The Role of Soil Biota in Soil Fertility and Quality, and Approaches to Influencing Soil Communities to Enhance Delivery of These Functions

Department for Environment, Food and Rural Affairs Research project final report

Project title The role of soil biota in soil fertility and quality, and approaches to influencing soil communities to enhance delivery of these functions.

Sub-Project A of Defra Project SP1601: Soil Functions, Quality and Degradation – Studies in Support of the Implementation of Soil Policy

Defra project code SP1601

Contractor SKM Enviros organisations Cranfield University Rothamsted Research

Report authors Karl Ritz ([email protected]), Jim Harris, Phil Murray

Project start date October 2009 Project end date March 2010

The role of soil biota in soil fertility and quality, and approaches to influencing soil communities to enhance delivery of these functions.

Sub-project A of Defra Project SP1601: Soil Functions, Quality and Degradation – Studies in Support of the Implementation of Soil Policy

1. INTRODUCTION The importance of soil and the functions it performs are unquestionable. Soil is a living entity that needs to be maintained and managed in a sustainable way. Soils are highly complex systems, both literally in that they are constituted of vast range of constituents that show great spatial heterogeneity across some ten orders of magnitude, and in the more formal construct of complexity science (Ritz 2008). Factors that contribute to effective soil fertility, i.e. the production function, are diverse and concomitantly complex (Gregorich & Carter 1997; Mader et al. 2002). However, it is apparent that the soil biota contribute substantially to effective soil functioning from many perspectives (Bardgett 2005), including the basis and maintenance of sustainable agricultural fertility (Kibblewhite et al. 2008). The soil biota can be conceived of as the 'biological engine of the earth' (Ritz et al. 2004) driving and modulating many of the key process that occur within soils. The biomass typically only constitutes a small proportion of the total mass of soils, but has a hugely disproportionate effect upon soil functions. For example, Jenkinson (1977) appositely describes the biomass, which is predominantly microbial in constitution, as the "eye of the needle through which all organic materials must pass". However, the soil biota consists not just of the microbes but of a myriad of larger multi-cellular organisms, and the entirety interacts via series of complex food-webs (Van der Putten et al. 2004). Microbes function as primary decomposers and biochemical transformers at the core of such webs, and larger organisms provide higher-order ecosystem services such as organic matter comminution, decomposition, and ecosystem engineering. It is important to take an holistic systems viewpoint when attempting to understand the complex interactions in the soil which affect the soil biota. For example, the addition of fertilizers can have direct impact on the soil biota, but also can have an indirect influence via the plant and the two are inextricably linked (Murray et al. 2006). Whilst the mineralogy, physics and chemistry of the soil system provides the context, and sets the boundaries in which the soil biota operates, the unique feature of the biota is that it is adaptive to changes in environmental circumstances, which occur by processes of natural selection, in ways that the abiotic systems of the soil are not (Kibblewhite et al. 2008). This has important implications for the way in which soil systems function, and the ways they can be manipulated and managed. Whilst the emphasis on the production function is to maximise yield1, and this was historically perceived as the primary goal for agriculture, it is becoming increasingly recognised that the production function has to be reconciled with provision of other ecosystem goods and services to avoid degradation of the wider environment and detriment to society. Given the imperative to produce sufficient food to support a global population currently projected to exceed 8 billion by 2030 (FAO 2006), this is an extremely challenging task. Agricultural systems can be classified within a conceptual space that varies in many factors that include the origin of energy sources, nature and intensity of fertiliser use, complexity, biodiversity, cultural tenets, etc. These can be broadly categorised, for example, as a

1 Yield is here defined to mean the mass of agriculturally prescribed product. For example this can be grain cereal or legume or herbage forage for stock or biomass for energy crops.

Page 1 spectrum of industrial – integrated – organic – biodynamic1, accepting this is not an entirely comprehensive list. However, it is important to move away from some of the more extreme caricatures of these different approaches to production, to recognise the spectrum of practices adopted, and to avoid presenting “conventional” and “sustainable” farming as opposites, incapable of being mixed (Shennan 2008). When environmental problems occur with agricultural production they usually hinge around poor management, and not the mode of agriculture per se (Trewavas 2004). In essence, the soil biota underpins five key ecosystem services that are fundamental to agricultural productivity, viz. carbon cycling, nutrient cycling, soil structural integrity and dynamics, biotic regulation and mutualism. Agricultural systems utilise or circumvent soil biota to differing degrees depending where they fall in the management spectrum above. Industrial agriculture, for example, typically substitutes services provided by the soil biota in other systems by industrially-derived substitutes such as inorganic fertilisers, synthetic biocides and ploughing. This distorts the natural balance of the ecosystem and may compromise the output of other environmental services (Kibblewhite et al. 2008). If production is taken as the sole aim of the system, then it can be seen as ‘efficient’, but there is likely a trade-off with other ecosystem services being compromised, such as water storage and biodiversity. The aims of this review are to briefly explain how soil biology operates with respect to production function, and to explore potential strategies for the management of the soil biota to maximise outputs whilst minimising inputs and impacts on delivery of other ecosystem goods and services.

2. THE ROLES THE BIOTA PLAY IN SUPPORTING THE PRODUCTION FUNCTION OF SOILS The soil biota underpin five key ecosystem services that are critical to the ability of soils to produce crops, viz. carbon cycling, nutrient cycling, soil structural integrity, biotic regulation and mutualism. These will be reviewed in turn, followed by a systems-level consideration. Carbon cycling Soil organic matter (SOM) originates from primary production, and in large part from terrestrial vegetation. There are strong links between SOM, soil functions (Tate 1992) and agricultural sustainability (Magdoff & Weil 2004). Carbon fixed by photoautotrophs organisms that carry out photosynthesis enters the soil via deposition in roots, rapidly in the form of soluble exudates that emanate from growing roots, and more slowly by the deposition of cells and tissues. Above ground, plant parts are deposited on the soil surface as they senesce, and en masse in the case of annual crops, unless such residues are harvested and removed from the field. In cropped grassland systems, such as for hay or silage, litter return to the soil surface is restricted by such harvesting, whilst in grazed pastures, part of the herbage consumed by livestock is deposited on the ground surface as dung. A managed return of organic matter to the soil can also be made, using such materials as slurry, manure, compost, industrial waste such as sewage sludge, food processing by-products, biochar, etc. A standing crop of vegetation can also be deliberately utilised as source of organic matter, for example in the case of green manures. All of these practices have short term outcomes, and long term effects (McLauchlan 2006). Organic material in soils is more or less continuously transformed by a very wide variety of chemical and biochemical mechanisms into a diverse range of compounds, with the majority of such transformations being carried out by the soil biomass (Marschner & Rengel 2007). The rates of such transformations range from seconds to centuries, as do the residence times of the associated compounds. Soil organic matter (SOM) contains energy- rich bonds which are the primary energy source for the soil biota. Soil organisms have appropriate biochemistries and life strategies to assimilate such energy for their growth and

1 The ‘industrial’ end of this spectrum tends toward a substitution of biotically-mediated processes with substantial use of fossil-fuel based energy consumption to create fertilisers, biocides and extensive soil disturbance via tillage. At the other end , ‘biodynamic’ type systems seek to avoid the use of any such sources and are founded on what are asserted to be holistic tenets. There are then a range of intermediate systems that are not straightforwardly discriminated.

