer until water extraction could be accomplished. Shoot material for each sample was dried and weighed. Frozen root samples were macerated in a cold room (4.5 C) using a mortar and pestle, which had been previously sterilized. One mortar was used for each of the two plant species. The crushed roots were rinsed with 50 to 100 ml of sterile water onto Buchner funnels with glass microfiber filters and vacuum was applied to extract water and the soluble compounds into the suction flask. The volume of the collected solution was determined. Small samples (0.5 ml) of the solution were taken from each for analysis of soluble carbohydrates by HPLC (high pressure liquid chromatography). Remaining solution was bulked with that of same plant species and filtered through 0.2 um membranes for resterilization. The root solutions were transferred in dose volumes (100+ ml, i.e. 20 ml x 5 replications) to plastic bottles for storage in the freezer (-20 C). The contents of each Buchner funnel (root, sand, glass microfiber filter) were transferred to a crucible for 46 drying, weighing, combustion, and reweighing in order to determine root dry weight. S911_Extragtign. For the extraction of soil solution from the treated soil, individual pressure cells, similar to Tempe cells (SoilMoisture, Santa Barbara, CA), were developed (Figure 3.1). The advantages of the new soil treatment cell (STC) compared to the Tempe cell are that it allows water extraction to 0.5 MPa, is larger, and allows sterilization by autoclave; compared to a large pressure pot, STC's allow collection of extracted solution from individual samples. The main components of the STC (top,‘ bottom, and cylinder) were constructed of Delrin plastic. A ceramic plate (0.5 MPa bubbling pressure) was fitted into the bottom, which had drainage channels and an outlet for the extracted solution. The top piece had a fitted opening for the application of air pressure to the cell. O-rings aided in sealing the cells, and long bolts through the top and bottom held all parts together when sealed. Fifteen cells were made and linked to a air pressure manifold system. The flow rates were variable, probably due to the polishing of the ceramic plates. Also small leaks were evident by bubbling but were largely eliminated by further tightening of the bolts. Treatments. The soil treatment cells (STC's) were filled with 200 grams of low organic matter, loam-textured soil from the E horizon of the Marlette series (fine-loamy, mixed, mesic Glossoboric Hapludalf) sieved through a 2 mm 47 .Aoamv dado ucoaucoue awom e no sew> soda can acuuomm nacho 853.. 25.5: \\\\\ §N\\\\\\\\._ .\ \“\ . ><:<»:o\mo_m 3m.) QOhxc‘OPHOm 48 screen. One STC had 224 g soil due to a miscalculation. Deionized water was initially added to saturate the soil. Each STC was sealed, and 0.3 MPa pressure was applied. Treatments consisted of repeated applications (20 ml) of either deionized water (control), alfalfa root solution, or ryegrass root solution. A completely randomized design was employed with five replications for each treatment. The solutions were applied to the soil through long syringe needles inserted into the top opening. An equilibration period of 15 to 18 h was allowed after application of the root solutions or water, and then a pressure of 0.3 MPa was applied to the soil for a period of 2.5 to 3 d. The treatments were applied eight times. The total period of time involved for soil treatment (eight cycles) was 36 days. The water forced from each STC was collected for quantification and frozen (-20 C) for later carbohydrate analysis. Each STC was weighed before and after each cycle to estimate the water content changes of the soil. S911_Analy§§§. At the completion of 8 treatments, soil was removed from each STC and broken while moist to pass through a 6 mm sieve. Soil was air dried on brown paper for 3 d. Size distributions of the aggregates were determined by dry sieving; size ranges included <1 mm, 1 to 2 mm, 2 to 4 mm, and 4 to 6 mm. Aggregates in the largest range (4 to 6 mm) were tested for crushing strength (Dexter, 1975) by a computer-controlled press. Statistics were performed on the means of 15 aggregates for each 15 STC. Subsamples of the 1 49 to 2 mm fraction were pre-wetted at 50 cm H20 tension and subjected to wet-sieving (Kemper and Rosenau, 1986) and turbidity tests (Williams et al., 1966) for further assessment of aggregate stability. Significance of treatment differences was tested by analysis of variance; Fisher's protected least significant difference (LSD) was computed at the 0.05 level if the analysis showed P<0.05. figlutign_gnaly§1§. The original solutions and the soil solution leached from each STC were prepared for HPLC analyses of soluble carbohydrates. A Dionex Carbopak Column and Pulsed Amperomeric Detector (Dionex, Sunnyvale, CA) were used. The eluent was 100 mM NaOH at a flow rate of 0.8 ml min'l. Sample preparation included filtration through 0.22 um membranes and Sep-Pak C18 cartridges. The C18 cartridges had to be pre-wetted with methanol, but approximately 2 ml leachate was rinsed through before the final 0.5 ml sample was collected to minimize/equalize methanol contamination and dilution. The external standard included 10 mg 171 each of rhamnose, arabinose, mannose, glucose, galactose, xylose, fructose, and sucrose. Because galactose, glucose, and xylose (and sometimes mannose) were not completely resolved, the actual concentrations of these compounds could not be determined separately; however the concentration values were presented to provide relative measures of the peak size. Sgil_Qrganig_Mat_gr. Organic matter content of the treated soil was determined using gravimetric loss on ignition (Jackson, 1958). A subsample of aggregates (1 to 2 50 mm) from each STC was placed in a tared crucible and covered. After drying at 110 C the crucibles were cooled in a dessicator and weighed. They were then transferred to a muffle furnace and heated to 350 C for 6 to 7 h. They were removed to cool in the dessicator and then reweighed. The weight loss occurring between 110 and 350 C was considered to be due to organic matter. The organic matter is expressed as a percentage of soil weight. Results and Discussion Elant Grog; . Ryegrass seedlings generally did very well in sterile sand culture. Alfalfa had poorer germination and slower initial growth. Alfalfa seedlings also tended to have several centimeters of root growth above the sand surface, possibly due to aeration problems or impedance to thick (relative to ryegrass) root growth. Therefore, root yields for ryegrass were much greater than for alfalfa. Sand samples from the sterile jars were plated onto agar at the time of harvest. A large number tested positive for bacterial or fungal populations. Whether this was because of ineffective seed sterilization or contamination during growth or harvest was uncertain. However, only in the case of pathological infection were the root samples discarded. Otherwise, it was a reasonable assumption that the vast majority of biochemicals extracted were root- derived. Pojasok and Kay (1990) also had difficulty 51 achieving sterile conditions for extraction of root exudates, but the microbial counts for their "sterile" treatments were much lower than inoculated treatments. a s s. The extraction process worked well. Since the samples were processed individually, the amount of roots varied and water required to rinse was arbitrary. The solution samples taken for carbohydrate analysis were often too dilute or too concentrated to give accurate information, but samples of the bulked root solutions were analyzed with appropriate adjustments to the instrument. The results of carbohydrate . analyses for bulked root extracts are summarized (Table 3.1). All sugars in root extracts were not resolved by carbohydrate analysis, and so it was impossible to estimate, based on either formula (Glucose+Mannose):(Arabinose+Xylose) (Oades, 1984) or M:(A+X) (Baldock et al., 1987), whether the primary source of carbohydrates was plant- or microbial- derived. WWW- STC test runs resulted in a water desorption curve (Figure 3.2). It was found that at 0.3 MPa pressure, the soil water content was 'approximately 9.0%. The addition of 20 ml of solution then increased the soil water content in the STC to approximately 19%, except for the STC containing 224 9 soil, where the water content was 17.9%. Extraction rates of STC leachate were monitored and recorded. The cumulative outflow over the dry-down periods 52 .moucESmo Eco v.3 acoficbcoocoo Emma ”3.on 00203.5: “concave mcEEoo mx\c_0\_so 0:6 once—:62 season 03»: acoSsbcoocoo J 2950 so.“— .u._cmsm 05 «o moo-toned 535:5 one monsoon?— soboeaoo 25.53% 3 can 39:52. do: 0.... cozubcoocoo 9.80.85 .8160.“ 00330.5... 3:32am.— bQEUEco Etc—co 05. I I I I I I $3003— I I I I _ 0.0 I 6:32 I I I I I I 33:00 a 298 .3288. saw I I 8.0 I 3 .0 I mmcuuoam I I I I 0N0 I «:32 00.0 00.0 N“ .0 00.0 A.— .0 I .obcoo n 298 .3858. saw I no.0 «0.0 I bud I mmcswohm I 00.0 00.0 I 0N0 I 3.53 I 00.0 0N0 I 0N0 I 33:00 m 296 .3233. saw 8.6 an." 88 8... SS 823.0. 3.6 m3 and 8.8 3.6 «:82 3.6 2: Que 2.6 we... .828 _ £96 .3203. mom 3.6 88 S .o I 8 .o I 3243:. I new :8 8.6 3 .o 83 2.82 I I I I I I —O.5GOU 38.5mm Loom I.