The Root-Soil Interface 

Changes in soil properties in the rhizosphere

Because of root activity, the soil close to the root may be modified greatly compared the bulk soil. Some of the differences can be described according to the information already available from earlier parts of the course. Others will become evident as we consider other factors associated with root growth and activity (Marschner pp 537-555, 561-564). 

Q Based on information previously discussed, what are the main soil properties that are likely to be influenced by root growth and activity ? 
 

• Bulk density - if roots have to enlarge pores in penetrating the soil, the surrounding soil will be compacted by compression and re-orientation of particles. 

• Organic matter - the sloughing of cells from the root cap, the secretion of plant mucilages, the release of organic compounds, and the turnover of fine roots will all add to the organic matter in the soil around a root. Mucigel has also been shown to bridge clay particles thereby increasing the cohesion of soil particles and leading to the formation of micro-aggregates. 

 
• Moisture content - in most agricultural soils during the growing season the water content around a root will be less than that in the bulk soil because of the plant demand due to transpiration. However, water can also move out of the root, and some 'pumping' of water from deep in the subsoil can result in localized re-wetting of dry soil around the roots of desert plants. 

Root mucilage consists 99.9% of water, the water potential of the fully hydrated gel is about -7 kPa. A clay-mucigel combination will hold more water than will the clay alone. 

• Nutrient concentration - for immobile nutrients there will tend to be a concentration gradient that increases with distance from the root. This gradient will tend to be much less pronounced for mobile nutrients, and the concentration may decrease from the root surface if the nutrient moves to the root faster by mass flow than it can be absorbed. 

 
• pH - net H⁺ extrusion into the apoplasm will lead to pH decreasing in the rhizosphere. In contrast, the net release of HCO₃^- into the apoplasm or net uptake of H⁺ will lead to an increase in pH. 

The effective change in pH of the rhizosphere will depend on the anion:cation uptake ratio. 
anion:cation > 1 pH increases 
anion:cation < 1 pH decreases 
pH at the root surface can change as much as 3.5 units. 

Studies using soil infiltrated with agar containing a pH indicator have been used to demonstrate key effects. 
The pH change will be often be determined by the form in which N is supplied: NO₃-N or NH₄⁺-N. 
 

N is the only nutrient that is absorbed in both the anion and cation forms (eg K⁺ is always absorbed as a cation). 
NO₃is the most common form of mineral N in the soil.  N_org => NH₄⁺           2NH₄⁺ + 3O₂ =>2NO₂+ 2H₂O + 4H⁺  (the release of protons causes the pH to decline) 2NO₂ + O₂ => 2NO₃   
• Redox potential - roots absorb oxygen, thereby generating a concentration gradient away from the root surface. However, in waterlogged soils the internal transport of oxygen can be sufficient that leakage into the rhizosphere is sufficient to maintain an oxygenated zone around the root. In rice, the oxidation zone may extend up to 4 mm from the root surface. 

• Micro-organism population - the microbial population in the rhizosphere will usually be much greater than in the bulk soil due to root exudates and cells or root hairs that have been sloughed off by the root. 

The likely differences in soil properties between the bulk soil and soil from the rhizosphere 
 

One thing is obvious from the table the size and direction of the change in soil properties may vary widely depending on the soil, the crop and their interaction. 
 

pH changes 

Phosphorus availability and pH effects in the rhizosphere of corn 
 

Acidification increases P solubility and the proportion absorbed. 
 

       pK_a = 7.2               pK_a = 12.3 
H₂PO₄^- <=> HPO₄^{2 -} + H⁺ <=> PO₄^{3 -} + H⁺ 

Q What are the implications for fertilizer recommendations ? 

• In neutral or alkaline soils: 

If N can be maintained in the NH₄⁺-N form, this will enhance the uptake of P. 
So for banded fertilizers, including NH₄⁺-N will increase P availability. 
Increasing anion uptake will increase pH, and this can lower P availability. 

 Effect of enhanced anion uptake on P uptake in Brassica napus (After Marschner) 
 

However, in acid soils, where P is adsorbed to Fe or Al oxides, enhanced availability of P can result if the phosphorus can be exchanged at these sites with bicarbonate ions. 