Page 2 reproduction; in doing so, the compounds are further transformed and cycled between compartments (Paul 2007). Importantly, this process yields stable SOM which contributes to structural development and buffering. Obviously, for such processes to occur, OM must be brought into physical contact with organisms. Many soil organisms are motile, and move though the soil matrix foraging for such substrate. However, motile bacteria, protozoa and nematodes are confined to water films to realise such passage. Filamentous fungi, by virtue of the hyphal growth form are not constrained in this manner, and are capable of bridging pores of several mm in dimension. Biological perturbation (bioturbation) is critical in the genesis and maintenance of soil structure and function (Wilkinson et al. 2009). Invertebrate fauna such as worms, ants and molluscs are capable of physically displacing the solid phases of the soil matrix to enable their passage, and in so doing can physically move substantial quantities of soil material, effecting a mixing action. Earthworms play a key role in temperate systems (Edwards 2004). There are three ecological guilds of earthworm, with complementary functional attributes. Surface-dwelling epigeic worms inhabit the upper litter-rich layers of soil and consume organic matter directly, comminuting it and thus accelerating degradation and nutrient cycling. Surface-casting anecic earthworms form vertical, persistent, burrows and directly incorporate organic matter present on the soil surface into the soil matrix by pulling such material into the burrows, ingesting it and excreting the material deeper in the soil. A third guild, the endogeic worms, form lateral burrows in foraging for organic materials, that are of necessity back-filled, and their passage further combines organic material with, and mixes, the soil matrix. The adverse effects of cultivation on invertebrate populations has been widely reported (Smith et al. 2008) and partial solution by, for example, reducing depth of cultivation do little or nothing to mitigate these effects (Metzke et al. 2007). Improved aggregate stability and increased incorporation of organic matter into these aggregates has been demonstrated when moving from conventional through reduced tillage to no-tillage systems (Kasper et al. 2009). The importance of earthworms in producing such aggregate structures and the stabilisation of organic matter in earthworm casts has been reported (McInerney & Bolger 2000) This suggests the possibility that enhancing the invertebrate “ecosystem engineer” population would increase C- sequestration and enhance buffering capacities, minimising export of C and other materials out of the system. Baker et al. (2006a) report on the impacts of accidental releases of non- native earthworm species into Australian agricultural lands which results in a net increase in incorporated carbon. Introduction of non-native species into ecosystems is an approach littered with a history of unintended consequences. In North America, invasive earthworms outcompete native millipedes and increase carbon losses from soils by respiration (Snyder et al. 2009). In arable systems, plant residues were traditionally incorporated into soils by mechanical means such as ploughing, which also serves as a weed-control strategy. However, there is now an increasing trend away from this approach because of rising costs, particularly for fuel, and the increasing demonstration that such incorporation is not generally necessary to maintain a productive system (Baker et al. 2006b). Instead, zero-till and conservation tillage systems are being more widely adapted by farmers. Here, biotically-based systems of residue incorporation are founded on worms and fungi, but conversion from intensive cultivation systems may require many years, or even decades, before the soil invertebrate community recovers sufficiently to carry out this function (Johnson-Maynard et al. 2007) but recent work suggests that this is principally due to the slow accumulation of SOM than the removal of physical disruption (Simmons & Coleman 2008). In grassland systems, the biota prevails as the principal SOM incorporation and mixing mechanism. It has been suggested that wide scale adoption of no-till agriculture would result in such systems becoming significant carbon sinks (Bernacchi et al. 2005), for which there is increasing evidence in the literature e.g. (Halvorson et al. 2002; Ussiri & Lal 2009). Thus plants, and therefore crops, are the primary producers of fixed C in production agriculture systems, and microbial respiration balances the net ecosystem flux – as is the case in all terrestrial ecosystems (Schulze 2006). Therefore the impacts of the soil biota, and its potential manipulation, on yield gains will be related to the supply of energy, associated nutrients, and providing adequate oxygen moisture and structural conditions.

Page 3 However how much increase in efficiency can be achieved will be strongly dependant on the production and characteristics of the soil organic matter.

Nutrient cycling Nutrient cycling is a fundamentally important process to the ecological functioning of all the Earth’s biomes. Conceptually, nutrient cycles involve a defined, typically bounded, compartment which nutrients enter and leave via a range of pathways, and within which they are transformed via a myriad of chemical and biochemical reactions. In soil systems, a large proportion of these transformations are mediated by biota. Organisms require nutrients to build structural components of which they are comprised, and for the biochemistry that underpins metabolic processes. Nutrient cycling transformations invariably involve the capture, storage or release of energy. In agricultural systems, if the quantities of nutrient elements removed by crop offtake and lost via other pathways exceed the input rates of such elements, then the system is essentially not sustainable, since pools of such nutrients will in time be depleted. The timescale here will be governed by input and output rates, and pool size, all of which can to some extent be managed. The compartment of immediate concern in this respect is the minimal management unit, typically the field, but in a policy context, the concept is pertinent at larger scales such as the farm, catchment, region or arguably national and international. The corollary also applies – if inputs exceed outputs, then there will be a net accumulation. Since such concepts are time-dependent, it may be possible to run systems in “deficit” for periods, followed by accumulative phases, notwithstanding that there may be variable efficiencies associated with this, and that tipping points, where recovery is not possible, are not exceeded. Nitrogen

The contemporary atmosphere of the Earth contains 78% N2 gas, which is unavailable to the majority of organisms in this form. The conversion (‘fixation’) of gaseous N2 into + ammonium (NH4 ), a form that is available for uptake by plants and microbes, requires high energy inputs and can occur naturally, for example by lightning discharge in the atmosphere, or be manufactured in industrially-based fixation systems, such as by the Haber-Bosch process. There are also biological mechanisms that fix N. In industrial and integrated farming systems, N is typically applied to crops in inorganic forms of ammonium or nitrate, or as chemically simple organic compounds such as urea. Global quantities of such fertiliser application amount to some 140 Mt per annum (FAO 2008). Organic-based farming systems prohibit the use of such industrially-fixed N and these together with extensive farming systems tend to rely instead upon biologically fixed sources, predominantly via plant:microbe symbioses, particularly based upon legumes. Free-living cyanobacteria and algae that colonise soil surfaces where there is adequate light to support them also fix N (Stewart 1969), but the quantities are relatively small and would not sustain a high level of production. Global estimates for biologically-derived inputs of N to agricultural systems are 50-70 Mt (Herridge et al. 2008). Biological N fixation is considered further in Section 2.5.1 and 2.6.2. Other sources of N in cropping systems include by-products of production systems such as manures and slurries, and industrial wastes streams such as sewage sludge and composts. The decomposition or mineralization of soil organic matter, which may include plant and animal derived fractions, is driven by microorganisms that require carbon for growth and development. The release of ammonium is often a consequence (Sprent 1987) and this is then becomes available for uptake by plants and other organisms. Ammonium, being positively charged a cation, is prone to absorption onto soil particles, particularly clays, and hence is relatively immobile. Nitrification is carried out by a relatively restricted number of microbes. The most studied process involves the sequential oxidation of inorganic ammonium to nitrate via two distinct functional groups of proteobacteria, namely the autotrophic ammonia oxidisers and autotrophic nitrite oxidisers. However, this relatively

Page 4 simple view of nitrification has been challenged by increased understanding of the diversity of autotrophic nitrifiers, the discovery of the anammox process1 and activities of ammonia- oxidising archaea, as well as the fact that certain fungi can oxidise reduced forms of organic N to nitrate and nitrite (Prosser 2006). Nitrification has major consequences for - plant N acquisition since nitrate is an anion (NO3 ) and as such is not prone to absorption by soil minerals, is highly mobile in water, and hence can be delivered to plant roots rapidly. The networks of interdependency between SOM and the myriad of soil biota are termed food webs, and involve complex interactions between different trophic levels, and the transfer of energy between them (e.g. Hunt et al. 1987; de Ruiter et al. 2005; Banasek- Richter et al. 2009). Such ‘turnover’ of N and indeed other nutrient elements is an important basis for fertility and where organic sources of N predominate, the soil biota play crucial roles in the provision of N to crop plants. The role of the soil fauna in decomposition has received considerable attention (Curry 1994) but is still not adequately defined. The animals in the soil may exert a major influence on the decomposition through interactions with the microflora. (Curry 1994) showed that the available data on faunal contribution to N- mineralization indicated a larger role than their metabolic rate would suggest. This may be due to their low production efficiency and hence their low N to C requirement (Anderson et al. 1981). This means that excess N is returned to the soil in excreta informs that are readily available to the microbial biomass. Subsequently, this biomass provides substrate for the microbiverous fauna, particularly protozoans and nematodes, resulting in the rapid turnover of a small pool of considerable importance for plant growth (Coleman et al. 1983). Bardgett & Chan (1999) showed that the presence of collembola and nematodes enhanced soil N mineralization relative to defaunated controls. Earthworms also play a large part in nutrient cycling by direct N excretion and indirectly via the processing of plant residues. As less than 10% of ingested material is assimilated the worm casts contain organic matter that is not changed chemically, but is subject to physical change that may make it more available to other organisms (Syers & Springett 1984). Pathways of N loss from agricultural systems are predominantly by nitrate leaching another - consequence of the high aqueous mobility of NO3 and denitrification. Denitrification is a - microbially mediated process involving the reduction of NO3 to N2, under anaerobic - conditions, the intermediaries being NO2 , NO and N2O (Firestone 1982). Denitrification may be considered beneficial if completed to N2, however, the intermediates are potential pollutants (Bouwmann 1990). For denitrification to take place there needs to be a readily available supply of substrates and low oxygen concentrations (Munch & Velthof 2006). Efforts to increase denitrification through to N2 may prove useful in reducing some of the negative impacts of excess nitrogen in soils (Murray et al. 2004a; Murray et al. 2004b). Nitrification inhibitors restrict the microbial conversion of ammonium to nitrate and therefore restrict the output of N2O. There are a number of studies on the development and effects of these inhibitors (Chien et al. 2009) with three now available on a commercial basis (Nitrapyrin, DCD and DMPP) (Edmeades 2004). There are also natural nitrification inhibitors present in some plants, for example the tropical pasture grass Brachiaria spp. has been shown to inhibit Nitrosomonas function and therefore suppress N2O emissions (Subbarao et al. 2009). It is thought that there is potential to develop improved forage grasses for low nitrifying potential given the significant genetic variability within the Brachiaria spp. Subbarao et al. (2007a; 2007b) also propose that introducing high biological nitrification inhibition capacity from wild wheat Leymus racemosus could be an option in the near future. Using crops and pasture plants that have a high biological nitrification inhibition capacity or commercially available nitrification inhibitors will result in production systems that benefit both agriculture and the environment (Edmeades 2004; Subbarao et al. 2009). Phosphorus There are no natural equivalents to C- or N-fixation relating to phosphorus (P), not least because there are no substantive gaseous forms of this element in the atmosphere. In natural systems, sources of this element for plant growth are founded upon the solubilisation of P-bearing mineral constituents of soils, such as various forms of apatite. P

1 Anaerobic ammonium oxidation (anammox) involves the oxidation of ammonium under anoxic conditions with nitrite as the electron acceptor and nitrogen gas as the main product (Mulder et al.1995).