\mE umosoam cacao—fl..— .mx\20\_co 03532 235992 80:52:. .mobozoofl 20m 05» muombxm boo.— E mocsodaoo 00:52:03 00 cofimbcmwocoo 4.0 03o.- 53 A 35 1 ter . wa N « added Drying pressure . 30 j. (29%) 0 experiment > I 2 r I 00 25 - v a +9 20- C‘. I 3 15 3 Ci 1 O 1 0 10-_- S I .I.> 5 -: It; 1 3 O " fl I I I j I 'T I air-dry wetted 68 136 204 272 340 408 Soil condition/Drying pressure (KPa) Figure 3.2. Water desorption curve for the disturbed. <2 mm loam soil obtained by Soil Treatment Cells (STCs). 54 was averaged across drying cycles for each treatment (Figure 3.3). Soil in the control (water) treatments appeared to drain more slowly than alfalfa or ryegrass treatments. There are several possible explanations. Since all treatments ultimately (at 0.3 MPa) reach the same level, the pore volume for size range >0.5 um is the same, however, changes in the micropores within that size range may have affected the rate of outflow. However, randomization of treatments should have eliminated any differential initial pore size distribution. For the same reason, it is unlikely that ceramic plate flow rates could be responsible. Tortuosity of pores might decrease flow rates, but it is unclear why tortuosity in the control might be greater than in the other treatments. Final water content at 0.3 MPa was not significantly different by treatment. There was a trend for increased water retention with successive cycles, however (Table 3.2). Note that the final measurement was determined directly by the gravimetric analysis of soil subsamples, while the others were estimated by STC weights. The change indicates a shift in pore size distribution; the overall change of pore diameters from >0.5 um to <0.5 um amounted to a volume of 6 cm3. The change was not unexpected; the initially loose, aggregated soil became cohesive and massive by the end of the treatment period. The results of carbohydrate analyses for extracted soil solutions are summarized with those of the bulk root Volume of leachate (m1) 55 15" 15%... / _, - "9 ‘ a gg/jbs—1-11; vv/ - / ”V - // Rf _ 9” /v -I V 5‘ J 1?, V—V Control ‘ o—o Alfalfa ' 0-0 Ryegrass O "WI"”'IHWIIWIIII'”IWHII'II'I'H'II'I'ITI"H'IHHII O 6 12 18 24 3O 36 42 48 54 6O 66 Time (hours) Figure 3.3. Mean cumulative outflow from Soil Treatment Cells (STCs) during dry—down periods. 56 Table 3.2. Mean gravimetric water content of soil after each STC dry-down cycle. stle Eater_sontent_at_9l2_nza % 0(water) ‘:~imtnow»aaw omenoooooococom .00000000 ~14~momm~u>q LSD(0.05) 0.4 *Water content for cycle 8 was determined by soil subsample._ Fisher's protected LSD procedure was used at the 0.05 level. extracts (Table 3.1). They demonstrated that sugars added were increasingly removed from the soil solution. Even the control soil with additions of only water had leaching of some sugars. Those sugars either had been protected in the soil from the time of drying, or the control soil was producing or releasing them from decomposition of soil organic matter when wetted. Aggregatg_Analy§g§. According to dry aggregate sieving, there were no treatment differences in aggregate size ranges (Table 3.3). The value of this evaluation is limited, however, because of the massive nature of the soil after treatments (with little indication of true aggregation) and the subjective process of breaking the soil into pseudo-aggregates. On the other hand, it demonstrates 57 Table 3.3. Size distribution of soil aggregates after 8 cycles of treatments. $1_mm 1:2.mm 2:1.mm. A:§_mm % % % % Control 37.6 15.4 16.8 30.2 Alfalfa 36.8 15.6 16.4 31.2 Ryegrass 36.7 15.7 16.5 31.1 LSD(0.05) NS NS NS NS Fisher’s protected LSD procedure was used at the 0.05 level. that all treatments were handled similarly during the breaking-down process. The results of the crushing tests (Figure 3.4) showed that the average aggregate strength of the control treatment was less than that of the other treatments, but the difference was not significant due to the high variability (C.V.=23.5%). Besides the inherent heterogeneity of soil, the variability in crushing strength can also be attributed to the range of sizes (4 to 6 mm and <0.1 to 0.3 g) and shapes of aggregates. Wet sieving analysis showed a tendency for aggregates from the alfalfa treatment to be less stable in the saturated condition (Table 3.4), but differences were not significant. Variability was reasonable, CV=12%. It is possible that use of air-dry aggregates rather than pre- wetted aggregates would have resulted in greater differences with reduced stability (Skidmore et al., 1975; Monroe and 58 7 Ar . . 0........0..........9 axxxxxxxxxxxxquxxxxv .aaafimaaaaafifiwafiafiaafiv .32V..0...:V......... LSD“: 174 9 600- _ _ a . . O O O O O O 4 3 2 1001 _ O O 5 Amv oocmamwmos wcfimdso Alfalfa Ryegrass Control Treatment Figure 3.4. Mean crushing resistance (CR) for 4 to 6 mm aggregates after 8 cycles of treatments. 59 Table 3.4. Mean wet aggregate stability (WAS) of soil aggregates after 8 cycles of treatments. Eflfi % Control 48.3 Alfalfa 44.1 Ryegrass 50.5 LSD(0.05) NS Fisher's protected LSD procedure was used at the 0.05 level. Kladivko, 1987). Pojasok and Kay (1990) found that extracted exudates of bromegrass increased wet aggregate stability of calcareous silt loam soil aggregates, especially with water-extracted exudates. Turbidity analysis also found no statistical difference in soil aggregate stability between treatments (Table 3.5). Transmission measurements after 2 min of end-over-end Table 3.5. Mean transmission of soil suspensions following 2 min (T2) or 20 min (T2 ) of end-over-end shaking of 1 to 2 mm aggregates. Turbidity ratio, TR=T20 T2 . 12 120 IR % % Control 19.1 7.1 0.374 Alfalfa 19.4 7.1 0.369 Ryegrass 21.0 7.1 0.339 LSD(0.05) NS NS NS Fisher's protected LSD procedure was used at the 0.05 level. 60 Table 3.6. Organic matter (OM) content of soil aggregates (1 to 2 mm) after 8 cycles of treatments. QM % Control 1.28 Alfalfa 1.29 Ryegrass 1.30 LSD(0.05) NS Fisher's protected LSD procedure was used at the 0.05 level. shaking were very close for all treatments. The mean transmission values for treatments after the 20 min of shaking were identical. The ratio of the two readings, TR = 1&0 Tz’l, showed no differences either. Q;ganig_m§tter. Organic matter content was not significantly increased by root extract solution treatments (Table 3.6). The amounts of carbon added to the soil through treatments was small relative to the weight of the soil, and the solubility of the sugars made them readily leachable when the soil solution was extracted. Effects of root extract solutions on soil structure and aggregate stability under the conditions of this experiment were not found. A greater number of drying cycles or drying to a greater extent may be necessary to measure differences in soil aggregate stability. Utomo and Dexter (1982) found that the response of soil to wetting and drying cycles depended on the initial state of soil, and they hypothesized 61 that an equilibrium state of aggregation exists for each soil which was approached with increasing cycles of wetting and drying. A series of sampling dates throughout this experiment may have revealed such a change. Information about soil respiration may have been valuable to compare microbial biomass estimates and determine the fate of the added carbohydrates. Microbial activity is generally considered beneficial to soil structure (Lynch and Bragg, 1985). Soluble sugars added via root extracts in this study appeared to be increasingly removed from the soil solution with repeated cycles. Initially, sugars were leached with the extracted soil solution even for the control. Then with increasing microbial activity, perhaps the added soluble sugars were readily metabolized with few by-products to contribute to stabilizing the soil. Soil aggregate stability analyses suggested that sorption of organic compounds to soil particles which would stabilize aggregates did not occur to any extent. Pojasok and Kay (1990) found that water- extracted exudates decomposed more rapidly but sorbed more readily to soil than longer chained compounds extracted by acetone, but bromegrass exudates extracted with water were the most effective in improving soil aggregation while corn exudates, which were more decomposable than bromegrass exudates, did not affect soil aggregation. Conclusions The results of this study suggested that soluble root extracts have little direct effect on the development of soil structure over a limited time and degree of drying. Perhaps the short-term effects of alfalfa and ryegrass roots on soil structure in the field result from more complex rhizodeposits (insoluble exudates or decomposition products) which cannot be extracted by water, although this is contrary to the findings of Pojasok and Kay (1990), who found that the bromegrass root exudates extracted by water were most effective in stabilizing soil aggregates. Perhaps the physical action of roots contributes significantly to the formation and/or stabilization of aggregates. 