The change in the uptake ratio of anions to cations in the example given in the last table was induced by P deficiency in the plant. Some plants (e.g. rape seed - Brassica napus) have been shown to lower the pH in the rhizosphere of a P deficient soil, even when N0₃^- was the source of N. If soil P is sufficient no change occurs. This effect is induced by alteration of the cation-anion absorption balance. The lowering of pH in P deficient soils has been used to account for the observation that nutrient absorption models work reasonably well for soils well supplied with P but underestimate P absorption by rape and some other crops from soils deficient in P. 

Studies using soil infiltrated with agar containing a pH indicator have demonstrated interesting effects. 
 

1. Rhizosphere pH was reduced by chickpea but increased by sorghum when both were growing in the same pot supplied with N0₃^--N. This is still a function of the imbalance of anion and cation uptake. Cereals tend to take up much more silicon than do dicots, especially legumes. The net result is that with N0₃^--N as the nitrogen source there will be a greater tendency to raise the pH of the rhizosphere in cereals, but the greater demand for cations in legumes will still tend to result in further acidification. 
2. Rhizosphere pH was increased along the main axis and decreased along lateral roots of corn growing in a soil supplied with N0₃-N (see Marschner et al. Root induced changes in the rhizosphere. Importance for the mineral nutrition of plants.Z. Pflanzenernähr. Bodenk. 1986. 149:441-456). Localized differences in the anion:cation uptake ratio appear to be important. 

Release of organic compounds 

Plants may secrete specific compounds that complex Fe - siderophores. These Fe-complexes may be absorbed intact by the plant increasing the supply of Fe to the plant. 
 
 

Q What affects the release of free compounds? 

• Mechanical impedance
• Nutrient deficiency 

• Most are organic acids, so they will affect pH in the rhizosphere.
• They may complex with ions in the soil. 
• There is chelation of Fe, Mn and other metals by organic acids. The mucilage of citrus species selectively binds Cu²⁺. 

Phytosiderophores are specific chelators of Fe³⁺. They are mainly organic acids such as citrate or phenolics. 
In calcareous soils these are important in iron nutrition since the concentration of iron in solution is very small. 
Fe deficiency results in the secretion of siderophores. 

In chelating Fe or Al, P can also be released. 

The availability of P, Fe and Mn is affected by a number of different secretions from roots. Their relative importance is indicated in the following table. 

Root secretions and nutrient availability 
 

Effects on other soil properties 

The growth of roots affects many soil properties because of the changes in soil water and nutrient contents. A number of these effects in combination can have a significant effect on soil structure and structural stability. 

Soil micro-organisms 

The concentration of soil micro-organisms in the rhizosphere can be either beneficial or detrimental to the plant depending on the species that dominate. Microbial activity may increase availability of nutrients by mineralization of organic forms or by increasing the solubility of mineral forms (Marschner 566-582). 

Availability of N and P, as well as nutrients like Mn have attracted research into soil microbiology. 

Contribution of grazing protozoa to availability of mineralized-N 
 

For Mn there are particular bacteria that oxidise the metal ions to form oxides. 

For P there are solubilising bacteria, which have been exploited in China and E. Europe. 

There are also P solubilising fungi eg Penicillium balaji strain (Provide/PB50). This is used on the prairies for wheat and canola. 

The most important microbes for P are mycorrhizal fungi that form associations with plant roots. 
The majority of plants establish such an association with certain types of soil fungi; this association is known as a mycorrhiza ("fungus-root"). 
Mycorrhizas are generally mutualistic. Carbohydrate is passed from the plant to the fungus, and in return the fungus facilitates increased nutrientuptake, particularly of phosphorus, from the soil to the plant. 
 

There are two main types of mycorrhizas found in association with agricultural and forest plant species: 

Ectomycorrhizas 

Ectomycorrhizas, in which the fungus does not penetrate the cells of the root, are most prominently associated with woody species, and are most prevalent in northern climates, but they are also found in tropical and sub-tropical regions. eg vital in pine forests. Often need to inoculate roots before planting eg in Australia 

Mycorrhizas characterised by the sheath (mantle) covering the root apex, and the network of hyphae growing in the intercellular spaces of the root cortex (hartig net). 

Rhizomorphs penetrate into the soil from the sheath. 

Endomycorrhizas 

Endomycorrhizas, in which the root cell walls are penetrated by the fungus, form arbuscules between the cell wall and the plasmalemma of cortical cells. They therefore capture material that leaks into the apoplast. This material would otherwise move out into the soil. 