Page 5 inputs proper i.e. imports to the soil compartment are via deposition of particulates from the atmosphere and from animal incursion such as birds defecating on the soil and other animal-related activities1. Such mineralisation and import rates tend to be relatively slow and cannot support a high rate of production. As such, agricultural systems require substantial inputs of fertilising P to promote plant growth. These can be inorganic e.g. rock phosphate or organic such as manures, composts, sewage sludge, etc. Biotic processes which regulate P availability to plants and microbes include solubilisation to phosphate PO4 ions via excretion of organic acids, which can be mediated by bacteria, fungi and plants themselves. Soil microbial population represents a large store of P in organic forms that is a potential source of inorganic P for crops – either directly or by replenishing the inorganic pools. This has previously been demonstrated in research by Brookes et al. (1984) but its significance has tended to be overlooked as many studies on soil P have focused on agricultural systems receiving large inputs of inorganic P fertilizer leading to inorganic pools dominating crop P supply. But in systems with low concentrations of soil P, as in many developing countries and tropical soils, organic P sources can be dominant and their better management is essential. Such low concentrations can arise by design as in low-input systems, due to lack of access to fertilizers, or rapid fixation of added inorganic P by soil phases. Although there is a general understanding that some organic P will be mineralised as cells die, the rates, precise mechanisms and controlling factors have been poorly investigated. For N mineralization the role of faunal grazing as a mechanism of accelerating N release has been demonstrated (e.g. Bonkowski 2004) but, this has not been apparently been demonstrated in relation to P, although it seems logical that it should occur. Factors controlling soil P supply to plants are vital in controlling the fertility of both natural e.g. woodland and agricultural ecosystems. The soil microbial biomass plays a key role by acting as a reservoir of potentially plant-available P which becomes available during the process of biomass turnover, whereby microbial biomass P is mineralised. The amounts of P immobilised in the biomass are surprisingly large. For example, in UK grassland and woodland soils, the biomass P pool may exceed 100 kg P ha-1, about 75 % smaller in arable soils. This gives a mean biomass P flux of around 7 kg P ha-1 in UK arable soils and 23 kg P ha-1 in grassland (Brookes et al., 1984). This compares with annual crop or grass P offtake of around 20 and 12 kg P ha-1 respectively (Brookes et al. 1984). Thus the biomass P pool only represents the potential of the system to supply P to plants; the critical factor is the rate of flux of nutrients through the microbial biomass. P is typically very immobile in soil and diffusive supply to roots or microbes extremely slow. This means that it is generally more efficient for organisms to grow or migrate into new regions of soil to acquire P than to rely upon diffusive supply alone. Plant root systems achieve this by elongation and branching, and root hairs in particular play a key role in P acquisition by increasing the surface area of roots. Mycorrhizal fungi form mutualistic associations with plant roots, where the fungus derives C from the host plant and forms extensive mycelial networks which ramify through the soil, absorb P and transfer this directly to the plant. Since such hyphae are typically of the order a few µm in diameter, they are able to explore and exploit considerably larger volumes of soil per unit C than plant roots. These are considered in more detail in Section 2.5.2. The role of the larger meso (>100um) and macro (>2mm) fauna (Swift et al. 1979) has been ill defined. Parfitt et al. (2005) showed that grazing on the soil microbes by these groups caused increased plant available P in New Zealand pastures. Setälä & Huhta (1990) carried out macrocosm studies of artificial forest floors and found increased leaching + 3- of both NH4 and PO4 was enhanced by the presence of soil fauna and also differences in the mineralization of N and P in both humus and mineral soil horizons. Both of these studies report correlations between density of soil meso-fauna and available P concentrations. There are potentially two pathways by which the fauna may enhance P mineralization, firstly through the natural fluctuations in population size of the fauna, with dead animals being broken down. Mckercher et al. (1979) showed that although soil

1 In N American forests, bears have been shown to play a key role in importing P to land via salmon they extract from rivers and the carcases they deposit on the forest floor (Gende & Quinn 2006).

Page 6 invertebrates account for a relatively small proportion of the total soil biomass their role in P transformations may be relatively more important. Secondly, mineralization of P through the microbial loop may be enhanced by soil meso-fauna directly grazing on the microbial biomass, which in turn may be controlled by higher trophic groups (Parfitt et al. 2005). In the case of richly P-fertilised soils, the microbial biomass may only make a relatively small contribution to crop P nutrition. However, in grassland and arable soils with relatively smaller fertiliser inputs, or soils where soil P reserves are declining, plant nutrition will likely become be much more dependent upon P supply from the microbial biomass. P losses from agricultural systems are predominantly via erosion, i.e. a loss of particulate soil material which has P associated with it. Hence control of erosion will conserve P, and as discussed below, the soil biota have roles to play in mitigating erosion via soil structural. The crucial mechanisms that regulate P supply are those that render it available to biomass and plant roots – much of the soil P pool is essentially biologically unavailable. Other elements The soil biota are also involved in the cycling of other nutrient elements of pertinence to agricultural production systems including S (Kertesz et al. 2007; Eriksen 2009), Fe (Weber et al. 2006), Mn (Tebo et al. 2004), Zn (Lasat 2002) and Se (Losi & Frakenberger 1998; Meyer et al. 2007). There are a range of microbes which utilise forms of these elements in oxidation and reduction reactions to glean energy from such transformations. These can be of particular significance in the rhizosphere with respect to plant uptake. Mycorrhizae can also influence the uptake of a range of elements by plants, including S, B, K, Ca, Mg, Na, Zn, Cu, Mn, Fe, Al and Si (Clark & Zeto 2000). Manipulation of such populations can be achieved by altering the redox state of the soil, via the water status, and there is potential for their manipulation by altering rhizosphere properties as discussed below. Soil pH can also affect solubility and availability of these metals to a great extent, which can in turn be managed by liming. Relatively little attention has been paid to biotic optimisation of cycling of such elements from a production perspective. Coupling to C cycle Many of the nutrient cycles in soil are coupled to the C cycle. This is because by definition, all nutrients associated with living organisms are to some extent associated with them in their cells and tissues. Many are chemically bound into organic molecules, and such elements are then de facto assimilated and transformed during decomposition and ingestion processes. Similarly, organic materials entering soils by natural or managed routes will contain many nutrient elements associated with organic molecules. These represent a potential energy source to the biota and during transformations of such compounds, associated nutrients are also transformed.

Soil structure The basis of soil structure The solid phases of soil are comprised of a diverse mixture of inorganic and organic components. Soils are produced by gradual processes of biogeochemical transformation including ‘weathering’, a range of erosive chemical, physical and biological mechanisms that produces a variably-sized population of mineral particles. The small nanometre, medium and large millimetre components are classified as the clay, silt and sand fractions respectively. These mineral constituents combine with organic materials, and aggregate to form larger units (Tisdall & Oades 1983). Generally, the forces binding such aggregates together are greater at smaller size scales, and hence there tends to be a greater stability of soil structure at these smaller scales. These small units then aggregate further to create larger structures, with an associated hierarchy of scale and structural stability. Since the units are non-uniform, their packing creates a porous matrix, and since the constituents carry such a wide size range, the porous network is heterogeneous across a concomitantly wide range of scales. It is the pore network that comprises the physical, and primary, habitat for all soil organisms, representing a form of ‘inner space’ in which the entirety of belowground life inhabits and functions. The nature of the pore network imparts a structural

Page 7 organisation to soil communities, and strongly influences the way they function and interact (Young & Ritz 2005). Six et al. (1998; 2002) have proposed the “aggregate dynamic model” that directly links the cycle of aggregate formation, breakdown and reformation to the turnover of soil organic matter to the activity and interaction of the soil biota, without which aggregate, and therefore structural, stability degrades and collapses. Organic inputs are also critical in this respect (Abiven et al. 2009). How microbes affect soil structure Whilst soil structure strongly affects the distribution and functioning of microbes and microbial communities, the microbiota also play important roles in soil structural dynamics (Brussaard & Kooistra 1993). Microbes create soil structure by a number of direct and indirect processes, including: (i) moving and aligning primary particles along cell or hyphal surfaces; (ii) adhering particles together by the action of adhesives involved in colony cohesion, and other exudates, such as extra-cellular polysaccharides (EPS); (iii) enmeshment and binding of aggregates by fungal hyphae and actinomycete filaments, and associated mycelia; (iv) coating pore walls with hydrophobic compounds, particularly by fungi which produce such polymers to insulate their mycelia, which have a relatively large surface area:volume ratio. These basic processes also operate to stabilise soil structure, noting that they all require the provision of energy to be manifest, and are linked to soil, vegetation and management type (e.g. Miller & Jastrow 2000; Bronick & Lal 2005). This explains why there is a relationship between SOM and what is perceived as ‘good’ soil structure. Soil structure is also destroyed by the action of microbes, since much of the organic material which serves to bind soil particles together is also potentially energy-containing substrate which microbes will decompose if they can gain access to it. This is the reason why frequent soil disturbance, such as where repeated tillage is applied to soils, typically leads to a degradation of soil structure and a loss of soil C (Conant et al. 2007). Undisturbed soils have a high proportion of physically-protected organic matter in them. When these are disturbed, such stabilising material becomes available to microbial assimilation, is decomposed, and a proportion of the C is lost as respired CO2. The role of “ecosystem engineers” There is ample evidence for the role of invertebrates in generating and stabilising soil structure (e.g. Lavelle et al. 1997; Folgarait 1998; Meysman et al. 2006; Jouquet et al. 2006). Earthworms are often cited as the principal agents of this in European temperate systems (Brown et al. 2000; Uvarov 2009), but other groups are just as important in other areas, such as millipedes in North America (Snyder et al. 2009). The actions of these organisms generate soil structure by processes involving comminution, incorporation of OM, burrowing, compacting and transporting of soil, often on relative large size scales, such that they termed ‘ecosystem engineers’. Management of such groups by simple interventions, such as the re-introduction of a single earthworm species are unlikely to be successful in achieving favourable soil structural and functional outcomes for production, as these are heavily dependant on earthworm community structure (Uvarov 2009). How soil structure affects microbial function The exceptional heterogeneity of the soil pore network imparts some significant properties to the soil system from the perspective of the microbiota. Firstly, it provides a huge surface area for potential colonisation. Many soil bacteria are adapted to adhere to surfaces, and occur as colonies on the surface of pore walls. Filamentous fungi are also well-adapted to grow though soil pore networks by the process of hyphal elongation and branching, but also require some direct contact with substrates in order to absorb water, and nutrients released by the action of the extra-cellular enzymes they produce (Ritz 2005). It is hypothesised that the extreme structural heterogeneity of soils is one basis for the exceptional diversity found in the soil microbiota, since it provides an extreme range of spatially isolated micro-habitats

