Biodiversity of soil microbial communities in ... - Springer Link

Biodiversity and Conservation 5, 197-209 (1996)

Biodiversity of soil microbial communities in agricultural systems C.E. P A N K H U R S T , * K. O P H E L - K E L L E R , B.M. D O U B E , * and V.V.S.R. G U P T A Cooperative Research Centre for Soil and Land Management, PMB 2, Glen Osmond, South Australia, 5064, Australia

Received 25 November 1994; revised and accepted 27 January 1995 The productivity and health of agricultural systems depend greatly upon the functional processes carried out by soil microorganisms and soil microbial communities. The biodiversity of the soil microbial communities and the effect of diversity on the stability of the agricultural system, is unknown. Taxonomic approaches to estimating biodiversity of soil microbial communities are limited by difficulties in defining suitable taxonomic units and the apparent non-culturability of the majority of the microbial species present in the soil. Analysis of functional diversity may be a more meaningful approach but is also limited by the need to culture organisms. Approaches which do not rely on culturing organisms such as fatty acid analysis and 16S/18S rRNA analysis have provided an insight into the extent of genetic diversity within communities and may be useful in the analysis of community structure. Scale effects, including successional processes associated with organic matter decomposition, local effects associated with abiotic soil factors, and regional effects including the effect of agricultural management practices, on the diversity of microbial communities are considered. Their impact is important in relation to the minimum biodiversity required to maintain system function. Keywords" ecosystem function; taxonomic diversity; functional diversity; nucleic acid analyses;

agricultural practices

Introduction The activities of the soil microbiota are integral to the long-term sustainability of agricultural systems. This is because individual species and consortia of soil microorganisms have key roles in the many functional processes that support such systems. These functional processes include the acquisition and recycling of nutrients required for plant growth, the maintenance of soil structure, the degradation of agrochemicals/ pollutants and the biological control of plant and animal pests (Parkinson and Coleman, 1991; Lee and Pankhurst, 1992). However, despite the importance of these functional processes in agricultural systems, very little is known about the biodiversity of the soil microbial communities that support them. This is partly due to (i) the technical difficulties we have in sampling and quantifying the diversity of soil microorganisms, and (ii) the fact that in many intensive agricultural systems some of the functional processes carried out by soil microbial communities are overridden by the use of agrochemicals and mechanical tillage. Agronomic measures of crop production over a cropping season of several months also integrate and obscure the short-term events that are influenced by the specific *Also at CSIRO Division of Soils, PMB 2, Glen Osmond, South Australia, 5064, Australia. 0960-3115 © 1996Chapman & Hall

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activities of soil microbial populations (Anderson, 1994). It is important to understand when and under what circumstances are the functional attributes of biodiversity of soil microbial communities really important for maintaining the productivity of agricultural systems. Microbial biodiversity may be described as a measure of the range of significantly different kinds of microorganisms within a natural community or habitat (Atlas, 1984). However, it has been argued that it is diversity at the functional level rather than at the taxonomic level that is important for the long-term stability of an ecosystem (Walker, 1992). Measures of taxonomic diversity are impracticable because of the numbers of taxa involved, and are confounded by difficulties in defining suitable taxonomic units for the different kinds of microorganisms present (O'Donnell et al., 1994). Analysis of functional diversity within a community is based on differentiation of guilds of organisms with similar characteristics (Walker, 1992) and would ultimately enable definition of those species which are of paramount importance to the maintenance of the functional processes within the microbial community. In this review, we propose to focus on issues which impinge on (i) our capacity to sample, measure and quantify biodiversity at the taxonomic and functional levels within soil microbial communities, (ii) ecological aspects of biodiversity including scale effects and the effect of management practices, and (iii) the importance of soil microbial biodiversity to the stability of agricultural systems.

Approaches to measuring the biodiversity of soil microbial communities The large number of organisms present (estimates of 10~ bacteria, 1()~ actinomycetes, 10" fungi, 104 algae and 105 protozoa per gram of soil (Miller, 1990)), their high diversity (estimates of 4000 different genomes per gram of soil (Torsvik et al., 1990b)) and the problems of adequately defining species of different microorganisms, all lead to tremendous difficulties in measuring soil microbial diversity. As well, most soil microbes cannot be isolated in culture and identified (Hawksworth and Mound, 1991). The techniques of molecular biology and fatty acid analysis have increased the ease with which we can identify organisms without the requirement for isolation in pure culture and these techniques will have an impact on the measurement of diversity at the community level. Community diversity can be measured by a variety of indices which include numerical and abundance data - species richness, dominance and evenness or combined indices such as the widely-used Shannon index (Atlas, 1984: Magurran, 1988). Ideally the measurement should include not only the number of different taxonomic groups or species but also the extent to which they differ genetically. However, the lack of appropriate criteria for the definition of a species (especially for bacteria and fungi) means that diversity at the species level may not be useful in estimations of biodiversity and there is a need to define a more suitable unit to measure diversity (O'Donnell et al., 1994). TAXONOMIC APPROACH Most published studies of microbial diversity have utilized microbial isolation as the starting point. One approach has been to group organisms using a variety of morphological criteria (colony morphology, cell shape, intracellular structures) and biochemical tests