62 LIST OF RHBRBNCBB List of References Baldock, J.A., and B.D. Kay. 1987. Influence of cropping history and chemical treatments on the water stable aggregation of a silt loam soil. Can. J. Soil Sci. 67:501- 511. Baldock, J.A., B.D. Kay, and M. Schnitzer. 1987. Influence of cropping treatments on the monosaccharide content of the hydrolysates of a soil and its aggregate fractions. Can. J. Soil Sci. Soil Sci. 76:489-499. Barber, S.A. 1959. The influence of alfalfa, bromegrass, and corn on soil aggregation and crop yield. Soil Sci. Soc- Am. PrOC. 23:258-259. Bathke, G.R., and G.R. Blake. 1984. Effects of soybeans on soil properties related to soil erodibility. Soil sci. Soc. Am. J. 48:1398-1401. Dexter, A.R. 1975. Uniaxial compression of ideal brittle tilths. J. Terramechanics 12:3-14. Fahad, A.A., L.N. Mielke, A.D. Flowerday, and D. Swartzendruber. 1982. Soil physical properties as affected by soybean and other cropping sequences. Soil Sci. Soc. Am. J. 46:377-381. Grieve, I.C. 1980. The magnitude and significance of soil structural declines under cereal cropping. Catena 7:79-85. Kemper, W.D., and R.C. Rosenau. 1986. Aggregate stability and size distribution. pp. 425-442. In A. Klute (ed.) Methods of Soil Analysis, Part 1. Physical and Mineralogical Methods. Agron. Monograph no. 9, 2nd ed. ASA-SSSA, Madison, WI. Lynch, J.M., and E. Bragg. 1985. Microorganisms and soil aggregate stability. Adv. Soil Sci. 2:133-171. Monroe, C.D., and E.J. Kladivko. 1987. Aggregate stability of a silt loam soil as affected by roots of corn, soybeans, and wheat. Comm. in Soil Sci. Plant Anal. 18:1077-1087. 63 64 Oades, J.M. 1984. Soil organic matter and structural stability: Mechanisms and implications for management. Plant and Soil 76:319-337. Pojasok, T., and B.D. Kay. 1990. Effect of root exudates from corn and bromegrass on soil structural stability. Can. J. Soil Sci. 70:351-362. Reid, J.B., and M.J. Goss. 1982a. Interactions between soil drying due to plant water use and decreases in aggregate stability caused by maize roots. J. Soil Sci. 33:47-53. Reid, J.B., and M.J. Goss. 1982b. Suppression of decomposition of 14C-labelled plant roots in the presence of living roots of maize and perennial ryegrass. J. Soil Sci. 33:387-395. Reid, J.B., and M.J. Goss. 1981. Effect of living roots of different plant species on the aggregate stability of two arable soils. J. Soil Sci. 32:521-541. Reid, J.B., M.J. Goss, and P.D. Robertson. 1982. Relationship between the decreases in soil stability effected by the growth of maize roots and changes in organically bound iron and aluminum. J. Soil Sci. 33:397- 410. Skidmore, E.L., W.A. Carstenson, and E.E. Banbury. 1975. Soil changes resulting from cropping. Soil Sci. Soc. Am. PrOC. 39:964-967. Tisdall, J.M., and J.M. Oades. 1980a. The effect of crop rotation in a Red-Brown Earth. Aust. J. Soil Res. 18:423- 433. Tisdall, J.M., and J.M. Oades. 1980b. The management of ryegrass to stabilize aggregates of a Red-Brown Earth. Aust. J. Soil Res. 18:415-422. Tisdall, J.M., and J.M. Oades. 1979. Stabilization of soil aggregates by the root systems of ryegrass. Aust. J. Soil Res. 17:429-441. Utomo, W.H., and A.R. Dexter. 1982. Changes in soil aggregate water stability induced by wetting and drying cycles in non-saturated soil. J. Soil Sci. 33:623-637. Williams, B.G., D.J. Greenland, G.R. Lindstrom, and J.P. Quirk. 1966. Techniques for the determination of the stability of soil aggregates. Soil Sci. 101:157-163. INFLUENCE OE ALEALEA AND RYEGRASS ROOT GROWTH 0N SOIL AGGREGATION IE GREENHOUSE STUDIES Abstract The mechanisms of root effects on soil structure are not clear. Two greenhouse studies were conducted to determine the effects of root growth and cycles of wetting and drying on soil aggregation. Alfalfa (Medicago sativa, L.) and perennial ryegrass (Lolium perenne, L.) were the plant treatments compared to the unplanted soil, which underwent similar drying cycles. Plants were grown in the greenhouse for four months in the first experiment (8H1) and for one month in the second experiment (6H2). The secondary factor for the first experiment was position of soil in the pot (top vs. bottom), and for the second experiment, two drying cycles were imposed by different irrigation schedules. Soil aggregation was measured by four methods: dry aggregate sieving, wet aggregate sieving stability (WAS), crushing resistance (CR), and turbidity tests. In GHl, aggregation under ryegrass appeared to be more 65 66 developed and stronger than the control when in a dry state, but when wet, was less stable than alfalfa aggregation. Soil organic matter was increased by plants compared to unplanted soil. In GH2, similar results or trends were found by WAS and CR as compared to 6H1. The two experiments had contrary results and trends with dry aggregates < 1 mm and with turbidity tests, which may be due to the periods of treatment. The soil position factor of GH1 was significant for dry sieving and T20, and the irrigation factor of GH2 was not significant. Root dry weight (RDW) was positively correlated with turbidity measurements in both studies. The‘ correlation of RDW with soil <1 mm was negative for GHl but positive for 6H2. In 6H2, negative correlations with RDW occurred for crushing strength and for the percentages of aggregates 2 to 4 mm and >4 mm. Limited root length (RL) data from GHl indicated negative correlations with WAS and turbidity measurements after 2 min (T2) and 20 min (T20) of shaking. A positive correlation between RL and the ratio of the two turbidity measurements (TR) was found. Introduction Effects of various plant species on soil structure have been observed in the field. Improved aggregation and stability of aggregates resulted with the growth of perennial ryegrass (Tisdall and Oades, 1979), bromegrass (Barber, 1959; Baldock and Kay, 1987), and alfalfa (Barber, 1959; Reid and Goss, 1981). Other crop species, such as soybeans (Bathke and Blake, 1984; Fahad et al., 1982), cereal grains (Grieve, 1980), and corn (Reid et al., 1982), have been associated with detrimental effects upon soil structure. In these studies, the plant roots have been regarded as possible causes of soil structure change. Laboratory studies (Monroe and Kladivko, 1987; Reid and Goss, 1981) also have been undertaken to investigate the possible mechanisms of root effects on soil structure under controlled conditions. The root processes that may effect soil structure change are exudation of organic compounds (Reid and Goss, 1981; Pojasok and Kay, 1990), root extraction of water (Reid and Goss, 1982a; Monroe and Kladivko, 1987), support of VAM fungi (Tisdall and Oades, 1979; Miller and Jastrow, 1990), mechanical binding (Soane, 1990), and/or contribution of organic matter to soil through decomposition (Tisdall and Oades, 1979; Elustondo et al., 1990). The objective of these greenhouse studies was to investigate the effects of roots of a grass species and a 67 68 legume species on soil aggregation with particular interest in soil water content and root additions to the soil. Materials and Methods Two studies were completed in the greenhouse; they are referred to as GHl and GH2. The main treatments in both GHl and GH2 were growth of alfalfa or ryegrass or no plants (unplanted). Soil from the E horizon of a Marlette loam (fine-loamy, mixed, mesic, Glossoboric Hapludalf) was dried, ground, and sieved through a 2 mm sieve. This soil has low organic matter content and is leached of CaCO3 (Cremeens, 1983). Plastic, 15 cm pots were used with a thin layer of gravel (approx. 2 cm) on the bottom. gal. This study was planned as a split block design with three replications, the split being a depth factor to account for differences in root length density and soil water content. Within each block were three pots each of unplanted soil, alfalfa, and ryegrass for analysis. Alfalfa and ryegrass were planted 7 October 1989 in three pots per block at high seeding rates (alfalfa: 13.4 kg ha'1 or 0.075 g pot-1; ryegrass: 24 kg ha"1 or 0.125 g pot-1). A trickle irrigation system with a timer was used to facilitate watering; the system provided 7.4 ml min”1 water for 15 min and was turned on manually based on the subjective determination of need. During seedling establishment, little water was taken up, and 111 ml water was adequate for a 3 day period or more. With increasing shoot growth, TI‘\\ 69 greater amounts of water were transpired, and watering for 15 min was necessary more frequently. Ryegrass plants were clipped to 5 cm height 44 days after planting, and all planted pots were overseeded at that time with the same rate as the initial seeding due to poor germination. The plants were grown for approximately 123 days. At the time of harvest, shoots were clipped near the soil surface, dried and weighed. Soil was removed from each pot as a cohesive unit; the top, bottom, and circumference were trimmed off (about 1 cm) with a knife to remove edge effects which may have developed at the plastic-soil interface. The soil was divided into top (1 to 6 an original depth) and bottom (6 to 11 cm), and each sample was split vertically to provide samples for both root quantification and soil aggregate analyses. Each subsample was weighed and further subsampled to determine soil water content. Samples for root quantification were frozen and stored at -20 C until they were washed at a later date. Samples for aggregate analysis were manually broken and air-dried on brown paper in the greenhouse. The soil samples were further broken by hand, if necessary, to pass a 6 mm sieve and stored in an air-dried condition until further analyses. 