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The rhizosphere: the key functional unit in plant/soil/microbial interactions in the field. implications for the understanding of allelopathic effects

Margaret McCully

Division of Plant Industry, CSIRO, GPO Box 1600, Canberra ACT, Australia 2601 margaret.mccully@csiro.au

Abstract

The rhizosphere is the environment in which roots function in the soil. This environment, created and managed by the roots themselves, is spatially and temporally heterogeneous. Rhizospheres develop, mature and senesce in parallel with developmental changes in adjacent regions of the subtending root, and they remain as relics after root death, often as biopores, which in hard soils are occupied by many of the roots of the subsequent crop. To use allelopathy as an effective biological control in particular field environments, it is crucial to know more about how the production and effectiveness of specific allelochemicals are affected by developmental changes in the root and rhizosphere, and by the placement of roots of donor and target plants in relation to their respective current and relic rhizospheres.

Media summary

Root placement, and the unique properties of the soil environments adjacent to roots (rhizospheres) can affect chemical interference between plant species in the field.

Key Words

Rhizosheaths, root clustering, buckwheat, sorghum, root hairs

Introduction

All allelopathic reactions in the soil, whether positive or negative, involve passage of the allelochemicals through the rhizospheres of the target plants, and, where the chemicals have originated from roots of the antagonist plants, they will also have passed through the rhizospheres of these plants. Rhizospheres are those narrow zones (a few mm or less in width) within the soil matrix surrounding roots. These zones are greatly modified from the bulk soil by the activities of their associated roots and microbial and faunal inhabitants. Knowledge of the nature and development of rhizospheres is, therefore, of importance for the understanding of the passage of allelochemicals within the soil matrix. Furthermore, many of the commonest classes of such compounds have profound effects not only on the growth of other plant and microbial species, but also on the physical and biological properties of rhizospheres, resulting in marked indirect effects on plant growth and survival in the field environment.

It is now 100 years since the initial concept of a rhizosphere was formally introduced by Hiltner (Curl and Truelove 1986), as that narrow region of soil within which soil microbe populations are stimulated by root activities. This original concept is now generally extended to include that portion of the soil surrounding a root which has been modified physically, chemically and biologically by the associated root (and as will be discussed below, frequently by the activities of earlier roots in the same location). It is only now being recognized that the rhizosphere of a single plant greatly changes character with the development of the root system of that plant. In each individual root system roots of different origin, position in the soil, function and longevity are produced through the lifetime of the plant. Also developmental changes occur along each individual root in the system. This plant heterogeneity is mirrored in the heterogeneity of the associated rhizospheres.

This paper aims to provide some insight into what is known (and unknown) of rhizosphere properties and of root activities in relation to rhizosphere development, particularly in agricultural crops which may be of importance for the understanding of the environment in which allelopathic interactions are occurring in the field.

The rhizosphere as a functional domain

Roots have been described as soil ecosystem engineers whose activities create a unique functional domain in the soil (Lavelle 2002). This terminology aptly describes the rhizosphere. This domain is ultimately created by the physical, chemical and physiological activities of roots, their growth through the soil, their uptake of water and nutrients, their developmental changes, senescence and death. Their contribution of energy and carbon to the rhizosphere by exudates, lysates and structural tissue components is the metabolic driving force for the complex interacting community of microbes and fauna that develop there and which further shape the properties of the region. The rhizosphere is the boundary across which flows all of the water, nutrients, exudates and information exchanged between the plant and the soil community influenced by the presence of the roots. Like all boundaries the rhizosphere is complex in character, probably the most complex environment on earth (Belnap et al. 2003).

What does the rhizosphere look like?

Rhizosheaths

The most detailed observations and experimental investigations of the development of a rhizosphere have been with a particular type of rhizosphere, the soil sheaths (rhizosheaths) that form on young regions of many grasses, other monocotyledons and some dicotyledons (McCully 1995, 1999). These sheaths are intimately attached to the roots at anchor points on distorted root hairs, separate cleanly from surrounding bulk soil and remain attached to excavated roots, thus facilitating their study (Figure 1). Rhizosheath soil is strongly coherent, and work in a model system (Watt et al. 1993) suggested that this coherence is due to mucilages exuded both by root cap cells and by specific bacteria that colonize the sheath. The rhizosheath first forms at the site of early root hair development. Mucilage released from peripheral root cap cells and abscised cap cells remain in situ as the root tip grows past them, then mingle with the new elongating root hairs and bind soil particles to each other and to the root surface. One of the most surprising findings (Vermeer and McCully 1982) has been that detached root cap cells can remain alive in the rhizosheath under field conditions for at least several days totally detached from the root. Subsequent extensive laboratory studies of these cells (now called root border cells) suggest that their release is closely controlled by exogenous and endogenous signals, that they are a major component of the young rhizosphere, and may be acting as outlying scouts attracting or repelling root invaders (Hawes et al. 1998). In an artificial rhizosphere these living cells differentially attracted or repelled different bacterial species (Gochnauer et al. 1990), also suggesting a role for them in rhizosphere microbial management.