Page 8 which can lead to adaptive radiation (Zhou et al. 2002). Secondly, it governs the distribution and availability of nutrient resources to the biota. Unlike in aquatic systems, where most constituents are relatively well mixed by virtue of existing in a fluid matrix, the physical structure of soils essentially renders nutrient resources into spatially distinct patches, many of which will then be physically protected from being accessed by organisms. This can occur as a result of coating with soil minerals, or by being located in soil pores smaller than the physical size of potential consumer organisms. Such physical protection mechanisms can equally apply to dead organic matter or potential prey in the case of active predators such as protozoa. The connectivity and tortuosity of the pore network governs the movement of gases, liquids and associated solutes, as well as particulates and organisms, through the matrix (Crawford et al. 1993). Sessile organisms such as attached bacterial colonies rely upon the delivery of substrates to the colony, usually in water phases, as well as oxygen for aerobic respiration, via such pathways. The foraging distances taken by motile organisms when searching for substrate or prey - and hence the amount of energy consumed – will also be affected by path-lengths for movement, which are related to these properties. There is a strong interaction between the pore network, water and microbial activity, fundamentally linked to the relationships between the matric potential of a soil and the associated distribution of water between differently sized pores. Bacteria and protozoa require water films to move in, and their passage will be curtailed where there is no continuity in such features. Fungi are less constrained since hyphae can grow extended distances through air-filled pores. Since oxygen diffuses some four orders-of-magnitude more slowly in water that air, water- filled pores – or narrow necks to larger pores - effectively act as valves preventing the passage of oxygen and hence its availability to organisms for aerobic respiration. In these circumstances, many so-called facultative microbes can switch their metabolism to alternative biochemical pathways involving anaerobic processes. These can include denitrification and methanogenesis.

Biotic regulation In natural systems, the size of populations of organisms tends to be constrained by a combination of resource availability and interactions within the trophic webs whereby no single group predominates because it is regulated by other organisms competing for similar resources, or by predation. There is a fundamental relationship between biodiversity and the propensity for pest or disease propagation, since more diverse systems contain a greater proportion of such potential regulatory mechanisms. In agricultural systems, where monocultures are often adopted, the superabundance of a particular plant form even to the extent of a clone is conducive to the development of pest or disease epidemics. Industrial agriculture substitutes biotic control mechanisms with pest- or disease-specific synthetic biocides. Soil-borne pests and pathogens can be particularly difficult to control by such means since biocides added to soil tend to be absorbed or dispersed in the soil matrix and do not make contact with all targets, or can be degraded by microbes resistant to such compounds which essentially utilise them as a C-source. Broad-spectrum biocides, which partially sterilise the soil, can be pernicious insofar as they can have a major impact on non-target groups and can impair many soil functions. Where a crop is grown for an extended period, and disease levels initially increase, agents which attack the pathogen can subsequently proliferate and biological control mechanisms start to operate. An example of this phenomenon is in relation to take-all decline, where the fungal root pathogen Gauemannomyces graminis is attenuated after several years of continuous wheat growth e.g (Andrade et al. 1994). The controlling agents in this case are not readily identified, although pseudomonad bacteria have been implicated (Sanguin et al. 2009). This is a classical predator:prey cycle phenomenon, which is a particular form of trophic interaction, of which a further consequence is the liberation of nutrients when predators consume prey and excrete materials to maintain homeostasis. It is not necessarily the case that the same control agents operate in all circumstances, nor that the control is effected by

Page 9 a single organism; there is some evidence that community-level controls operate to attenuate disease propagation (Toyota et al. 1996). Insect feeding on plants stimulates the production of secondary compounds that provide protection from further herbivory. Root feeding insects generally induce strong responses by the plant in both the shoots and the roots, in contrast to foliar feeding which tends to only induce effects in the shoots (Kaplan et al. 2008). Inter-organism signalling is important in biotic interactions in the soil (e.g. Bonkowski et al. 2009) and is implicated in regulation of some pests of crops. The interactions between nematodes and their hosts has been one focus of investigation in recent years, both where the nematodes are the pest species or where they act as a biological control agent. Potato cyst nematode Globodera pallida is an important pest of potatoes in the UK and there are a number of emerging biotic mechanisms to limit their impact on crops. The wild Solanum species Solanum sisymbriifolium has shown considerable promise in regulating the nematode. The roots of the plant are invaded by the nematode, but the infective juveniles fail to mature (Blackshaw & Kerry 2008). Interactions between rhizosphere and endophytic microorganisms and potato cyst nematode are also thought to regulate infection (Kerry 2000). Williamson & Gleason (2003) have shown that root feeding nematodes locate host roots by chemical gradients, however root border cells can misdirect the nematodes until the vulnerable root tip has outgrown the pest (Rodger et al. 2003). Root-feeding nematodes may secrete an array of signal molecules and other compounds which are specifically targeted to downregulate host defence responses (McKenzie Bird 2004). Root feeding insects are important pests of many crops and are responsible for reduction in crop yield and sustainability For example, the citrus root weevil Diaprepes abbreviatus is an important pest of citrus in Florida but nematodes antagonistic to insects (i.e. entomopathogenic types) can be a useful means of controlling this pest (Goldson SL & Gerard PJ 2008). Johnson et al. (2004) demonstrated that the larvae of the weevil Sitona lepidus are clover-specific pests and use CO2 gradients to locate roots generally, but then use the presence of the compound formononetin (an isoflavenoid) to distinguish their host. Work is ongoing to develop of biological control methods such as entomopathogenic fungi (Goldson & Gerard 2008) or insect parasites (Goldson et al. 2005) to control this pest. There are also reported instances of tri-trophic interactions, for example Rasmann et al. (2005) showed that maize roots, when attacked by the larvae of the root feeding beetle Diabrotica virgifera virgifera, emit volatile compounds that attract entomopathogenic nematodes that effectively kill the larvae and so indirectly protect the plant. A fundamental assumption of biological control is that damaged plants are less ‘fit’ and compete poorly. However, Centaurea maculosa, an invasive plant in North America, exudes greater quantities of an allelopathic chemical known to affect native plants, when attacked by the larvae of two species of root boring biocontrol insects (Thelen et al. 2005). In addition to plant-pest or pathogen interactions some plants are able to manipulate the soil community to provide services to the plant. Horiuchi et al. (2005) reported that the nematode Caenorhabditis elegans transfers the rhizobium species Sinorhizobium meliloti to the roots of the legume Medicago truncatula in response to plant-released volatiles that attract the nematode. The study of natural plant defences has a practical application and it appears timely to consider the study of the rhizosphere ecology as a multidisciplinary task to improve plant breeding efforts (Bonkowski et al. 2009). Mutualism Mutualism is a specific form of symbiosis where both partners derive benefit in evolutionary terms, improves their fitness from the association. In agricultural systems, there are two principal mutualisms – both between plants and microbes - that are of significance: N-fixing symbioses