Mycorrhizas may reduce the oxygen consumption close to the rhizoplane, because less material is available to free-living microbes. It is also likely that the decrease in the exudate entering the soil from the root can reduce Mn availability. 

The most common type of endomycorrhiza is the vesicular-arbuscular mycorrhiza (VAM). Almost all crop species worldwide have VA mycorrhizas, with the exception of members of the Brassicaceae, eg canola, and Chenopodiaceae, eg pigweed, which are mostly nonmycorrhizal. 

Example of mycorrhizal hyphae from a colonized root (Courtesy of D. Stribley)

Not all endomycorrhizal fungi produce vesicles (these have a large lipid content), so best general name for non-ericaceous mycorrhizas is arbuscular mycorrhizas (AMs). 

Formation of the mycorrhiza. 

The fungus forms an appresorium at the root surface behind the zone of maturation, penetrates the tissue and invades the root cortex. Fungal hyphae enter the root cortex and develop finely branched arbuscules in cells. In a cell which contains an arbuscule the plasmalemma of the cell is not punctured, but greatly increases in surface area as it is pushed inward. Arbuscules are the site of exchange between host and endophyte. Vesicles are also formed in the root cortex, and appear to be a resting stage. Finally it develops a dense external hyphal network through the soil. 

Example of mycorrhizal colonization of soybean root. Arbuscules are seen in a number of cells, and the dark stained rounded structures are vesicles.

Tillage can produce lots of potential pieces of inoculum, but the rate of colonisation is slower. 

The hyphal network can survive over winter, and can colonise new roots of the following spring-sown crop. 

Fungal colonization of the roots is affected by the phosphorus status of the plant. The proportion of root length infected is reduced by either very low or very high levels of phosphorus in the plant. 

Importance of mycorrhizas for mineral nutrition 

It is hyphae external to the root that are important for nutrient acquisition. The mechanism by which this occurs is a combination of increased surface area for absorption from the soil solution, and inward translocation of phosphorus from beyond zones of depletion around roots. Phosphorus moves more quickly in hyphae than it can diffuse in soil. 

Without mycorrhizas, the depletion zone of P around a root is cir 1 mm, but mycorrhizas can extend for 100 mm, thereby greatly increasing the zone from which P is absorbed. 

There is evidence of rapid transport of immobile nutrients to the root via the hyphal network. 

In addition to the larger volume of soil explored, the kinetics of adsorption may also be increased, C_min being smaller, and I_max or K_m also changing. 

Uptake of P in the early stages can be enhanced by as much as 3 fold, and Zn 2-fold when a well established external net is in place. 

Disrupting the external network offsets any advantage of the system. Thus tillage would be disadvantageous. 

In principle less P should be required to be banded with the seed for no-till corn. This is a recommendation in Maryland. However, the mycorrhizal association does not change the total P requirement of the crop. 

Roots of more than one species may be connected by the fungal hyphae. Some transfer of N from legumes to grass through this route has been identified. 

AMs can reduce Mn availability. Mycorrhizal fungi can increase the availability of Zn to the root. 

Other effects of arbuscular mycorrhizas 

The higher plant does carry a significant cost from the carbon supplied to the fungus, although not as much as the total C used by the fungus, since much of the carbon would be released into the rhizosphere in any case. 

AMs have been shown to increase drought tolerance. Possible explanations include changing the content of growth control substances such as ABA and cytokinins, increasing the rate of transport to the root in very dry soil because of better soil structure, and improved P nutrition. 

AMs also are important in soil structure development and maintenance. Macro-aggregate stability has been show to relate to the hyphal length. 
 

 

Relative impacts of soybean roots and mycorrhizal fungal hyphae 
 

The importance of the external hyphal network 

Miller and his co-workers in the Department of Land Resource Science have provided the most significant information. They established that: (1) disturbance of no-till soil reduces VAM infection and P absorption by corn. In their experiments, soil disturbance, sufficient to cause disruption of the external hyphal network, reduced early P nutrition in wheat and corn, but not in canola and spinach. 

The fungicide, benomyl, and -radiation caused the same effects as soil disturbance. (2) corn yields following a non-mycorrhizal crop (canola) may be smaller than those following a mycorrhizal crop due to reduced effectiveness of the mycorrhzal symbiosis 

There is also a clear benefit for N₂-fixation by soybeans associated with keeping the external hyphae intact 

A major limitation to the introduction of AMs to fields under large field management is the mass of inoculum required. 