The extent of rhizosheath development depends both on the plant species and soil moisture conditions. An alternating wet and dry cycle (as occurs diurnally at the root surface) is necessary to expand the mucilage among the soil particles. During drying the mucilage contracts greatly and cements soil particles into stable aggregates, which are not dispersed by further wetting (Watt et al. 1993). Rhizosheaths do not stabilize in very wet soil (Watt et al. 1994). Stable rhizosheaths can form in soil separated from a growing root by mesh that excludes root hairs but allows passage of root cap mucilage and other root exudates (McCully 1999). This further emphasizes the active role of the root in engineering this soil region. Measurements of maize rhizosheaths developed in silty soil indicated significantly higher aggregate strength and lower friability compared to bulk soil (Czarnes et al. 2000). In dry soil rhizosheaths of Opuntia are tightly aggregated and have low water permeability (Huang et al. 1993).

As long as the root epidermis and cortex are alive and intact the rhizosheath remains attached to excavated roots. In maize and wheat at least, the position along the root where excavated roots become largely bare of soil sheath coincides with maturation of the major xylem conducting elements. Thus this position coincides with the start of full attachment to the transpiration stream and the lowered water status of the bare portions of the root (McCully 1995, 1999). In wheat, rhizosheaths have a higher moisture content than surrounding bulk soil (Young 1995) probably reflecting the higher water content of the subtending root region and the altered soil structure of the rhizosheath. The transition from sheathed to bare, more-mature regions can occur many centimetres from the root tip in rapidly extending roots.

Most of the properties of rhizospheres have been investigated in rhizosheath-forming species, where physical and biological properties of soil adhering to young root regions to those of bulk soil have been compared. In many species of dicotyledons rhizosheaths do not form, and the reason for this lack of attachment to the immature regions of the roots has not been determined. Presumably, other properties of root-managed soil, obvious in rhizosheaths, also apply to the rhizospheres of these unsheathed young roots. In general, research has indicated that rhizosphere soil has higher bulk density than the surrounding soil as a result of circumferential expansion of roots (Young 1998) with increases of 20% or more.

‘Mature’ rhizospheres

Rhizosheaths of mature regions of plants like grasses become detached from the still living inner root tissues with the normal developmental death of the epidermis and outer cortical cells. These mature rhizospheres incorporate the degenerating root tissues and compounds liberated from them, but possibly receive no further actively exuded material. Little is known of the properties of these rhizospheres or whether they remain as active sites of exchange between roots and the soil and soil microbes but it has been shown in maize that these mature rhizospheres support a very different microflora than do the rhizosheaths (Gochnauer et al. 1989).

Only recently has any attention been focused on the development of rhizospheres which parallels root development (McCully 1999; Jones et al. 2004). The latter authors propose a four stage rhizosphere developmental sequence, juvenile, developing, mature, and relic. This important recognition that all rhizospheres are not equal even when associated with the same plant is of crucial consideration for all sampling and comparisons of rhizosphere characterizations.

‘Relic’ rhizospheres

Root-formed biopores are the only relic rhizospheres that have been investigated. Biopores are created as roots grow through the soil, compressing the immediately surrounding soil. When outer tissues of the roots senesce, they, along with their rhizospheres, are added to the periphery of the pores, and with root death the decay-resistant vascular tissues remain in the pores. These special relic rhizospheres are of particular importance in hard and duplex soils, and under minimum tillage crop management, because they provide preferred channels for the passage of new roots of subsequent crops (Rasse and Smucker 1998; Pankhurst 2002; McCully 2004). As many as 80% of new roots were located in root-formed biopores in red duplex Kandosol soils in NSW, Australia. This occurred in fields where canola followed lucerne (Pankhurst et al. 2002), or in minimum-tilled fields of the same soil type with a long-term wheat/canola rotation where roots of the current crop shared biopores with remnants of roots from at least two previous crops (McCully 2004). The rhizospheres of the living roots in these biopores present a largely unexplored environment. Pankhurst et al. (2002) reported that microbial populations and chemical properties of soil from the biopore periphery differed markedly from that of the bulk soil. There is no information on how roots function in this relic rhizosphere of biopores. In many field situations the majority of roots of the current crop may be located in biopores, or in cracks and other irregularities in the soil, together with root remains from previous crops where the traditional concept of a rhizosphere does not apply.