Page 10 Certain bacteria and algae are able to fix N2, some in a free-living state and others in symbiotic associations of varying intimacy with other organisms, particularly green plants (Sprent 1987). In a UK agricultural context, the mutualistic association between legumes and some bacteria including members of the orders Rhizobiales and Burkholderiales are of particular significance since substantial quantities of N can be fixed and effective yields of protein-rich grain or herbage obtained without additional N fertilisation of crop or soil. These symbioses are generally so effective due to the highly intimate relationship between plant host and bacterial symbiont where the latter infects the hosts’ roots and there is an endogenous transfer of fixed N into the plant tissues. Other plant:microbe systems of pertinence to the UK context that fix N include so-called actinorhizal associations between actinomycete Frankia spp. and woody plants such as alder (Lechevalier & Lechevalier 1979). Cyanobacteria and algae that colonise soil surfaces where there is adequate light to support them also fix N (Stewart 1969), but the quantities are relatively small and would not sustain a high level of production. Nitrogen-fixing organisms are particularly important in grassland systems where considerable effort is being focussed on exploiting the potential of white clover in animal production systems which can meet the financial and environmental requirements that are likely to prevail within the UK and the rest of Europe. In the UK by far the most important forage legume is white clover. It is included in 75% of grassland seed mixtures. However, although the presence of white clover in swards is desirable, both for its N-fixing capability (Newbould et al. 1982) and its enhanced feeding value for livestock (Bax & Schills 1993), surveys have shown that white clover makes a significant contribution to the botanical composition in, at most, only 20% of UK swards (e.g. Forbes et al. 1980; Hopkins et al. 1985; Hopkins et al. 1988). The failure of clover to thrive in pasture systems is, in part, due to the impact of pest and diseases on the seedling crop (Clements et al. 1990), as well as being suppressed due to high inorganic N fertilisation rates. Optimisation of N-fixing symbioses to maximise the quantities of nitrogen fixed by legume crops as been a long-term goal in agricultural research for many decades, and logically should remain a priority (Herridge et al. 2008). Proposed strategies (e.g. Sessitsch et al. 2002; Hardarson & Atkins 2003; Stacey et al. 2006) include: (i) A more precise matching of symbiont and host, since there is wide variation both in the specificity of such associations and their efficiency i.e. amounts of N fixed. From the perspective of the plant this can be achieved by selective breeding. In relation to the symbiont, it may be achievable via the identification of more infective i.e. extent to which hosts are colonised and effective i.e. amounts of N fixed strains of bacteria, which also show greater survival in soils when in free-living modes. Genetic engineering of such microbes clearly offers potential in this regard, but needs to be founded on a fundamental understanding of the molecular and ecological mechanisms involved in these traits. (ii) (ii) Optimisation of inoculation technologies to introduce preferred symbionts to crop roots, whereby the longevity of the bacteria is increased, the delivery systems are more effective. (iii) (iii) Application of appropriate agronomic practices to avoid unfavourable climatic or edaphic factors. Strategies to introduce N-fixing capabilities into non-legumes, particularly cereals, have also long been vaunted, but progress has to date been restricted (e.g. Ladha & Reddy 2003; Cocking et al. 2005)). There is likely more potential in the short term to develop more efficient associative N-fixing systems than any deeply integrated symbioses, notwithstanding advances in genomics and genetic engineering. Mycorrhizae The majority of vascular plants form associations with mycorrhizal fungi in natural systems, and there are several basic forms of mycorrhizal association which predominate exclusively

Page 11 amongst woody species, ericaceous species and orchids (Smith & Read 1997). Arbuscular mycorrhizae (AM) are the form that predominate in herbs and grasses, and hence the roots and soils of agricultural crop plants. The most recognised beneficial effect of this symbiosis with respect to the plant is in relation to the acquisition and uptake of nutrients that are typically immobile in soil, particularly P, and several micronutrients (Jeffries et al. 2003). AM can also enhance N uptake where ammonium is the predominant ionic form of nitrogen, and hence diffusive supply to roots can be restricted, or where soil moisture status is low such that the transport of nitrate is curtailed (Javaid 2009). Mycorrhizal plants may also be less prone to single or multiple stresses such as drought, pests, diseases and toxins (Smith et al. 2010). AM modulate the interactions between plants within communities (van der Heijden et al. 1998; Smith et al. 1999) including by affecting the competitive ability of their hosts for nutrients, and there is some evidence that plant root systems may be directly connected to each other via common mycorrhizal networks (Heap & Newman 1980; Finlay & Read 1986). This is hypothesised to allow the direct transfer of nutrients between plants, although for AM-based systems, most studies suggest that the quantities involved are insufficient to have any major impact upon plant growth or nutrition (Ritz 2006). As well as strongly influencing plant growth, nutrition and community interactions, AM affect soil structural dynamics by mycelial enmeshment of soil particles and through the production of a hydrophobic glycoprotein called glomalin (Rillig & Mummey 2006; Treseder & Turner 2007). In terms of plant nutrition, mycorrhizae have little role to play in industrial agriculture since inorganic fertilisation suppresses them and any selection pressure for their action in nutrient acquisition. Tillage practices also compromise them due to the associated disruption of their mycelia, and application of biocides and growth of non-mycorrhizal crops principally Cruciferae are detrimental to them (Gosling et al. 2006). However, in systems relying on nutrient provision from organic sources, they likely have a stronger role to play, and the potential application of mycorrhizae in temperate and tropical systems is extensively rehearsed (e.g. Hart & Trevors 2005; Cardoso & Kuyper 2006; Gosling et al. 2006; Smith et al. 2010). There is a very large body of literature considering the potential importance of mycorrhizae in non-industrial agriculture spanning many decades, and persistent discussion about their likely utility. However, whilst there are number of commercial mycorrhizal inocula systems, little of this promise has to date – and certainly at the larger field or arable scale – been realised. Hart & Trevors (2005) consider that this is due to the inherent complexity of the symbiosis and mycorrhizal functioning. As Piotrowski & Rillig (2008) point out, there is apparently far more selectivity of AM community association with host plants, and dependence upon particular host:fungus combinations with respect to the nature of the mutualism than hitherto thought. They suggest that the goal should be to manage mycorrhizal systems at a community scale, and a more ecologically-focused environmental match of AM species and ecotypes with local conditions (Piotrowski & Rillig 2008). Mycorrhizae interact with other soil organisms, particularly within the immediate zone of their hyphae, termed the mycorhizosphere. This has implications for the optimal exploitation of mycorrhizae in agricultural systems. There have been many studies into bacteria associated with mycorrhizae but precise mechanisms of interaction remain poorly understood (Johansson et al. 2004). Jordan et al. (2000) consider that mycorrhizae could play a significant role in weed management. They posit that weed:mycorrhizal interactions may reduce crop yield losses to weeds, limit weed species shifts and increase positive effects of weeds on soil quality and beneficial organisms (Jordan et al. 2000). Other biotic associations The rhizosphere The zone in the immediate proximity of plant roots, termed the rhizosphere (Lynch 1990; Cardon & Whitbeck 2007; Jones & Hinsinger 2008; Raaijmakers et al. 2009), generally contains a greater concentration of organisms than in the bulk soil. This is because of the deposition of carbon and nutrients emanating from the roots as direct exudation and as a result of root cell sloughing. Microbes therefore colonise the rhizosphere rapidly and higher- order trophic levels soon develop subsequently. As such trophic interactions develop, nutrient cycling in general can be accelerated, including the mineralisation of N as result of

Page 12 the consumption of microbes by predators and grazers. The phenomenon, termed the ‘rhizosphere priming effect’ (Clarholm 1994) has been demonstrated experimentally to result in significant increases in plant growth and nutrient content e.g. (Clarholm 1985; Kuikman et al. 1991). Bonkowski et al. (2009) point out that soil fauna also interact beneficially with plant roots to a greater extent than is generally considered, and stress that soil food webs are not necessarily principally driven by above-ground inputs. Hence many such biotic rhizosphere interactions operate in a general sense, and there may be opportunities to manipulate them by affecting root exudation properties of crop plants by selective breeding or genetic manipulation. This is in addition to selecting or engineering more specific root exudation traits focussed on unitary compounds such as signalling agents, discussed elsewhere. Plant growth promoting rhizobacteria Associative rhizosphere microbes interact with plants in a variety of modes ranging from neutral, through beneficial to antagonistic with respect to the growth or health of the plant. These relationships are not deemed symbioses as such, since it is unclear as to the extent to which both partners ‘benefit’ directly from such relationships in a manner which is rather clearer than for true symbiotic associations discussed above. There is a very large literature that considers the potential plant growth-promoting characteristics of such microbes, particularly the so-called plant growth-promoting rhizobacteria (PGPR), with decades of research seeking to understand the mechanisms behind such effects, and optimise their application to stimulate plant growth (e.g. reviews by Dobbelaere et al. 2003; Lucy et al. 2004; Zahir et al. 2004; Avis et al. 2008; Ryan et al. 2008; Hardoim et al. 2008; Spaepen et al. 2009). PGPR effects can be direct or indirect and modes of action include: (i) Via production of plant growth-promoting substances phytohormones. The production of such compounds, by bacteria including auxins, cytokinins, gibberellins, ethylene and abscisic acid has been reported for many species, but direct evidence that such production in the soil situation affects plant growth is actually scarce (Spaepen et al. 2009). This is partly due to the difficulty if discriminating between such compounds derived from the plant as opposed to an associated microbe. Amongst the strongest evidence is for auxin-induced effects upon root morphology by Azosprillum brasilense (Dobbelaere et al. 1999). (ii) Via biological N fixation. A variety free-living N-fixing bacteria, may proliferate in the rhizosphere or even endophytically within roots (Elmerich & Newton 2007), and the N that they fix can be transferred to the associated plants. These so-called associative diazotrophic relations are deemed more significant where plants such as sugar cane and maize are involved, possibly due to the higher rates of carbon delivery to the rhizosphere in these species. However, such transfers are less efficient than in the case of legumes and generally quantities of N transferred in the field are low, typically of the order 10 kg N ha y for grain and forage grass crops (Okon 1985). There has been much research into such associative fixation, but despite hundreds of studies, most have not shown a substantive and more significantly consistent contribution to increasing plant growth (Dobbelaere et al. 2003). Based on the evaluations of over 20 years of field applications of Azospirillum brasilense and A. lipoferum across a range of countries, (Okon & Labanderagonzalez 1994) concluded that application of Azospirillum can increase crop growth and yield by 5 to 30% depending on soil and climate conditions. A UK- based study also did not demonstrate significant effects of inoculation of cereals with Azorhizobium spp. (Cooper 1999). Whilst such pathways of N fixation may of ecological significance in terms of importing N into the ecosystem, they cannot apparently support enhanced or sustained production since the amounts involved are too low (Giller & Merckx 2003). Exploitation of this relationship may then be restricted to lower-productivity systems. (iii) Via increased solubilisation of phosphate. A large proportion of soil-inhabiting bacteria appear to be capable of solubilising P, both from inorganic forms by producing organic acids, and from organic forms via phosphatases, at least under laboratory conditions (Gyaneshwar et al. 2002). However, an unequivocal