932. This study was limited to a period of 37 d after planting of alfalfa and ryegrass in order to minimize root death, since exudation was the major root contribution of interest. Soil water contents were monitored by weighing each pot throughout the study, and evapotranspired water was 70 replaced according to one of two watering regimens: the soil water was maintained (control) at a relatively high content in half of the pots by watering daily to a water content of 24.1%, and the soil in the rest of the pots was allowed to dry down to 21% water content before water was replaced to 24.1%. The plant factor (unplanted, alfalfa, ryegrass) and the drying cycle factor were combined in a 3 by 2 factorial, randomized complete block design with 6 replications. The tare of each pot with gravel was determined and 1300 9 soil was added to the pot. After initial wetting by‘ capillarity (approximately 13 cm water tension), seeds of alfalfa and ryegrass were planted at rates of 0.75 9 pct-1 (134 kg ha'l) for alfalfa and 1.25 g pot'1 (240 kg ha'l) for ryegrass. An additional 100 g of soil was added to all pots to cover seed in planted pots. A white mulch (3 mm deep) was established by spreading approximately 24 g of Perlite on the soil surface in order to reduce evaporation at the soil surface and prevent significant gradients of water content within the potted soil profile. The pots were covered with foil during the 3 d seed germination period. Damping off of alfalfa seedlings, killing approximately 50 to 95% in each pot 7 d after planting, necessitated the removal of seedlings and reseeding of those pots only. Each pot was weighed daily to determine water evapotranspiration and remaining soil water content. The pots designated as controls for the drying cycle factor were 71 watered daily to a soil water content of 24.1%, which was approximately equivalent to -0.033 MPa matric potential as determined by desorption studies of this disturbed soil (Figure 4.1). The other pots were allowed to dry to 21.0% soil water contents, approximately equivalent to -0.30 MPa matric potential, before being watered to 24.1%. The drying periods varied according to environmental conditions and plant growth. By the end of the experiment, soil of control pots was drying down as much as soil of the dry-down treatment, and dry-down pots were being watered daily as the control pots were. At the end of the experiment, the soil water content was determined by weighing each pot, and a pot was harvested when the soil water content reached approximately 15%, although the range was approximately 8 to 18%. In the end, there were 36 or 38 d of growth for ryegrass, 28 to 29 d of growth for alfalfa, and 37 or 38 d of incubation for unplanted soil. Plant shoots were cut near the soil surface, dried at 70 C, and weighed. The soil was removed from the pot, and approximately 1 cm of soil was removed from the top, bottom, and circumference with a knife to remove edge effects. The remaining sample was split vertically to provide subsamples for root quantification and aggregate analyses. Each sample was weighed to determine soil volume, assuming bulk density of 1.4 kg m'3 (Murphy and Smucker, 1991b). Aggregate samples were sealed in individual plastic boxes. Aggregate analysis samples later .zom 032.52 .0095me 23 Lou ciao :oSQsomop some; 4+ 0.2%...”— Ammv: coflmco... his? 72 oo_2 2.5 2.5 of. owe 0me 2.; 2.5 cam 2.: me P h P b p b n L p p m A: w .I 9 l Ina O O U 1. Iom m 1 «mm am 9 .A IS aw Imm 73 were broken manually to < 6 mm while moist and spread on brown paper in the lab to air dry for 3 days. These samples were then sealed in air-tight jars. Samples for root quantification were frozen (-20 C) and stored until a later time. BQQ§_Qngntifigg§1gn. Roots were separated from the soil by the elutriation method (Smucker et al., 1982). Samples frozen for root quantification were removed from the freezer, and the frozen soil was inundated with sodium hexametaphosphate solution (50 g L'l) and thawed to absorb the dispersing solution for at least 24 hr. The washed roots were stored in 15% methanol solution at 5 C until further processing. Portions of GHl roots were analyzed for length by the line intersect method (Tennant, 1975) and/or video image analysis and are reported elsewhere (Murphy and Smucker, 1991a). After length measurements were completed, all roots were dried at 70 C and weighed to four decimal places. SQil_Analy§1§. Soil aggregates were analyzed by four different techniques: dry aggregate sieving, wet aggregate sieving, crushing (tensile) strength, and turbidity measurements. Dry aggregate sieving provided information on the size distribution of aggregates. For GHl, only the size fraction of aggregates <1 mm was weighed and reported. For GH2, the fractions 4 to 6 mm, 2 to 4 mm, 1 to 2 mm, and <1 mm were quantified and reported. sieves (15 cm in diameter) were shaken by hand. 74 A method for determining aggregate strength by unconfined compression has been described by Dexter (1975) and was used here. A digital balance measured the stress applied to an air-dry aggregate (4 to 6 mm size) by a plate pressed slowly and steadily on top of the aggregate (Figure 4.2), and a computer constantly read the weights and graphed the input on a monitor. The sudden decrease in strain associated with rupture of the aggregate formed a peak in the graph. The peak, reported in grams, defined the crushing resistance (CR) of the aggregate. A series of small peaks often complicated the analysis, but the peak associated with rupture of the whole aggregate was selected to quantify the aggregate’s strength. Wet aggregate sieving has been a standard technique of determining soil aggregate stability for many years (Yoder, 1936), with various modifications. The one-sieve (250 um) method (Kemper and Rosenau, 1986) was used with a 25 9 sample of 1 to 2 mm, rewetted (50 cm water tension) aggregates. In this method, aggregate stability is defined as the percentage of aggregates remaining on the sieve after gentle agitation in water. Turbidity of soil suspensions also has been used as a measure of soil aggregate stability upon immersion in water and agitation (Williams et al., 1966). For this analysis, 0.5 9 samples of 1 to 2 mm, rewetted aggregates were used. Each was added to a calorimeter cuvette with 20 ml of water, and five samples at a time were mechanically rotated (4 rpm) 75 .moumvoumms Mach «0 “mow mossumwmou madcmsuo on» usacaauouoo you maven unmanasvm «OH H 20: a «whnmzuu mmufluamwnflufl < mmmmm Alllllmmm mm .mé 0.33m «aha: mummwhm WI." e”IIJ E .RNwsSRbS§V_§Gbottom), and the difference at the top position between unplanted and planted pots may have occurred because of drying solely by evaporation in the surface of the unplanted soil whereas less evaporation and various degrees of root uptake were responsible for drying in the planted pots. With regard to the bottom position, the ryegrass treatment produced the lowest percentage of soil <1 mm, which was similar to the unplanted soil but significantly less than alfalfa treatment soil. A possible explanation for this is the greater length 79 Table 4.2. Proportion of soil (by weight) found in the size range <1 mm by dry sieving for GHl. Plant $.1Emm Bosition $.13mm None 41.4 Top 39.7 Alfalfa 37.5 Bottom 35.7 Ryegrass 34.3 LSD(0.05) 5.36 3.51 Fisher's protected LSD procedure was used at the 0.05 level. of ryegrass roots at the bottom position compared to alfalfa, but other possibilities could include differences in degree and type of root exudation, microbial populations (type, activity), and root water uptake. The plant factor was significant (P<0.01), indicating _that aggregates from the ryegrass treatment showed the greatest resistance to crushing (Table 4.3). Fine roots of ryegrass were often observed transecting aggregates of the ryegrass treatment, which may have contributed to their tensile strength (Soane, 1990). The pot position was not significant. There was a slight plant by position interaction (P<0.10) because aggregates from the bottom position of unplanted and ryegrass treatments exhibited greater resistance than those from the top position, but the reverse was true for the alfalfa treatment (data not shown). Others have used different measures to compare resistance to 80 Table 4.3. Mean crushing resistance (CR) of 4 to 6 mm soil aggregates for GHl. 21311:. 