Survival of inoculum is not certain, because it depends on the competitive nature of the introduced strain compared with other organisms in the soil. 

Survival also depends on the cropping system. 

'Long-fallow' syndrome in Australia is believed to be due to the die-off of mycorrhizal fungi. 

As AMs are not host specific, they can infect all crops in the rotation, unless these include non-mycorrhizal forming crops eg canola. 

Colonisation of corn roots after canola was less than in corn after corn. 

Colonisation by AM-fungi can be reduced in soil given high levels of P, such as in a fertilizer band. However, bands 5 cm to the side and 5 cm below the seed do not offset effects of disruption of the external hyphae. 

The P concentration in the shoot of corn at the 4-5 leaf stage is critical for yield. 

Less P in shoots at this stage in corn after canola than in corn after corn. The greater P content of the shoot increased the harvest index of the second corn crop 

Effect of previous crop on corn yield and harvest index 
 

In practice, ridge-till and no-till should gain more benefits from AMs than conventional tillage. 

At Ridgetown, the benefits of these conservation practices disappeared 30 days after planting. 

For crops given less than 50 kg P ha^-1, no-till out-yielded conventional tillage. 

With larger applications the yield benefit was reversed. 

Symbiotic Nitrogen Fixation 

N₂-fixation is carried out by a variety of prokaryotic organisms that reduce atmospheric N₂ gas (which higher plants cannot assimilate) to NH₄⁺, a form of N readily incorporated into organic molecules. Many of the N₂-fixing prokaryotes are free-living, but those that have the greatest agricultural significance form symbiotic associations with plants. 

Nitrogen fixing systems 
 

Most studied of these systems are in legumes such as soybean (Glycine max L. Merr.), pea (Pisum sativum L.) and clover (Trifolium repens L.). 

Estimates of crop N derived from N₂-fixation by legumes 

• The rhizobia infect cells within the roots of the host plant, causing the formation of root outgrowths called nodules. 

Nodules on soybean (Courtesy of D Hume)

The first stage of infection by the microbe involves recognition of the host. Particular phenolic compounds, flavonoids such as lutein, are released by the roots, and act as a signal for chemotaxis of the rhizobia in the rhizosphere. 
As the rhizobia get closer to the root surface the increased concentration of the flavonoid stimulates expression of the genes for nodulation (nod genes), which is required for the production of lectins (proteins involved in the recognition of foreign molecules) and attachment of the bacteria to root hairs. 
The bacteria release signals, thought to be cytokinins, that cause the root hair to curl around the bacterium. Next, part of the cell wall of the root hair is degraded, and this allows bacterial entry into the hair cell. 
The cell produces a thread-like structure, the infection thread, which consists of an infolded and extended plasma membrane together with new cellulose formed on the inside of the membrane. At the same time division of some cortical cells is induced in the root, forming a nodule meristem. 
The bacterium undergoes extensive division within the thread, which extends inwards penetrating through and between cells of the cortex. 
In the inner cortex the bacteria are released into the cytoplasm of dividing tetraploid cells. 
The bacteria become devoid of a cell wall, and are several times larger than their original size, becoming bacteroids. There may be as many as 20,000 bacteroids per infected cell. The bacteroids usually occur in groups of about four surrounded by a peribacteroid membrane formed by the host cell, initially being derived from the plasma membrane. The membrane also encloses the peribacteroid space. 



Freeze-etched sections of nodules showing the dense array of bacteroids within the cells, and the groups of bacteroids within the peribacteroid membrane (Courtesy of D Hume). 
 
 

Transformation of the bacteria to bacteroids is closely related to the synthesis of enzymes required for N₂-fixation. 
The bacteroid-infected cells are located in the central region of the nodule, and are surrounded by layers of uninfected cortical cells. 

The cortex contains numerous vascular traces that branch from the adjacent stele of the root. 
Phloem elements provide photosynthate, in legumes this is in the form of sucrose, to the nodule as an energy source. 
The xylem removes the end-products of fixation (amides and ureides) which are determined by the plant. Typical amides are glutamine and asparagine: the latter is commonly produced by legumes of temperate origin (eg clover, alfalfa and lupins). 