Page 13 demonstration of the role of phosphate solubilization in bacteria:plant associations is still not evident (Jones et al. 2004; Spaepen et al. 2009). There are many studies which attempt to enhance P supply to crops by inoculating soils with phosphate- solubilising bacteria PSB. However, results in the field are typically very variable, possibly related to survival and colonization of inoculated P-solubilising microorganisms in the rhizosphere, and their competition with native microbes; the variety of contexts presented by the nature and properties of soils, plants and cultivars; and insufficient resources in the rhizosphere to engender adequate organic acid production (Kucey et al. 1989). (iv) Via mobilisation of iron. Although iron is typically abundant in most soils, it is generally unavailable for biotic assimilation as it occurs mainly as Fe3+ oxides with a low solubility. Bacteria have developed a strategy for efficient uptake of iron by producing and secreting low-molecular-weight iron-chelating molecules, known as siderophores (Bossier et al. 1988; Raaijmakers et al. 1995) Upon binding of iron to these molecules, they are transported back into the cells, readily available for microbial metabolism. There is some suggestion that bacterial siderophores may be taken up by plants and serve as an iron source, but direct evidence for this is sparse (Marschner & Romheld 1994). (v) Via production of phytoactive volatile organic compounds (VOCs). A number of studies have demonstrated stimulatory effects of VOCs upon plant growth, at least in vitro (e.g. Ryu et al. 2003; Cho et al. 2008). There is thus some suggestion that PGPR could be effective in promoting plant development via such mechanisms. However, as for other similar concepts, there are issues of whether such effects are direct, and if they could be sufficiently consistent to be an effective strategy. (vi) Via suppression of plant pathogens. PGPR can act directly to control disease by acting as biological control agents, but such bacteria may also confer indirect benefits via the mechanisms described above, which can be manifest in the absence of any pathogenic agents (Avis et al. 2008). (vii) Via a reduction of plant stress. A more general concept vaunted by Dimkpa et al. (2009) suggests that PGPR can increase plant resistance to both abiotic and biotic stress factors, and that systematic identification of bacterial strains providing cross- protection against multiple stressors would be an appropriate strategy. A multiple- component approach to PGPR has merit in that it avoids the focus on single actions, which are demonstrably variable in their outcome. There has been a recent trend to term rhizosphere colonising bacteria antagonistic to pest and pathogens as 'plant probiotics' (Picard & Bosco 2008), as a concept synonymous with probiotics in human and animal systems, where the term refers to live microorganisms, which when administered in adequate amounts, confer a health benefit to the host. Spaepen et al. (2009) regard this as potentially useful since both these plant and animal contexts have common basic scientific questions microbiological community structure and dynamics, identification of the probiotic factors and host responses, and there could be useful synergy between these lines of research. Allelopathy Allelopathy is defined as "any process involving secondary metabolites produced by plants, algae, bacteria and fungi that influences the growth and development of agriculture and biological systems” (Roger et al. 2006). On this basis, some of the mechanisms described above fall into this purview, but there are classes of other interactions that have significance and potential in relation to manipulation of the soil biota (Anaya 1999; Mallik & Williams 2009). These include the use of rhizobacteria in weed control, and the use of plant-derived allelochemicals to modulate the activity of specific soil organisms. Anaya (1999) points out that the majority of work in relation to allelopathic interactions between plants and microorganisms has concentrated on symbiotic N fixers and mycorrhizal associations. There is likely scope in expanding such approaches to other components of the soil biota. The role of VOCs in biotic interactions in the soil, discussed in relation to some faunal interactions and PGPR above, has received relatively little attention to date and warrants

Page 14 further consideration. Soil microbes can produce a wide variety VOCs, and a significant proportion of such compounds released from soils and litters appear to be derived from microbes (Stahl & Parkin 1996; Isidorov & Jdanova 2002). The production of VOCs by soil microorganisms may have important influences on soil processes and the interactions between organisms. Studies have shown that the presence of specific VOCs in the soil atmosphere can alter the rates of microbial processes such as nitrification (Wheatley et al. 1996; Paavolainen et al. 1998), nitrogen mineralization (Smolander et al. 2006), denitrification, and methane oxidation (Amaral et al. 1998). VOCs may also regulate microbial interactions by inhibiting or stimulating the growth and activity of soil fungi and bacteria (Mackie & Wheatley 1999; Xu et al. 2004). The range of VOCs emanating from soils and litters can be very diverse (Leff & Fierer 2008) and hence there may be interactive effects of such compounds at a community scale. Since such compounds are by definition volatile, their transport through the soil may occur at rates that far exceed those manifest by solutes being transported in soil water and hence their delivery through the matrix extensive and at greater spatial scale.

3. MANIPULATING AND MANAGING THE SOIL BIOTA: SYSTEM-LEVEL CONSIDERATIONS Soils are literally and formally complex systems, which have evolved in the context of being part of a connected and coherent earth system. It follows that a more effective management will take account of this fact, particularly where a number of functions are required to be optimised in space and or time – something which occurs unaided in natural ecosystems during succession and under the guidance of evolution. In terms of the production function, it is important to recognise that agricultural systems are artificially arrested successions – we need to understand which evolutionary and ecological processes we need to replace and manage to bring them to maturity i.e. stable and efficient, and arguably a pseudo-maturity in ecological terms, whilst maintaining productivity. Role of the habitat All components of the soil biota, from individuals through populations to communities, live and function in the context of their habitat, and one of the principal ways of modulating their activity is via manipulation of this construct (Elliott & Coleman 1988; Young & Ritz 1998; Young & Ritz 2005). The soil habitat is essentially comprised of the fundamental physical framework, defined by the soil pore network; the physico-chemical properties of its constituents; the biotic context provided by the resident communities; and environmental factors such as temperature and moisture. Soil architecture Soil structure, defined by the pore network, provides the inner space in and through which all soil processes occur. Given the pivotal role that biota play in soil functions, it is appropriate to consider soil structure from the perspective of that of soil architecture, which emphasises the interaction between organisms and the physical construction of their environment. The precise definition of an optimal architecture for biotic function in its totality remains elusive, not least because of the diversity of functions, but must be related to an appropriately wide range of pore sizes, associated with the connectivity between such pores and their tortuosity; all of these factors serve to modulate processes by governing the transport of gases, liquids, solutes and organisms, access to resources and something else. Quantification of these features in intact systems and three or four dimensions is now feasible using X-ray tomography (Young et al. 2008; Taina et al. 2008; Lombi & Susini 2009), and hence the potential definition of appropriate soil architecture becomes feasible with due study. Soil architecture can be practically managed at a field scale via combinations of appropriate use of crops, application of organic matter, controlled tillage and drainage, based upon knowledge of the mechanisms by which structure is created and destroyed. This relies upon a system-level consideration and management of what affects structure. If such inputs and practices are appropriate, then biological mechanisms of soil structural dynamics may