93 9 None 602.4 Alfalfa 686.8 Ryegrass 847.7 LSD(0.05) 89.0 Fisher's protected LSD procedure was used at the 0.05 level. crushing, such as energy-based values (Boyd et al., 1983) and tensile strength (Dexter, 1975; Braunack et al., 1979).. Though more resistant to crushing forces, aggregates from the ryegrass treatment were less stable in the wet sieving test compared to the alfalfa treatment (P=0.04, Table 4.4). Aggregates from the unplanted treatment were intermediate in wet aggregate stability (WAS) tests and not significantly different from either plant treatment. The position factor was not significant. The difference between ryegrass and alfalfa could be the amount or composition of root exudates produced, or the solubility of these or other stabilizing agents. Several researchers have indicated that aggregate stabilizing agents are water soluble (Pojasok and Kay, 1990; Haynes and Swift, 1990). Haynes and Swift (1990) suggested that the water extractable pool of carbohydrates was from mucigel of microbial origin, but Cheshire and Mundie (1990) distinguished root mucigel from root exudate 81 Table 4.4. Mean wet aggregate stability (WAS) for 1 to 2 mm soil aggregates of GH1. 21101 118$ % None 64.6 Alfalfa 69.8 Ryegrass 62.2 LSD(0.05) 5.20 Fisher's protected LSD procedure was used at the 0.05 level. by mucigel's insolubility in water and said that mucigel may remain associated with mineral surfaces after soil dispersion. Pojasok and Kay (1990) found that water- extracted compounds of roots sorbed more readily to soil but decomposed more quickly than longer-chained exudate compounds extracted with acetone. In turbidity tests, transmission of soil suspensions also indicated that stabilizing agents from ryegrass may be more water soluble than those from alfalfa and, in this case, the unplanted soil (Table 4.5). The lower transmission values for ryegrass and unplanted treatments compared to alfalfa after two minutes of end-over-end shaking (T2, P=0.05) indicated greater dispersion of aggregates. The pot position was not a significant factor for T2, but became very important for transmission values after 20 minutes of end-over-end shaking (T20, P<0.00). Values of T20 for the top position were greater than those 82 Table 4.5. Mean transmission of soil suspensions following 2 (T2) and 20 minutes (T20) of end-over-end shaking of 1 to 2 mm soil aggregates for GH1. 21401 12 120 29511120 120 % % % None 33.9 8.3 Top 8.5 Alfalfa 37.5 8.6 Bottom 8.2 Ryegrass 33.1 8.1 LSD(0.05) 3.45 0.41 0.14 Fisher's protected LSD procedure was used at the 0.05 level. for the bottom position, and T20 for alfalfa were greater than for ryegrass and the unplanted treatment (P=0.05). Although recommended by Williams et al. (1966) as a measure of soil aggregate stability, the ratio (TR) of T20:T2 gave no significant differences and so was ineffective in distingushing between treatments in this study. Determination of organic matter demonstrated increased levels in planted treatments (P<0.00, Table 4.6). The soil position was an insignificant factor. Fragments of roots may account in large part for the organic matter increase in alfalfa and ryegrass soil compared to the unplanted treatment. Root exudates, root decomposition products, and the microbial biomass in the rhizosphere surely contributed to the soil organic matter pool as well. Root length density was determined by the line intersect method and by image anlysis using two algorithms; the second algorithm should improve the uncorrected value by 83 Table 4.6. Soil organic matter (OM) content of soil after GH1 treatments, as determined by low-temperature combustion. Elan; .911 % None 1.51 Alfalfa 1.55 Ryegrass 1.56 LSD(0.05) 0.035 Fisher's protected LSD procedure was used at the 0.05 level. attempts to account for organic debris in the sample. Root weight was positively correlated with T2 and T20 while root length estimates showed a negative correlation with T2 and T20 in addition to wet aggregate stability (WAS, Table 4.7). The turbidity ratio (TR) was positively correlated to root length density estimated by image analysis. The percent of soil <1 mm was negatively correlated (P<0.10) to root weight. Crushing resistance (CR) was not correlated with the quantity of roots by any method of measurement. Correlations among soil aggregate analyses results are summarized in Table 4.8. The fraction of soil <1 mm was negatively correlated with crushing resistance, which was not surprising since manual crushing forces were responsible for breaking apart the soil mass. The reason for a positive relationship between soil <1 mm and T20 was not as clear, but greater breakdown of aggregates (more soil <1 mm) may result in a greater proportion of stable aggregates in the 84 Table 4.7. Correlation coefficients for comparisons between root parameters and soil aggregate analyses for GH1. Line intersect and image analysis were two methods used to determine root length density; two algorithms were used for the image analysis, resulting in uncorrected and corrected values. Root length density Image analysis Aggregate Root Line Corrected 50512515 ' immersest. .Qnsgrrested 1:1;str15 <1mm -0.219 -O.246 -0.340 -0.199 CR 0.205 0.113 -0.043 -0.332 ** *** * was 0.015 -0.358 -0.597 -0.512 ** ** *** ** T2 0.305 -0.350 -0.717 -0.512 *** *** ** * T20 0.440 -0.507 -0.579 -0.524 *** ** TR -0.034 0.038 0.727 0.611 *p < 0.10 **P < 0.05 ***P < 0.01 size range used (1-2 mm) for T20 analysis. However, it was evident from previous results that different factors are responsible for the stability of wet and dry aggregates. T2 was correlated with WAS (P<0.10) and highly correlated with T20 and strongly correlated with T20, probably due to the similarity of processes occuring. The strong, negative correlation of T2 with TR was expected because they are inversely related by definition. The relation between T20 and TR was also negative, despite definition, because T20 85 Table 4.8. Correlation coefficients for comparisons among methods of soil aggregate analysis for GH1. sme 23 WAS 12 120 *** CR —0.507 - - - - was 0.177 -0.094 - - - * T2 0.172 -0.168 0.240 - - ** *** *** 120 0.253 -0.205 0.320 0.614 - *** *** TR -0.079 0.067 -0.131 -0.859 -0.326 *p < 0.10 **p < 0.05 ***P < 0.01 had a small range of values compared to T2, allowing T2 to be the more influential factor. Se29nd_Qrssnhouse.£xnsrinent_l§HZl- Mean dry weights of shoots and roots are presented in Table 4.9. Ryegrass had greater weights of both roots (P<0.00) and shoot tissues (P<0.00) than alfalfa. Root weight tended to respond to watering regime (P=0.09) as well; drydowns reduced the root weights compared to the water control especially in the ryegrass treatment (data not shown). An interaction of the plant factor with the soil water factor (P=0.06) resulted from reduced shoot weight of ryegrass alone with drydown periods. Root-to-shoot ratios were significantly affected by plant only (P<0.00), with ryegrass values being greater. The size ranges 4 to 6 mm, 2 to 4 mm, and <1 mm were significantly affected by plant treatment (P<0.00, P=0.02, and P=0.01, respectively; Table 4.10). Ryegrass had a 86 Table 4.9. Mean dry weights of plant shoots and roots for the significant factors in GH2. Root dry Shoot dry Elam; 1519.01? reign: R_:_§ mg 9 Alfalfa 190.96 0.63 0.311 Ryegrass 621.83 1.45 0.428 *Root dry weight was based on measured root weight density, assuming uniform root distribution throughout the potted soil. smaller proportion of soil aggregates in size ranges 4 to 6 mm and 2 to 4 mm than the unplanted treatment and a greater proportion in the range <1 mm. This contrasted with results from GH1, where ryegrass tended to have the smaller percentage of soil <1 mm compared to other treatments. The condition of soil (dry or moist) when it was broken apart may have accounted for the difference. Because of their small diameters and profuse branching, grass roots tend to Table 4.10. Proportions of soil (by weight) found in various aggregate size ranges by dry sieving for GH2. 4 to 2 to 1 to £150: §_mm 4_mm 2_mm $1_mm % % % % None 28.3 23.6 22.2 26.0 Alfalfa 28.7 21.8 24.0 25.4 Ryegrass 18.8 18.1 25.4 37.6 LSD(0.05) 4.13 3.79 NS 8.58 Fisher's protected LSD procedure was used at the 0.05 level. 87 be associated with finer aggregation. Alfalfa did not differ from the unplanted treatment with respect to aggregate size distribution. The soil water factor did not have an effect on aggregate size distribution. The mean crushing resistance of aggregates for each treatment is listed in Table 4.11. Ryegrass roots resulted in greater strength of soil aggregates of the 4 to 6 mm size range than alfalfa or the unplanted treatment (P=0.03); alfalfa was not significantly different than the control. As in GH1, ryegrass roots within aggregates of this size fraction may be contributing to their tensile strength. The soil drying factor did not have a significant effect on crushing resistance. There were no significant differences among treatments for the wet sieving aggregate stability (Table 4.12). There may be a trend for results similar to those found in GH1: ryegrass was the least stable and alfalfa was the most Table 4.11. Mean crushing resistance (CR) of 4 to 6 mm soil aggregates for GH2. 2.1.0.01; £3 9 None 371.5 Alfalfa 381.9 Ryegrass 476.2 LSD(0.05) 81.67 Fisher's protected LSD procedure was used at the 0.05 level. 88 Table 4.12. Mean wet aggregate stability (WAS) of 1 to 2 mm soil aggregates for GH2. 