Microbes in the Rhizosphere

Hiltner’s original concept of the rhizosphere as a region of root-induced enhancement of microbial populations relative to those in the bulk soil has been amply established in the intervening century. But the full complexity of the microbial interactions that probably occur there, the signalling back and forth between microbes and the roots within the rhizosphere, and among soil fauna (particularly nematodes), bacteria and roots is only now being revealed (Hirsch et al. 2003; Mathesius 2003; Bais et al. 2005). These studies are suggesting an astonishing interactive network of chemical signals linking roots to the other inhabitants of the associated rhizospheres, though most studies have been done in vitro and not yet confirmed in natural rhizospheres. The first of these interactions to be revealed in detail was the subtle exchange of signals between legumes and their nodule-forming rhizobia, initiated by flavonoids released by the root, which induce gene activity in the bacteria. This results in the production of lipochitinous molecules which initiate the plant developmental sequences that result in nodule development and the nitrogen-fixing symbiosis. Similar cross signalling is now known between roots and mycorrhizal fungi and roots and nematodes. Perhaps the most surprising signalling that has been revealed to occur in the vicinity of roots is that of quorum-sensing between root-associated bacteria (Bauer and Mathesius 2004). Signals between individual bacteria of the same species can initiate clustering (quorums) of these bacteria which, unlike the individual bacteria, are capable of interacting with roots either positively as growth promoters or negatively as pathogens, depending on the species. Roots, in turn, can release molecules similar to quorum-sensing signals which, depending on their exact structure, can either induce quorum formation, or confuse the interbacterial signals to prevent clustering and induction of pathogenicity.

Recent research that also highlights the complexity of root microbial interactions that may be played out in the rhizosphere (Bais et al. 2005) emphasises the potential for specific rhizosphere microbes to manage the release of potentially allelopathic chemicals by roots. In elegant lab-based experiments with Arabidopsis roots interacting with eight strains of the bacterial pathogen Pseudomonas syringae, only one strain caused significant disease symptoms or plant death. Not only was this strain resistant to the root exudates (10 different antimicrobial phenolic compounds were identified) but their amount was reduced to very low levels in the presence of the pathogen, and these failed to inhibit the otherwise non-pathogenic strains. Evidence was presented that factors translocated from the virulent strain into the host root cells were responsible for this suppression. In contrast, exudation was markedly increased in the presence of the non-pathogen strains. However, if this exudate was absorbed by activated charcoal placed next to the roots the non-pathogenic strains invaded the roots and killed the plants.

Some questions bearing on understanding root-mediated allelopathic interactions in the field

Do we know where, how and when allelochemicals from living roots are released into the rhizosphere?

It is generally assumed that small molecular weight root exudates diffuse passively out from surface cells, or are actively exuded through membrane channels, and that most originate close behind actively-growing root tips. The only well documented precise localization of where a proven allelochemical is exuded from roots is that of the hydroquinone sorgoleone (Czarnota et al. 2001; 2003). This hydrophobic, coloured compound, a known inhibitor of photosystem II in target plants, is synthesized in the endoplasmic reticulum of a population of root hairs of Sorghum spp. It then accumulates in deposits between the plasma membrane and the wall and subsequently forms droplets at the tips of the hairs. How and when it passes through the cell wall into the rhizosphere is not known. A mutant that lacks root hairs does not produce sorgoleone (Yang etal. 2004), confirming the strict localization of its production in the root. All the experimental work to date has been with laboratory-grown young plants, but cryo-microscopy of growing roots on mature plants in the field shows similar accumulation of hydrophobic droplets on root hairs (Figure 2). Environmental factors in the field which promote or inhibit root hair development will thus influence the effectiveness of sorgoleone-produced allelopathy.

Figure 1. The root/soil interface of a young branch root of buckwheat (Fagopyron esculentum) frozen in situ in the field with liquid nitrogen (LN2) and observed with a cryo-SEM. Note the root hairs extending into the rhizosphere and the variation in soil contact with the root surface. Arrow indicates the root epidermis. Bar = 600 m.

Figure 2. Droplets of exudate (arrows) on the tips of root hairs in the rhizosphere of broom corn (Sorghum sp.). The material was frozen in situ in the field with LN2 and observed with a cryo-SEM. Bar = 50 m.

Localization of a coloured naphthoquinone antimicrobial compound in the root hairs and border cells of Lithosperman roots in culture (Brigham et al. 1999) suggests the possibility that border cells in the rhizosphere may also be the source of allelopathic compounds. Apart from these two examples where the compounds are coloured, not much is known of the exact location of allelochemical entry into the rhizosphere.