Page 15 serve to maintain an appropriate habitat for optimised biotic function. This is based upon an emerging concept that when configured appropriately, soils are essentially self-organising systems (Young & Crawford 2004). This has major implications for effective management of the soil biota, since if correct then approaches based upon habitat-manipulation as opposed to direct intervention of biota will be much more effective. Whether resultant functions are then optimal for production is unclear. Physico-chemical environment The soil biota is strongly influenced and regulated by the physico-chemical and environmental properties of its habitat. A primary physico-chemical factor governing soil community structure appears to be pH (Fierer & Jackson 2006), and certainly individual organisms have differing pH optima (Killham 1994). This is likely a consequence of different soils each having a characteristic pH, and communities must necessarily be adapted to such circumstances. Cropping soils tends to lower pH, and in management terms this can be raised, at a field scale, by the addition of carbonates such as lime, which will likely result in community-scale re-adaptation to the prevailing pH. All life-forms have temperature optima, below and above which their function is modulated, and at extremes curtailed. As such, management practices which affect temperature, such as covering the soil surface with mulches or membranes can affect biotic activity. Solarisation, which involves elevating the temperature of the top layers of soil by covering the surface with plastic mulches can partially sterilise the soil and thus be used to control pests and diseases (Bonanomi et al. 2008) Thermally treating the soil with tractor-drawn heating devices can also be used for pest and weed control e.g. (Lague et al. 1997; Wszelaki et al. 2007). However, these approaches are non-selective and can have effects upon non-target organisms (Roux- Michollet et al. 2008). Soil moisture optima for the belowground biota vary between organism types. Bacteria, protozoa, fungal and oomycete zoospores, and nematodes rely upon moisture films for their transport through the soil matrix whilst filamentous fungi and legged fauna are not thus constrained. The distribution of water in the soil matrix also modulates the passage of gases through the soil, and as discussed above, water-occluded pores can result in the formation of anaerobic zones in the soil and the activation of anaerobic metabolism in certain soil microbes. Management of soil moisture status can thus be used to regulate biotic activity, notwithstanding it must also be reconciled with plant water requirements. Biotic context Soil organisms always exist in the presence of other populations and the diverse members of the biomass, and have always done so. Hence they have evolved in the context of communities, and a panoply of interactions emerge and evolve such that the prevailing community structure within soils has a strong impact upon the growth and function of individual organisms. This biotic context serves to regulate and affect the actions of individual members of the community, such that there are community-level controls upon organisms and processes (e.g. Janzen et al. 1995; Janzen & McGill 1995; De Nobili et al. 2001; Wheatley et al. 2001). This has important implications for the selection of putatively beneficial organisms for use in inoculation strategies, since if such selection occurs outwith such a context, then organisms are unlikely to be fit when introduced into extant and diverse communities in soils. Environmental factors The principal environmental factors which affect biological activity in soils are temperature and moisture and hence management of the biota via manipulation of these factors is tenable. Soil temperature can be altered by application of surface-applied mulches, which can serve to insulate the surface from excessive heat or cold (Horton et al. 1996). Plastic sheets can be overlain on the soil surface to increase temperature and hence partially sterilise the soil, albeit to a relatively small depth, which can be an effective means to control soil-borne diseases (Denner et al. 2000; Gallo et al. 2007). The albedo of the soil surface affects the absorption of solar energy, and the nature of crop residues and mulches can also affect this property (Horton et al. 1996). The addition of biochar to soils may have particular effects in the respect and appears to have be rarely considered in the research literature to date. Soil moisture is related to soil structure, and thus this links to the more

Page 16 general architecturally-based concepts above. Where water input to soils can be controlled via irrigation systems, there is some potential to manipulate the biota by this means. An example is the potential control of common scab of potatoes by optimising soil water content (Wilson et al. 2001). Flooding has been proposed as potential means to control nematode populations (Sotomayor et al. 1999; Nelson et al. 2002), but results have not always been successful (Asjes et al. 1996). Energy – the role of substrate Conceiving of the soil biota as the biological engine of the earth is a fair analogy in the sense that the biota carry out a myriad of processes which underpin soil function, all of which require the expenditure of energy, fundamentally via biochemical pathways. This is a form of work, in the literal physics-based sense in relation to soil structural dynamics, but in a broader sense too. It then follows that a primary means of controlling the soil biota in general is via the regulation of the amount and form of energy available to the biomass. As explained above, the principal - but not sole -source of such energy for the soil biota is in the form of fixed carbon and hence the management of the inputs of such fixed C to the soil is a key factor. Such inputs can be managed via the nature of the crop grown, residue management, and via the import of other organic material such as manure, slurry, compost or other waste streams. The availability of the energy contained in such substrates is pertinent. Readily-available forms will result in the rapid assimilation of such materials, an increase in biomass and metabolic rate, with accelerated carbon and associated nutrient cycling. Thus is conducive to a greater plant production rate since there will generally be greater pools of available nutrients, so long as these are underwritten by sources. This is akin to the early successional stage of an ecological system, and can be associated with greater losses i.e. more ‘leaky’. Such responses are underpinned by so-called r-strategists, organisms that are adapted to rapid growth and short life cycles. Biomass that develops under such conditions can only prevail if there is sufficient energy available to sustain it – if this is not the case, it will die, with an associated short-term release of some of the nutrients therein. Access to the energy bound into more recalcitrant materials requires a higher biochemical overhead and is released more slowly by organisms adapted to such modes, termed K-strategists. In fact, there is a continuum of lifestyle strategy amongst organisms along the r-K spectrum, which approximately relates to ecosystem succession from pioneer to climax ecosystems. A critical point here is that production-oriented systems are necessarily located at the r-end of this spectrum, and may be ecologically naïve to expect high production concomitant with inherently efficient and conservative systems. Organic matter, whether derived from the crop or other exogenous sources manures, composts, etc. represents an important source of energy for the soil biota. The role of OM in agricultural production has long been appreciated and its importance should not be underestimated in the future (Magdoff & Weil 2004). The potential utility of organic materials from industrial sources for soil management is well recognised (e.g. Shiralipour et al. 1992; Gajalakshmi & Abbasi 2008; Hargreaves et al. 2008; Farrell & Jones 2009), and the interactions between such materials and the soil biota needs to better understood to optimise their use. As Kibblewhite et al. (2008) discuss, an energy-based consideration of potential ways to manage energy flow in soil systems potentially offers much insight into a mechanistic basis for understanding how to rationalise the delivery of soil functions. An important principle arising from such considerations is that there will only be a finite amount of energy available within the soil system which can be manifest as ‘work’ i.e. the delivery of the range of goods and services, and therefore there needs to be a decision as to where this should be delivered, in any one context. Another perspective is that it may enable the prescription of how to optimise the configuration of the system, both in space and time, to improve efficiency of delivery to such functions. The role of the crop It is quite clear that the crop has a particularly significant role to play in managing the soil biota. The choice of crop type and, more precisely, the genotype, will affect the nature of C inputs to the soil system both from the perspective of gross quantities of substrate that are input to the soil system, and more subtle effects based upon particular allelochemicals. This

Page 17 considers the issue from the perspective of the individual crop, but there are also important potential interactions with respect to crop rotations, which essentially operate on the principal of ensuring heterogeneity in substrate input to the soil over time. Rotations have a long history in agriculture, based upon experiential evidence that likely has a sound ecological basis in actually managing soil biota via the contrasting nature of different crops. Much is known about the principal mechanisms responsible for these benefits, including effects on disease control, improved nitrogen nutrition and water supply, although researchers continue to be challenged by inexplicable "rotation effects" that have yet to be documented or fully understood (Kirkegaard et al. 2008). Most contemporary plant breeding programmes tend to operate in the context of the industrial-end of the production spectrum, and it is not necessarily the case that such cultivars will perform in contrasting production systems. To this end, alternative breeding programmes are being developed, for example within the context of organic farming systems (e.g. Wolfe et al. 2008). Such concepts are arguably critical if biotic interactions are to be exploited effectively. Rengel (2002) considers plant breeding related factors which could lead to more efficient symbioses. These stress the need for a deeper understanding of infection process by rhizobia and mycorrhizae, and the apparently crucial role of flavenoids as signalling molecules. Ryan et al. (2009) discuss potential strategies for engineering the rhizosphere to enhance plant growth, including those based upon pH manipulation, suppression of synthesis of stress-inducing hormones and stimulation of probiotic bacteria. Other examples of approaches being considered are in relation to the generation of new cultivars that could enhance the agronomic potential of AM associations (Sawers et al. 2008) or in relation to plant allelochemicals (Bertholdsson 2010). However, it is notable that these considerations are largely founded upon single traits within plants, with an emphasis on point-interventions in the system as opposed to a system-scale perspective. The broad concept of the crop ideotype, i.e. a model of a plant community where all necessary and beneficial traits for crop performance in a particular environment are combined (Donald 1968; Makela et al. 2008), is pertinent here and should be developed with due consideration of the soil biota in the environmental construct.