  

The two principal ureides in legumes of tropical origin (eg soybeans and cowpeas) are allantoin and allantoic acid, while citrulline is produced in the non-legume alder. 
 
 

 

The reduction of N₂ requires a lot of energy (about 960 kJ mol^-1 N₂-fixed), and in many cases, better crop growth and yield can be obtained with fertilizers than with N being derived from fixation. The overall equation for N₂-fixation can be summarized as: 

In reality about 25-30 moles of ATP are required. In this case H₂ should not be seen as a by-product of the reaction but as part of the chemistry of binding and reduction of N₂. 

The first step is the respiration of carbohydrate by the bacteroids leading to the reduction of NAD⁺ or NADP⁺ to NADH or NADPH. 
The next step is the reduction of the electron carrier, ferredoxin. The electron carrier donates electrons to nitrogenase. This enzyme consists of two oxygen-sensitive non-haem iron proteins, the larger unit is a Mo-Fe protein whereas the smaller unit is a Fe protein. 
The Mo is protonated and the Fe reduced in the Mo-Fe protein by the Fe protein accompanied by hydrolysis of ATP. Too much oxygen will oxidatively denature both proteins. 
Leghaemoglobin, a haem-Fe protein in the cytosol of the host cells partly controls the availability of O₂ to the bacteroid. Cobalt is essential for synthesis of leghaemoglobin. 
  Sections of soybean nodules. White non-fixing nodules (LHS) lack leghaemoglobin typified by the pink colouration of the fixing nodules (RHS)  (Courtesy of D Hume)
 

 

As shown on the diagram nitrogenase can reduce a number of substrates other than N₂, including H⁺, and acetylene (This has provided ways to measure fixation indirectly). H₂ can therefore be an inhibitor of nitrogenase activity. Within different strains of rhizobia, and in many other nitrogen fixing bacteria, there are those with an enzyme, uptake hydrogenase (hup). This enzyme re-oxidises H₂, thereby recycling electrons for use in N₂ reduction. Marschner argues that the benefits if this enzyme come from reducing the inhibitory effects of H₂ and the protection of nitrogenase from oxygen. In contrast other authors have suggested that the O₂ concentration is so highly sensitive that the hydrogenase activity could limit the oxygen supply to the bacteroids. Hume and Shelp (Can J Plant Sci., 70: 661-666) indicate that recycling of hydrogen would save up to one third of energy lost due to the hydrogen production. Rhizobia with the enzyme (+hup) release very little H₂ from nodules, but -hup bacteroids liberate lots of H₂ very rapidly. Marschner suggests there could be a cost of 2-3 Mg sucrose per hectare for fixation. 
In laboratory experiments it has been shown that nodulation with +hup strains can result in 16% more dry weight production in soybeans: total plant-N increased by 26%. However, the strain of Bradyrhizobium japonicum which has consistently given the superior crop yields in Ontario is -hup. 
 

Influence of rhizobium strain on yield of soybeans. Strains differ in several characteristics, including hup activty 
 

Greater N-fixation by soybeans inoculated with B.japonicum 532C than plants with nodules formed by a wild-type present in the soil.

The relative efficiency of N₂-fixation is determined as: 
 

with the evolution of hydrogen is measured in moles per unit fresh weight of nodule in unit time, and acetylene reduction is measured in terms of ethylene produced on the same basis. 

Q What other factors limit N₂-fixation ? 
 

Key factors are summarized in the following table.

Factor	Aspects concerned	Consequences
Photosynthate availabilty	Light  Leaf area  CO2 concentration  other sinks -seed production	Large demand for carbon to provide acids for NH3 assimilation
Temperature	>28C reduces fixation,  cool temperatures can impair nodulation
Water supply  -phloem is main source  -also water is needed to export of ureides in the xylem.	This can affect the photosynthate availability	Both excess water and water deficits appear to alter the water content of the intercellular spaces in the 1-5 cell layers if the outer cortex. Changes in water status results in increased water content and reduced O2
Soil pH	Rhizobia tend not to survive well in acid soils.  Also nodulation may be impaired
Nutrient supply	N: nitrate inhibits N2 fixation, probably by competing for carbohydrate to support nitrate reductase activity  P: large P requirement for nodulation  Mo: large requirement for nitrogenase enzyme  Fe and Co required for leghaemoglobin, Fe also for nitrogenase and electron transfer

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