21.9.01; 5.911.122: HAS 3 None control 65.9 drydown 62.9 Alfalfa control 65.2 drydown 66.6 Ryegrass control 62.6 drydown 62.5 LSD(0.05) NS Fisher's protected LSD procedure was used at the 0.05 level. stable in water. Some researchers have found that pre- wetted aggregates show less differences in stability than dry aggregates (Monroe and Kladivko, 1987; Grieve, 1980), but changing the initial condition of aggregates may also indicate a different order of stability (Skidmore et al., 1975, Grieve, 1980). Average light transmission values measured after 2 minutes (T2) and 20 minutes (T20) of shaking are given for the turbidity tests (Table 4.13). T20 measurements found significantly greater transmission for ryegrass treatments than for the unplanted treatment (P=0.03). T20 for the alfalfa treatment did not differ from either the unplanted or ryegrass treatment. The soil drying factor was not significant. T2 measurements were not different, nor did the turbidity ratio show a difference between treatments. Root dry weights (RDW) were compared to mean values of each soil aggregate analysis test (Table 4.14). Positive Table 4.13. 89 2 (T2) and 20 minutes (T20) of end-over-end shaking of 1 to 2 mm soil aggregates for GH2. Mean transmission of soil suspensions following Elan; 12 120 % % None 21.3 7.3 Alfalfa 22.6 7.5 Ryegrass 23.7 7.6 LSD(0.05) NS 0.24 Fisher's protected LSD procedure was used at the 0.05 level. Table 4.14. Correlation coefficients for comparisons between root dry weight and soil aggregate analyses for GH2. Size ranges (%) 4 to 6 mm 2 to 4 mm 1 to 2 mm <1 mm Crushing resistance (g) Wet sieving stability (%) Turbidity (% transmission) T2 T20 TR Root dry weight All r2255 *** -0.444 0.327** 0.470 -0.523*** 0.374::* 0.400 -0.217 Alfalfa 19225 -0.099 0.409 0.740 -0.612 0.718* -0.304 0.058 0.428 0.248 *** * 0.107 -0.178 -0.362 0.142 0.141 -0.113 0.438 0.630 -0.175 Ryegrass 229:5 *P < 0.10 **P < 0.05 ***P < 0.01 90 correlations of RDW occurred with T2, T20, and the size range 1 to 2 mm. Negative correlations of RDW were found with size ranges 4 to 6 mm and 2 to 4 mm, and with crushing resistance. For alfalfa roots only, RDW was positively correlated to the size range 1 to 2 mm and crushing resistance, and alfalfa RDW was negatively correlated to the size range <1 mm. The only significant correlation for ryegrass RDW was with T20 and was positive. Table 4.15 shows the correlations between different methods of soil aggregate analysis used in this experiment. There were several interesting comparisons to be found. The size range <1 mm was negatively correlated with all other size ranges. The correlation between the wet aggregate Table 4.15. Correlation coefficients for comparisons among methods of soil aggregate analysis for GH2. 1:5 2:4 1:2 51. 98 m 12 120 *** 2-4 0.509 - - - - - - - 1-2 -0.153 0.317 - - - - - - *** *** ** <1 -0.789 -0.866 -0.384 - - - - - *** ** *** T2 -0.108 0.204 0.332 -0.137 0.587 -0.287 - - ... ... T20 -0.024 0.063 0.136 -0.060 0.435 0.048 0.445 - .. .. ... TR 0.105 -0.166 -0.266 0.099 -0.402 0.326 -0.881 0.017 *P < 0.10 **P < 0.05 ***P < 0.01 91 stability and crushing resistance was negative, and correlations between crushing resistance and the turbidity measurements, T2 and T20, were positive. Turbidity measurements, T2 and T20, were not correlated with wet aggregate stability. Conclusions These greenhouse experiments demonstrated that plant roots affect soil structure. The use of several methods to quantify aggregate stability revealed that different mechanisms of stability operated under different conditions,- e.g. air-dry or saturated. In general, the ryegrass treatment had the strongest dry soil aggregates, and the alfalfa treatment produced aggregates most stable in water, compared to other treatments. In the dry state, soil aggregates from the ryegrass treatment might have been strengthened by more roots and/or fungi and more effective stabilizing compounds than the alfalfa and control aggregates. The very fine roots of ryegrass were observed more often growing through soil aggregates than were alfalfa roots. If the stabilizing compounds of ryegrass aggregates were more soluble in water than those of alfalfa, however, the stabilizing compounds would have been diluted, diffused outward, and/or more susceptible to microbial decomposition by the prewetting of aggregates for wet sieving and turbidity tests. The organic matter of unplanted soil 92 aggregates may be less in amount (GH1) but more stable, i.e. humified. Differences between soil aggregate measurements from the two experiments were probably related to the time period of treatments, but roots themselves were not solely responsible for the differences. The unplanted soil treatment appeared to be affected as well as planted treatments, and root weights were not necessarily greater for the longer experiment. The position factor of GH1 had an effect on some soil aggregate analyses, and that may have resulted more from soil drying by evaporation than from root contributions since root weights and lengths did not correlate well with those measurements. Although an increase in organic matter resulted from planted treatments, the increases were unrelated to soil position in the pot and so were not directly linked to root distribution. The solubility and therefore movement of some root exudate materials may partially explain this. The soil drying factor of GH2 had no significant effect on soil aggregate analyses. Greater differences in the drying cycles and the ability to maintain those differences would be expected to have an effect on soil structure, but the effect cannot be predicted. The need for further research is obvious from this study. More complex greenhouse experiments involving a range of root length densities, a series of harvest dates, 93 and more closely controlled soil water contents are suggested to clarify results of the present study. Improved methods of soil aggregation analysis may be necessary as well. Laboratory work should include characterization of root exudates, quantification of root material lost to the rhizosphere, and characterization of the soil microbial biomass and its metabolism. There is still much to be learned about plant root effects on soil structure. LIST 01' REFERENCES List of References Barber, S.A. 1959. The influence of alfalfa, bromegrass, and corn on soil aggregation and crop yield. Soil Sci. Soc. Am. Proc. 23:258-259. Baldock, J.A., and B.D. Kay. 1987. Influence of cropping history and chemical treatments on the water-stable aggregation of a silt loam soil. Can. J. Soil Sci. 67:501- 511. Bathke, G.R., and G.R. Blake. 1984. Effects of soybeans on soil properties related to soil erodibility. Soil Sci. Soc. Am. J. 48:1398-1401. ' Boyd, P.W., E.L. Skidmore, and J.G. Thompson. 1983. A soil-aggregate crushing-energy meter. Soil Sci. Soc. Am. J. 47:313-316. Braunack, M.V., and A.R. Dexter. 1989a. Soil aggregation in the seedbed: a review. I. Properties of aggregates and beds of aggregates. Soil Tillage Res. 14:259-279. Braunack, M.V., G.S. Hewitt, and A.R. Dexter. 1979. Brittle fracture of soil aggregates and the compression of aggregate beds. J. Soil Sci. 30:653-667. Cheshire, M.V., and C.M. Mundie. 1990.0rganic matter contributed to soil by plant roots during the growth and decomposition of maize. Plant and Soil 121:107-114. Cremeens, D.L. 1983. Argillic horizon formation in the soils of a hydrosequence. M.S. Thesis, Michigan State University. Dexter, A.R. 1975. Uniaxial compression of ideal brittle tilths. J. Terramech. 12:3-14. Elustondo, J., D.A. Angers, M.R. Laverdiere, and A. N'Dayegamiye. 1990. Influence of corn cropping and of meadow on aggregation and on organic matter associated with particle-size fractions of seven soils. Can. J. Soil Sci. 70:395-402. 94 95 Fahad, A.A., L.N. Mielke, A.D. Flowerday, and D. Swartzendruber. 1982. Soil physical properties as affected by soybean and other cropping sequences. Soil Sci. Soc. Am. J. 46:377-381. Grieve, I.C. 1980. The magnitude and significance of soil structural declines under cereal cropping. Catena 7:79-85. Haynes, R.J., and R.S. Swift. 1990. Stability of soil aggregates in relation to organic constituents and soil water content. J. Soil Sci. 41:73-83. Jackson, M.L. 1958. Soil Chemical Analysis. Prentice- Hall, Inc., Englewood Cliffs, NJ. Jastrow, J.D. 1987. Changes in soil aggregation associated with tallgrass prairie restoration. Amer. J. Bot. 74:1656- 1664. Kemper, W.D., and R.C. Rosenau. 1986. Aggregate stability and size distribution. pp. 425-442 In A. Klute (ed.) Methods of Soil Analysis, Pt. 1, (2nd ed.). Agronomy Monograph no. 9. American Society of Agronomy and Soil Science Society of America, Madison, WI. McKeague, J.A., C.A. Fox, J.A. Stone, and R. Protz. 1987. Effects of cropping system on structure of Brookston clay loam in long-term experimental plots at Woodslee, Ontario. Can. J. Soil Sci. 67:571-584. Miller, R.M., and J.D. Jastrow. 1990. Hierarchy of root and mycorrhizal fungal interactions with soil aggregation. Soil Biol. Biochem. 22:579-584. Monroe, C.D., and E.J. Kladivko. 1987. Aggregate stability of a silt loam soil as affected by roots of corn, soybeans and wheat. Commun. Soil Sci. Plant Anal. 18:1077-1087. Murphy, S.L.S., and A.J.M. Smucker. 