Little seems to be known about the timing of release of allelochemicals from roots. Recently, however, Melnitchouck et al. (2005) have shown a significant increase in water-soluble exudates that could be leached from the rhizosphere during the day compared to the night from 5 week-old maize grown in containers of unsterilised soil. These compounds included phenols and lignin monomers. Also of interest in this study was the finding that products of microbial processes dominated the exudates recovered during the night.

Many more studies, particularly under field conditions, are needed to clarify the location and means of allelochemical release from living roots.

How easily do allelochemicals move through the rhizosphere and how stable are they in this environment?

Allelochemicals may be rapidly deactivated in soil by microbial metabolism, adsorption to soil particles, or chemical oxidation. Some innocuous exudates can also be converted to toxins by specific microbes (Inderjit 2001). In young rhizospheres such as the compact rhizosheaths (Figure 1) these processes would be expected to be much more efficient than in bulk soil, not only because of the enhanced microbial activity, but also because of the physical constraints of the closely aggregated soil. This rhizosphere effect on allelochemicals would occur adjacent to roots of both donor and recipient plants. Many phenolic compounds are released from roots (Wu et al. 2001; Bais et al. 2005) and are well established as allelochemicals in laboratory and pot-based assays. Many of these compounds are rapidly inactivated in non-sterile soil and their ability to survive passage through rhizospheres in the field is problematic, and may depend on specific soil conditions. In contrast, sorgoleone was found to persist in unsterilised soil for up to seven weeks (Bertin et al. 2003). Trials of suspected allelopathic chemicals must be done in natural soil, preferably in the field (Inderjit 2001). In addition, consideration must be given to where the roots of the donor and recipient plants are placed in the field situation. Are they, for example, mainly in the relic rhizospheres that are biopores, and do they share the same biopores? How active are the microbial populations of biopores in interactions with allelochemicals?

Is root clustering and intermingling important?

This question does not seem to have been addressed directly in respect to allelochemical donor and target plants in the field. Indeed, there is little reliable information about crop root placement in specific field conditions. Besides the clustering of crop roots in biopores in hard soils, roots also cluster in cracks and on the surface of soil peds (Taylor 1974; Logsdon and Allmaras 1991), but it is not known if weeds share these clustering sites with crop roots. Because roots at these sites are closely intermingled and often not in close contact with soil, movement of allelochemicals between roots would not be hindered. Close juxtapositioning of rhizosheaths is common in crop plants and multiple roots occasionally share a rhizosheath.

Figure 3. The intermingling of the root systems of a wheat plant and a weedy mustard species. Reproduced with permission, and modified from Pavlychenko (1937). The red lines outline the axile roots of the wheat plant, numbered 1-4 by Pavlychenko.

Bormann (1957) showed that water exuded from the roots of one plant can be taken up by the closely entwined roots of a second plant. This observation strongly suggests that close juxtaposition of the roots of donor and target plants in an allelopathic relationship may be of crucial importance for the successful transfer of allelochemicals. The extensive excavations by Pavlychenko (1937) of crop root systems from fields where they were competing with weed species, shows very clearly that the roots of the crop are intimately interlaced with the root systems of competing weeds. For example, his Figure 4 (our Figure 3) strongly suggests that the weedy species (in this case wild mustard) has closely intermingled its roots with those of the crop plant (wheat). There is no knowledge of whether such intermingling of roots is necessary for successful allelopathy in the field.

How to move forward?

The potential for allelopathic studies to lead to effective biological control methods in crop production is now widely recognized (Inderjit and Keating 1999; Fitter 2005). Laboratory based research has led to very significant advances in the knowledge of allelochemical production and interactions with target plants and microbes.

These studies have also revealed some of the possible biological complexities that exist in the rhizosphere which will affect (or effect) biological control by allelochemicals. The laboratory “rhizosphere” is, however, not an adequate surrogate for the real environment of roots in the field. Field studies are difficult and some are impossible. But real attempts must be made to reveal the locations and interactions of allelochemicals in the heterogeneous root environments that characterize the field soil.

Two recently developed techniques for micro-sampling of chemicals in the rhizosphere, by micro suction cups (Vetterlein and Jahn 2004), and by “biomimetic” sorbent materials (Weidenhamer 2005), if combined with careful excavation, would make possible in situ field sampling of allelochemicals. Sorgoleone was successfully collected from soil of pot-grown sorghum by the sorbent method (Weidenhamer 2005). The importance of close juxtapositioning of roots of donor and target plants in the field should be assessed by selective excavation and soil coring.

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