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(Gamble et al., 1977; Williams and Crawford, 1983; Gilbert et al., 1993). The advantage of this approach is that isolates which cannot be identified to genus are not excluded from diversity estimation. After grouping the organisms, diversity indices are calculated using either the Shannon index (H') or rarefaction IE(S)I, as a measure of species richness. In a comparison of these two indices, Mills and Wassel (1980) concluded that the Shannon index must be used with caution on microbial communities because there is an assumption in its calculation that all members of the community are known and identifiable. On the other hand, rarefaction is useful because studies using different sample sizes can be compared. However, neither of these indices estimates the genetic divergence between groups within the population. Functional approach

Functional diversity can be examined from a variety of perspectives. A simple approach is based on substrate richness or the number of different substrates that are utilized by a microbial community in a defined habitat. Using the BIOLOG microplate identification system (BIOLOG, Inc. Haywood, CA, USA) which tests the capacity of bacteria to utilize 95 different substrates, Garland and Mills (1991), Winding and Hendriksen (1992) and Zak et al. (1994) have been able to obtain an assessment of functional differences between soil bacterial communities from a variety of habitats. Employing principal component analysis, it was possible to differentiate individual communities based on their patterns of substrate utilization. Zak et al. (1994) have taken this analysis one step further and examined community activity with selected groups of substrates, thus providing information at another level of functional resolution. Substrate-specific analyses facilitate detection of feeding guilds within communities that are obscured in overall analyses of substrate use. It is acknowledged that the BIOLOG system has many limitations, including its sensitivity to inoculum densities, a selection of substrates biased towards simple carbohydrates and its inability to determine fungal activity. Nonetheless, this methodology has the capacity to produce a rich data set that is ideal for detecting site-specific differences in the functional diversity of micoorganisms and for evaluating the relationship between biodiversity and the expression of function in a natural ecosystem. There is clearly a need for the development of this kind of approach for other soil microorganisms (fungi, actinomycetes, protozoa). APPROACHES WHICH DO NOT RELY ON CULTURING Several key studies based on analysis of RNA sequences (Giovannini et al., 1990; Ward et al., 1990) and D N A heterogeneity (Torsvik et al., 1990a) have estimated that the proportion of unculturable microorganisms in the soil is 90-99%. DNA-based methods together with fatty acid analysis are two non-culture dependent techniques which are currently being developed to examine soil microbial diversity. D N A Methodologies

Molecular techniques have greatly expanded our ability to look at taxonomic relationships between organisms and have revised systematics to a large extent. Identification and population structure of many organisms have been determined by molecular methods but the use of DNA techniques to look at community level diversity is still in its infancy. Two