1991a. A comparison of alfalfa and ryegrass root quantification. In preparation. (Dissertation Ch. 5). Murphy, S.L.S., and A.J.M. Smucker. 1991b. Soil stability analyses following repeated application of root extract solutions and drying cycles in the laboratory. In preparation. (Dissertation Ch. 3). Pojasok, T., and B.D. Kay. 1990. Effect of root exudates from corn and bromegrass on soil structural stability. Can. J. Soil Sci. 70:351-362. 96 Reid, J.B, and M.J. Goss. 1982a. Interactions between soil drying due to plant water use and decreases in aggregate stability caused by maize roots. J. Soil Sci. 33:47-53. Reid, J.B., and M.J. Goss. 1981. Effect of living roots of different plant species on the aggregate stability of two arable soils. J. Soil Sci. 32:521-541. Reid, J.B., M.J. Goss, and P.D. Robertson. 1982. Relationship between the decreases in soil stability effected by the growth of maize roots and changes in organically bound iron and aluminum. J. Soil Sci. 33:397- 410. Satterthwaite, F.E. 1946. An approximate distribution of estimates of variance components. Biometrics Bull. 2:110- 114. Skidmore, E.L., W.A. Carstenson, and E.E. Banbury. 1975. Soil changes resulting from cropping. Soil Sci. Soc. Am. Proc. 39:964-967. Smucker, A.J.M., S.L. McBurney, and A.R. Srivastava. 1982. Quantitative separation of roots from compacted soil profiles by the hydropneumatic elutriation system. Agron. J. 74:500-503. Soane, B.D. 1990. The role of organic matter in soil compactibility: A review of some practical aspects. Soil & Tillage Res. 16:179-201. Steel, R.G.D., and J.H. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical Approach, (2nd ed.). McGraw-Hill, Inc., New York. 633 pp. Tennant, D. 1975. A test of a modified line intersect method for estimating root length. J. Ecology 68:995-1001. Tisdall, J.M., and J.M. Oades. 1979. Stabilization of soil aggregates by the root systems of ryegrass. Aust. J. Soil Res. 17:429-441. Williams, B.G., D.J. Greenland, G.R. Lindstrom, and J.P. Quirk. 1966. Techniques for the determination of the stability of soil aggregates. Soil Sci. 101:157-163. Yoder, R.E. 1936. A direct method of aggregate analysis of soils and a study of the physical nature of erosion losses. A COMPARISON OF ALERLER AND RYEGRRSS ROOT QUANTIEICRTION Abstract Three methods were used to quantify roots of alfalfa (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.) from a greenhouse experiment: 1) root length density (RLD) by the line intersect method, 2) RLD by video/image analysis, and 3) dry root weight density (RWD). The plant species proved to be very important in the comparison between methods. Estimates of RLD for alfalfa agreed very well and were positively correlated with RWD. Little correlation was found between methods for ryegrass roots. The difficulty with ryegrass was related to the fineness, huge number, and total length of the root samples. 97 Introduction Many methods have been developed for quantifying or estimating root parameters (Bohm, 1979). Comparisons between methods are necessary for calibration and often reveal useful information. This report summarizes the quantification of the root systems of two plant species, alfalfa and ryegrass, by different methods of measurement. Materials and Methods Alfalfa (Medicago sativa L.) and perennial ryegrass (Lolium perenne L.) were grown in the greenhouse in individual 15 cm pots for a period of 4 no. They were surface irrigated with drip tubes daily. At the time of harvest, soil/root samples were destructively obtained by extracting the soil mass from the pot and cutting with a large knife. The soil was subsampled by cutting the soil in half top-to-bottom (one half being for soil analyses and the other for root quantification), trimming 1 cm soil from the top, bottom, and periphery to eliminate edge effects, and then cutting the bottom half from the top half. The top (approximately 1 to 6 cm depth) and bottom (approximately 6 to 11 cm depth) were analyzed separately to determine the influence of soil moisture content during the frequent drying cycles on soil conditions and root growth. Roots were washed from the samples using the hydropneumatic elutriation system (Smucker et al., 1982) and stored in 15% methanol solution at 4 C. The roots were 98 99 prepared for video imaging according to Smucker et al. (1987). This entailed staining the roots with Malachite green, spreading the roots in a (45 by 45 cm) glass tray, and positioning the tray on a light table. The robotic video camera mounted above the tray (with computer- controlled x and y motion) obtained 64 images (3.9 by 4.9 cm) of roots representing 64% of the tray area. Individual images from the resulting video tape were processed by an image analyzing computer (Vicom, Santa Barbera, CA) to determine root length and size class (diameter range). The magnification resulted in a resolution of 11.4 pixels mm'l. The tray of roots was placed over a 2 cm grid for determination of root length by the line intersect method (Newman, 1966, Tennant, 1975). In this method, intersections of randomly distributed roots with lines of the grid are theoretically and statistically related to the total length of roots by the equation L = (pi N A)(2H)'1, where L is root length, N is the number of intersections, A is the area of the grid system, and H is the total length of grid lines. Roots were collected from the counting tray, oven-dried at 70 C, and weighed to four decimal places. Results and Discussion During sampling, it was observed that soil containing ryegrass roots was bound together by the network of roots, while soil with alfalfa roots was more apt to crumble apart 100 because the root density was relatively low. The difficulty in cutting through alfalfa roots also contributed to the crumbling. In the process of washing roots from soil samples, it was necessary to use fine mesh sieves to retain the fine roots of ryegrass. Because of this, considerable organic debris was also retained. Repeated decanting was successful in separating the large majority of roots from the debris. Debris remaining in the sample was hand-picked from the sample as it was distributed in the glass tray used for counting. Difficulty of separating and spreading the fine ryegrass roots on the glass tray and the time consumed (5 h or more) led to the conclusion that every sample of ryegrass roots could not be analyzed by optical methods. Preliminary results also suggested that the resolution of the fine ryegrass roots was inadequate because significant portions of the root length were not detected and/or were subtracted as debris by the computer algorithm. Ryegrass root lengths determined by the line intersect method were greater than those found by image analysis and were not closely correlated (r2=0.090 for corrected values). The comparison of root length determined by the two methods (Figure 5.1) implied that some ryegrass roots were not detected by the image analyzer, underestimating ryegrass root length due to the fineness and overlapping of roots. On the other hand, operator fatigue may have contributed to 101 an overestimation by the line intersect method (Bland et al., 1989). Error also could have resulted from calibration of the line intersect method with roots of coarser morphology (Tennant, 1975). The fineness of the ryegrass roots and their profuse branching may have resulted in judgement errors of true intersections. Root length determinations of alfalfa showed much better agreement between the methods (r2=0.914 for corrected values), perhaps because alfalfa root morphology is more similar to roots routinely quantified by this method (Newman, 1966). A relatively large number of root pixels in the video recorded- ryegrass images was determined to be debris by the computer algorithm even though the samples were picked free of debris. This suggested that some of the ryegrass roots were too fine to be properly detected by the image analyzer. Table 5.1 demonstrates the mean root lengths calculated from pixel numbers before and after debris extraction for alfalfa and ryegrass. Amounts of debris found by the computer in alfalfa samples were relatively small (7.0%) compared to ryegrass (27.3%). Ryegrass debris in the smallest size class (<0.22 mm) represented 81% of the total. The maximum percentage of debris for alfalfa was 41.9%, which was found in the 0.22 to 0.55 mm size class. Low resolution of the fine ryegrass roots at the magnification used could lead to nondetection of fragments of roots, and the detected portions then may be insufficient in length to be judged as roots by the computer algorithm and be thrown out as debris. Root length by line intersect (cm g“) Figure 5.1. 102 RYE GRASS e Corrected IA 0 Uncorrected IA :Y = 0.404 + 0.877 X /.° Y = 0.047 + 0.872 X ”' ALFALFA <> Uncorrected IA 9 Corrected IA I'l' 'llel‘l‘ 12141618 0 2 Inj'III 4 6 810 Root length by image analysis (cm g”) The relationship between the line intersect method and image analysis (IA) to determine root length. 