4. THE WIDER CONTEXT It is clear that to achieve sustainable production based on establishing and maintaining “mature” agro ecosystems, we need to consider the lessons to be found across the whole range of terrestrial ecosystem research, and not just that focussed on agricultural production, a view receiving wider attention and calls for action (e.g. Jackson et al. 2007; Mulder & Lotz 2009). It is unlikely that interventions at one trophic level alone will bring about the desired increases to maximize production without consequences for other ecosystem services, we need to couple above and below ground system components (Van der Putten et al. 2009). In essence we need to reconcile the production function of soils with other ecosystem goods and services (Barrios 2007), by increasing biodiversity and ecosystem efficiency in a production context (Birkhofer et al. 2008). Although organic farming approaches are claimed to increase biodiversity, the benefits are unclear (Hole et al. 2005), and the changes in biodiversity are measured at the whole farm scale e.g. birds and not targeted specifically at enhanced biodiversity to increase productivity by increasing ecosystem efficiency. Some approaches to agriculture are, however, aimed specifically at enhancing biodiversity to improve ecosystem efficiency and greater production without compromising other ecosystem services. These approaches have local indigenous when referring to aboriginal peoples knowledge at their core (Handayani & Prawito 2010). Amongst these approaches permaculture (a term derived from “permanent culture”) is one of the most widely adopted. It is interesting to note that many of the central tenets of permaculture, current for many years, map directly onto ecosystem service and “natural capital” concepts. Permaculture has been remarkably little studied in a scientifically rigorous manner – however the system is predicated on a deep knowledge of local natural resources soil, water, biology set in a systems-thinking concept. It is perhaps easy to speculate that the more “mystical” elements of the approach in some areas are off-putting to scientists, but whatever the cause the

Page 18 outcomes appear to be beneficial, certainly with respect to delivering social capital. It is more difficult, however, to discover information as to yields, water quality and other more traditional outcomes of such an approach, because, perhaps of the difficulty in funding and prosecuting the inherently trans-disciplinary approaches required to produce a rigorous and transferable assessment. Barrios (2007) has outlined an astute and pertinent set of research opportunities and gaps linking soil biota to ecosystem function, ecosystem service provision and land productivity, including: (i) Integration of spatial variability research in soil ecology and a focus on “hot spots” of biological activity; (ii) Using a selective functional group approach to study soil biota and function; (iii) Combining new and existing methodological approaches to that link the temporal-spatial dynamics of soil organisms to the delivery of specific services; (iv) How these relationships might be directly or indirectly manipulated to enhance particular functions this will result in trade-offs of function; (v) How remote sensing of vegetation condition and composition could inform understanding of soil microbiological communities at a landscape scale; (vi) Developing land-quality monitoring systems that inform land users of ecosystem service delivery to aid policy and decision making. As Jackson et al. (2007) point out, the challenge is understanding the combined ecological and social functions of agri-biodiversity, determine its contribution to ecosystem goods and services and value for society at large, and evaluate options for the sustainable use and conservation of biodiversity across the agricultural landscape. The potential for a more holistic, ecologically astute, management approach is high, but dealing effectively with the inherent unpredictability of responses to management interventions requires an increased emphasis on the effective synthesis of complex and often apparently contradictory information and on field-based adaptive research, monitoring and social learning by farmer/researcher collaborations (Shennan 2008)

5. CONCLUSIONS Managing the soil biota It is clear that the soil biota is intimately involved in many aspects of soil functioning, and the delivery of the full range of ecosystem goods and services that soils support. However, the relative roles of biology in underpinning the production function differs between systems: in industrial agriculture it is relatively low, in integrated-style systems involved to some extent, and in organic farming and permaculture systems very high. In terms of manipulating the soil biota to enhance production there are two broad classes of intervention that operate at different scales, i.e. ‘point interventions’ that target specific, often monotonic, aspects or sub-components of the biotic assemblages or their environment, and more systems-oriented approaches that have a more holistic basis. Those approaches which target individual component organisms or restricted populations, and hence the processes that they underpin, is an approach which has been a primary focus of much research and is essentially based upon a reductionist approach. Examples of such direct intervention generally involve inoculation of organisms, for example biocontrol agents against disease, specific mutualists such as rhizobia or arbuscular mycorrhizae, or plant growth-promoting rhizobacteria PGPR. These represent a ‘point intervention’ strategy, in that only particular parts of the system are targeted, and their development – and application - tend to occur without much attention to other components of the system. Such approaches have shown some efficacy, but when taken in the round, they generally demonstrate short-term, non-persistent effects and are not always effective in all circumstances. This is because:

Page 19 (i) soil systems are inherently complex and tend to re-organise towards complexity – hence introduced organisms do not prevail at high concentrations; (ii) such organisms have often been selected for ‘desirable’ traits out of the ecological context in which they are required to operate; (iii) biotic interactions are invariably complex, since biological systems are founded upon variety and the adaptive potential that it provides, whilst these approaches are based upon the actions of single-species or even strains – this will inevitably result in inconsistency in effect, since no single organism will be optimally adapted for all circumstances; (iv) contemporary industrial agricultural systems tend to operate on the basis of monotony and uniformity, which is an approach inherently not conducive to optimisation of processes via biological systems. This is especially the case where multiple-outcomes are desired, such as the delivery of a range of goods and services. Despite this, many research papers persist in extolling the potential and virtues of such approaches, and the need to develop them. However, advances are only likely to arise if it is acknowledged that such strategies may only be effective at local scales, and that the context-dependency of such interactions are accommodated. Understanding of mechanistic bases of agriculturally beneficial symbioses and looser biological associations remains remarkably poor, despite a large body of work in this area. The potential for allelochemicals derived from plants, and optimisation by breeding to influence biotic processes is apparently strong, but the complexities involved – and consistency of responses – currently hamper their general application. System-level interventions are likely to be more successful and consistent since they accommodate the fact that soil organisms have evolved and operate in the context of an inter-connected physical, chemical and biological system, founded upon a myriad of interactions between these components. Primary amongst these are the manipulation of the two key factors which influence the functioning of the biota, viz. (i) The provision of energy-containing substrate, principally as organic matter. Such additions operate at two scales: (i) a general provision of energy to the biota, and hence a fuelling of all biotically-mediated processes; (ii) more subtle effects arising from specific compounds influencing particular processes. Organic matter inputs to the soil can be managed via the crop or introduction of exogenous materials. Crop-derived inputs, via the prescription of the species or cultivar grown are particularly amendable to management of subtle effects, notwithstanding the caveats above, and the use of on-field crop residues as a fundamental energy source. Crop rotations are founded upon a time-based variation in such inputs to the soil system and are a potentially effective way of managing biotic effects. Introduction of organic matter derived from off-field sources such as manures, composts, or other waste streams offers great potential, but can require specific management to avoid undesired side-effects such as pollutant accumulation or excessive greenhouse gas production. (ii) Optimisation of the architecture of the soil habitat. This is manifest at the scale from micrometres to the field, primarily as the soil pore network and distribution of substrate and organisms therein. It is notable that there is a connection between soil organic matter and soil structural integrity which further demonstrates both the connectedness of the components of the soil system, and that a key means to potentially manage such architecture is via organic matter in all its manifestations. Controlling tillage practices to avoid excessive disturbance of the soil, and encourage biological mechanisms of structural dynamics, is a further device. Definition of the precise ‘architectural configuration’ of any particular soil system - or indeed soil systems in general - that would reflect an optimal state is reasonable but not yet feasible, and is a key research requirement. This is essentially an issue of abstracting the key features of the highly complex soil system that relate to the capacity of the system to deliver

Page 20 prescribed goods and services. Whilst these will certainly not be solely based upon biotic properties, certain aspects of the biota may be an effective filter with which to assess the state of the system in this respect1. There is then a higher-order approach to managing the biota, which can be considered as optimisation of the larger-scale context. This is a system-level approach where the soil biota are managed at spatial scales of the field and beyond, and over timescales of years, such as is practised in permaculture-type approaches. Crucially, this is set within the context of the entire production system, or better still, the regional ecosystem, where the aggregated delivery of ecosystem goods and services are considered and optimised by an appropriate arrangement of production-oriented and other systems in the landscape, over space and time. The policy context The Soil Strategy for England was published in September 2009 (Defra 2009), and has at its heart the protection of soil resources. It is not yet clear whether these policy priorities will continue but, as this review explains, any such strategy must recognise that functional, healthy, soils are living systems founded upon the biota that reside within them. These concepts are recognised by the Soil Strategy for England within a number of the general and specific headlines. As such, life in the soil also needs explicit management and protection, across the piece. It is notable that there is more to this than an overarching need to maintain biodiversity per se, which bears little direct relation to function (e.g. Bardgett et al. 2005; Barrios 2007; Shennan 2008), rather there are likely requirements for specific communities to be fostered and maintained within the diverse range of soils across England and Wales, which provide the necessary functions required in such circumstances. Such soil health indicators should be of utility in policy terms, but the definition of such configurations - precise or general - are not yet definable and this is a gap and research need. In relation to the production function, which essentially relates directly to the aim of ‘better protection for agricultural soils’ within the Strategy, this review highlights possible means of optimising production via manipulation and management of the soil biota. However, realising the challenge of maximising the production function of soils whilst reconciling the delivery of other goods and services cannot be founded on optimising soil biology alone, it must be taken in the context of the wider system. In policy terms, a key issue is where the balance is struck between production and the provision of other goods and services. There is strong evidence that to truly optimise the role of soil biota to achieve this will require context-dependent approaches. Whilst there are general principles, which are increasingly being understood, there is no panacea. It is ecologically naïve to expect a soil to optimally provide all functions simultaneously, and hence a strategy founded upon optimal use of soils which are most suited to particular purposes is logical and outwardly sensible (Haygarth & Ritz 2009). This will potentially require offsetting some functions at the expense of others, and a sophisticated spatial management of soil systems at local, regional and preferably also at national scales. Management of systems holistically, aimed at manipulation of all system parameters – and notably not just the soil biota - have been hardly studied at all scientifically, much less with the aim of optimisation. This is possibly due to this approach appearing to be ‘fringe’ and not worthy of study, and because of the need for a genuinely coherent trans-disciplinary approach. This more holistic type of approach was founded on those principles now articulated in the “Ecosystem Approach” promoted by (Defra 2007) derived, in part, from the Millennium Ecosystem Assessment. Much will be gained by the ongoing connection of these strategies with the Soil Strategy.

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1 This is one of the tenets of the Defra project SP0534, “Assessing potential biological indicators of soil health”

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