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approaches have been taken; estimations of overall genetic heterogenity of a community via DNA reassociation kinetics and the use of ribosomal DNA libraries to determine community structure. Both techniques have been made possible by recent advances in our ability to extract and purify whole soil DNA (Holben, 1994) and their appeal is that they do not require isolation of organisms, Identification o f community members. One of the limitations of our ability to measure accurately community diversity is the difficulty in identifying community members. The use of specific DNA probes, but particularly the amplification of ribosomal genes and their subsequent sequencing allows us to place virtually any organism in a phylogeny. This use of DNA technology is an adjunct to conventional methods. A novel use of DNA probes to look at community structure was proposed by Voordouw et al. (1991) in a study of sulphate-reducing bacteria from oil-field samples. Whole soil DNA was labelled and used to probe filters to which DNA from bacterial standards had been applied. This allowed rapid detection of a range of bacteria which were present in the community. Estimation oftotalgenetic diversity. A rapid method for estimating total genetic diversity of soil DNA was developed by Torsvik et al. (1990b). The method involves bulk DNA extraction from soil, denaturation and spectrophotometric measurement of reassociation kinetics. These values are measured relative to a homogeneous DNA standard and an estimate can then be made of the number of different genomes present in the environmental sample and thus its genetic heterogeneity. A comparison of this technique with estimates of phenotypic diversity using bacterial isolation and identification using the API 20B system (API System S.A., Montalieu Vercieu, France) gave a good correlation (Torsvik et al., 1990a). Ribosomal R N A libraries. The creation of ribosomal RNA (rRNA) 'libraries' relies on the cloning either directly or after amplification via polymerase chain reaction (PCR) of DNA coding for rRNA from whole soil DNA. These cloned rRNA fragments create a 'library" of the organisms present in soil and theoretically constitute an unbiased representation of the community. The cloned rRNA fragments can be sequenced and compared with an extensive database of rRNA sequence information (Olsen et al., 1991 ) or they can be more rapidly and cheaply screened via dot blot hybridization to genus or species-specific rRNA probes. Several studies have used this method to look at whole soil communities (Liesack and Stackebrandt, 1992; Stackebrandt et al., 1993). The power of the technique to reveal unculturable members of a community was shown by the fact that most of the soil libraries isolated rRNA sequences which had not been found in cultured bacteria. In a study which compared an rRNA library with conventional isolation from soil (Stackebrandt et al., 1993), genera which were commonly isolated and dominant in the soil were not represented in the rRNA sequences. This points out not only the severe limitations of our ability to culture a representative part of the community but also raises the possibility that amplification and cloning contain their own biases. A study of different methods for DNA isolation and cloning revealed method-dependent differences in the composition of cyanobacterial libraries (Ward et al., 1990). Because the relative abundance of rRNA in uncultivated species is not known, the method has limited quantitative use. As well, PCR may preferentially amplify some sequences making quantitative comparison of sequence abundance unreliable (Embley and Stackebrandt, in press).


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An alternative to the laborious and expensive task of sequencing rDNA clones is the use of denaturing gradient gel electrophoresis (DGGE). This involves separation of amplified 16S rRNA on a denaturing gradient gel followed by hybridization to specific oligonucleotide probes. This allows identification of specific RNAs as well as crude differences in rRNA profiles between soils (Muyzer et al., 1993). To date, these methods have only been used for bacterial communities but theoretically it should be possible to use eukaryotic ribosomal primers to reveal unculturable members of the broader soil community. The method is valuable in its ability to detect and provide phylogenetic information on the bulk of the members of the soil community but it is untested for its usefulness in revealing community structure. Fatty acid analysis Measurements of fatty acids in soils have been used to estimate microbial biomass and have more recently been used to examine community structure (Tunlid et al., 1989; Baath et al., 1992, Haack et al., 1994). Ester-linked fatty acids in the phospholipid component of cell membranes are a useful tool for microbial identification, primarily of prokaryotes. Some bacteria have unique 'signature' phospholipid fatty acid profiles which can be measured by gas chromatography and this is the basis of a microbial identification system developed by Sasser (MIDI-FAME, Microbial I.D. Inc., Newark, Delaware). The system has been developed largely for bacteria but has been used in soil and is sensitive, detecting 30 to 50 different phospholipid fatty acids in soils and sediments (Tunlid and White, 1992). The method has been used for community analysis either to identify taxonomic groups in soil via signature fatty acid profiles (Tunlid et al., 1989) or to detect differences between communities by comparing fatty acid profiles of the whole soil (Baath et al., 1992; Haack et al., 1994). There are a number of cautions and limitations to the use of fatty acid analysis in soils. Signatures may vary with growth temperature or medium (Parker et al., 1982), making identification of signatures potentially unreliable in environmental samples. A thorough study of the potential use of FAME to compare soil community structures (Haack et al., 1994), found that although results were reproducible and could clearly separate communities, the method did not provide taxonomic information. Widely different taxa have similar fatty acid profiles and there is taxonomic variation in fatty acid yields. So although the technique may be very useful to look at shifts in community structure in a soil subject to a particular stress, it may not provide qualitative and quantitative information about the species present, the functioning of the community nor about genetic divergence between organisms.

SAMPLING CONSIDERATIONS Any method used to estimate soil microbial diversity must ultimately address the problem associated with the tremendous spatial and temporal heterogeneity of microbial communities within soil. Factors which influence microbial communities include: the availability of substrates, seasonal and diurnal fluctuations in temperature and moisture, pH, redox potential, oxygen availability, as well as the nature of the soil parent material (Lee, 1991). These factors may vary over very small distances (mm--cm) and will contribute not only to differences in the spatial distribution of microbial communities in the soil but also to the diversity of the microorganisms within such communities (Foster, 1988). These