103 Clumping of roots together also could have resulted in their classification as debris. This resulted in large signal to noise ratios which could be eliminated by using greater lens magnification during the video recording. Further testing has shown that greater magnification (26.3 pixel mm'l) is necessary for adequate image analysis of fine roots such as ryegrass roots (unpublished data). Table 5.1. Mean length of washed roots of alfalfa and ryegrass before and after applying algorithms to exclude debris. Root length Root length 2111050215 W m m Alfalfa 5.629 5.179 Ryegrass 13.524 9.827 Alfalfa root weights were equal to or greater than ryegrass root weights, despite greater lengths of ryegrass roots, because of the differences in root diameters. Alfalfa root dry weight was positively correlated with the root length determined by the line intersect method when the data is separated by position in the soil (Figure 5.2; r2=0.569, p=0.0073 for the top and r2=0.755, P=0.0005 for the bottom). Roots near the surface have greater root diameters and therefore greater weight per unit length. Ryegrass roots near the soil surface gave a negative linear relationship between root dry weight and root length by line 104 09:05 5000.005 0:: 05 .3 005500500 new—0— aoos 0:0 2903 20 Loos c0252. 222550.00 0f. .m.m 0.5951 :28 0 co: 0.5 5063 be room. co“ om on OK. om om 00 on om Nu _ p — P _ . _ . _ L _ _ P . — b O u r a U H T... D \II +1 m. D . I \\I\\-\\ I I m 3 H x mmod + an; H > \\\\ as H mm \I\ x ONTO + ~md H > - a 1. I IA: 0 I... H 1.9 - an... 4 .Imfi m m... Eofion .mmcsw0zm 4 . \\ H )0. Q3 .mmcnw0zm a \4\ WON QorA 86:66 6:82 _. . H % Q3 .0232 U H "H -mm (m 105 intersect (r2=0.595, P=0.1266); the correlation for ryegrass roots below 6 cm was positive but weak (r2=0.268, P=0.372). Alfalfa root dry weight was positively correlated to image analysis measurements of root length (Figure 5.3). The alfalfa roots below 6 cm had a greater increase of root length with root weight compared to the roots near the soil surface; this was expected since those finer roots require more length to achieve an equivalent weight. Ryegrass roots, on the other hand, showed a negative relationship between root dry weight and root length by image analysis. The relationship for roots below 6 cm was quite strong (r2=0.915, P=0.1884), while the regression for roots in the top volume of soil was very low (r3=0.102, P=0.7927). One reason for these incongruous results may have been the large errors resulting from the high number of crossover (unpublished data) and the clumping of the fine ryegrass roots. Crossover errors occured when two or more roots overlapped, since the pixel area of the crossover region is converted to root length only once. As amount of roots increased (by weight), the probability of crossover increased and the calculated root length decreased. It is doubtful, however, that this explanation can be wholly responsible for the decrease in observed ryegrass root length with increasing weight. 106 Ema—0:0 0mcE_ .3 00580000 35 5wfl0. Loos 0:0 gammy; mac boos :00309 3228520.. 05. do 0.532 9.38 0 co: 0.5 35:60 206; .660 2: cm om 2. cm w. _ . _ . — . _ _ O 1 B N Go .IN. a n V U0 .1 m I. \s .1. u o o \I\\ n W0 l\\\\\\ u ix. Wm rA 1... a ,, H $1.1 ,, Wm S a 2 . u 4E no 00304, egoctooco 6,, PO“ \qw/QO 2000.500 Eonfiam Bo:o: & 3, n m m... I I Eofion .mmcsm0zm . o. ,, UN“ )0. Q3 60030.3“ 4 . “a; 3A 8865 6:65. .. 6 m. ‘ s QOA .m:m:< 0 . o wwfi TmL _ Conclusions Alfalfa root weight and lengths determined by video recording/image analysis and line intersect methods agreed reasonably well. However, it became apparent during this experiment that current quantification methods were not adequate for ryegrass roots. When using the line intersect method for ryegrass roots, it would be advisable to sample smaller soil volumes which would reduce the density of roots in each analysis and minimize operator fatigue during counting. It may also be necessary to calibrate the line intersect method for use with grass roots. When using video recording and image analysis for ryegrass roots, higher camera magnification during the video recording is recommended to resolve all roots. This would necessitate more images and longer processing time unless a smaller tray area and random samples were video recorded, and so smaller samples and a smaller tray are also advised for this method. Finally, comparisons between the line intersect method and root video/image analysis could be enhanced if the same roots could be counted by the line intercept and robotic video recording system without moving the sample. Progress must continue in the development of image analysis of root systems because of its capability to provide root width, surface area, and volume as well as length, which are important measurements for a complete understanding of agronomy and soil ecology. 107 LIST 01' REFERENCES List of References Bland, W.L., and M.A. Mesarch. 1990. Counting error in the line-intercept method of measuring root length. Plant and Soil 125:155-157. Bohm, W. 1979. Methods of studying root systems. Ecological Studies 33. Springer-Verlag, Berlin. 188 pp. Newman, E.I. 1966. A method of estimating the total length of root in a sample. J. Appl. Ecology 3:139-145. Smucker, A.J.M., J.C. Ferguson, W.P. DeBruyn, R.L. Belford and J.T. Ritchie. 1987. Image analysis of video recorded _ root systems. ASA Spec. Pub. 50:67-80. Smucker, A.J.M., S.L. McBurney, and A.K. Srivastava. 1982. Quantitative separation of roots from compacted soil profiles by the hydropneumatic elutriation system. Agronomy J. 74:500-503. Tennant, D. 1975. A test of a modified line intersect method for estimating root length. J. Ecology 68:995-1001. 108 CHAPTER 6 CONCLUSIONS The soil treatment cell (STC) designed and built for this study was an excellent tool for drying treatments up to 0.5 MPa. Connected to a pressure manifold, a series of STCs made an effective experimental setup. Collection of soil leachate from individual samples was an advantage of the STCs, and the heat-stability of the plastic will allow autoclaving. Seedlings of alfalfa and ryegrass were grown in sterile. sand culture to obtain root extracts for soil treatment. Root extract solutions were not effective in developing soil aggregation under the laboratory conditions in this study. Therefore, the hypothesis that the root extract solutions would have a beneficial effect on soil structure was not confirmed. However, it is possible that different conditions and improved techniques would result in significant changes in soil aggregation such as those .reported in the literature. The water-extractable soil carbohydrate profiles were followed upon addition of the 109 110 root extracts or water, and they suggest changes in the microbial activity throughout the experiment. The plant treatments in the greenhouse experiments caused significant differences in aggregation. Soil exposed to root growth had improved aggregation according to at least one method of measurement. Ryegrass aggregates consistently had greater crushing resistance than aggregates of the unplanted or alfalfa treatments. Alfalfa aggregates generally tended to be more water-stable than those of the other treatments. Organic matter increases in soil of planted treatments was thought to be mostly from root fragments remaining after sieving. Correlation of soil aggregation indices was sometimes positively related to quantity of roots but also seemed related to the species of plant. The time period of treatment seemed to affect the level of differences found by aggregate analyses. Methods of soil aggregation analysis show the need for improvement and standardization. The dry sieving technique to determine aggregate size distribution was flawed by the need to break the soil mass manually to <6 mm. The crushing resistance test gave significant, consistent results for the greenhouse experiments. The wet sieving technique found a similar trend for the two greenhouse experiments, and greater differences may have resulted if aggregates had not been prewetted. Turbidity measurements showed promise as a method of aggregation analysis, but optimization of the test conditions (pretreatment of aggregates, period of end-over- 111 end shaking, settling period) with the soil(s) to be used may be required. Measurement of root length may require certain rules or algorithms dependent on the plant species. The current methods appear effective for roots such as alfalfa but seem inadequate for the fine roots of perennial ryegrass. Many questions are raised by this study. Characterization of root exudates would be valuable to more correctly simulate exudates for laboratory studies. Different soils, greater drying conditions, and sequential sampling of soil might reveal meaningful factors of soil aggregation. Soil respiration rates and other measures of microbial activity need to be made, as well as comparisons of sterile and nonsterile soil responses to root growth and root extracts (exudates). The soil organic matter might be wholly characterized to confirm differences in pools of the various fractions. Improved or different methods of soil aggregate analysis could be used. The possibilities for further research are endless, but these listed are the most important for the objective of determining the mechanisms of root influence on soil aggregation.