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considerations, and the difficulty of extracting some microorganisms from soil, because of their location within or interaction with soil particles, are major limitations to quantitative and representative sampling of microbial communities. There is thus a need to use dispersion or homogenization extraction techniques which improve quantitative extraction of microorganisms from the soil (Hopkins et al., 1991). Another constraint on quantitative and representative sampling of microbial communities is the lack of suitable selective isolation procedures. This problem is being approached using computer-assisted taxonomic methods as an aid to the formulation of selective media, and has been used successfully to isolate novel and uncommon actinomycetes from diverse habitats (Bull et al., 1992). Ecological aspects of the biodiversity of soil microbial communities Microbial diversity and its measurement can be interpreted in different ways depending on whether the perspective is ecological or taxonomic. In considering the ecological aspects of the biodiversity of soil microbial communities, scale effects, both temporal and spatial are of fundamental importance, not only to aid in the quantification of biodiversity but to assist in our understanding of how microbial communities are structured in time and space, how they respond to environmental stresses and how biodiversity is connected to function (Roper, 1993: Klug and Tiedje, 1994; O'Donnetl et al., 1994). Here we consider soil microbial diversity at three spatial scales; (i) successional processes associated with organic matter decomposition, (ii) local effects associated with edaphic factors, and (iii) regional effects including the effect of agricultural management practices. SUCCESSIONAL PROCESSES ASSOCIATED WITH ORGANIC RESIDUES IN SOIL The diversity of a soil microbial community will be determined by the nature of the substrate, and the kinds of microorganisms which by chance come into contact with the substrate in the soil (Killham, 1994). Initial colonization of a fragment of organic matter will be by microbes which exploit soluble organic compounds (e.g. carbohydrates, organic acids, amino acids) (Moody, 1993) and will be dominated by fungi such as Mucor, Pythium and PenicUliurn (Bowen and Harper, 1990: Moody, 1993) and bacteria such as Pseudomonas (Killham, 1994). The second successional phase in the decomposition of organic residues is dominated by species such as Trichoderrna, Fusarium and Chaetomium (fungi) and Bacillus spp. (bacteria) which decompose cellulose and hemicellulose (Moody, 1993). In the rhizosphere similar biological processes occur in response to root exudation and sloughed-off dead root cells (Rovira et al, 1983; Whipps and Lynch, 1986). This flush of primary decomposers induces corresponding increases in the abundance of secondary decomposers, organisms such as protozoans, bacterivorous and fungivorous nematodes which feed on the primary decomposers (Anderson, 1994). The third phase of decomposition of the organic residues is much slower and involves the slow metabolism of recalcitrant organic components with high ligin and/or polyphenol contents. Fungi such as white rot fungi (Lavelle et al., 1993) and basidiomycetes commonly dominate this phase of the successional process although cellulolytic fungi which can decompose delignified polysaccharides (e.g. Fusarium and Trichoderma spp.) may also be found at this stage (Raynor and Boddy, 1988; Bowen, 1990; Moody, 1993).


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In most agricultural systems there are strong seasonal patterns to the inputs of organic residues into soil. These pulses of introduced organic residues (especially if ploughed into the soil) will induce corresponding waves of decomposition with their associated microbial successional processes. Thus at any particular time, soil samples are likely to display radically different abundances and diversity of microorganisms. At the process level, little is known about the extent to which complex or simplified microbial communities are needed for organic matter transformations. In one such study, Bowen and Harper (1990) showed that eight species of fungi isolated from decomposing straw varied widely in their ability to degrade lignin. Combinations of these fungi resulted in rates of decomposition greater or smaller than the rates of decomposition shown by pure cultures of the most-effective decomposer (Bowen, 1990). The consequences of interactions between microorganisms during the initial stages of organic matter decomposition are unknown, but they suggest that microbial communities with different species composition are able to function in a similar manner (Anderson, 1994). EFFECTS OF EDAPHIC FACTORS The sensitivity of microbial activity to variations in edaphic factors is well recognized (Lee, 1991). Wide variations in microorganism-specific response to these factors, in association with the highly heterogeneous nature of the soil environment, will have a major effect on the diversity of microbial communities. Dry conditions generally favour fungi over bacteria, and favour Bacillus over Pseudomonas, while wet conditions favour anaerobes (largely bacteria). Similarly, pH commonly varies down the soil profile and with soil type and fungi are generally favoured over bacteria in acidic soils (Roper and Gupta, in press). Thus the relative abundance of microbial species or functional groups and hence the diversity of microbial communities will vary widely across even relatively small spaces. Robertson (1994) has provided an example of the use of geostatistical procedures (e.g. Semivariagram analysis) to analyse patterns of microbial activity at the field scale and showed that even within an apparently homogeneous field, rates of net N-mineralization were patchily distributed and varied by >5-fold over relatively short distances in both tilled and non-tilled fields. Whether such differences reflect differences in microbial diversity is not known, but these data illustrate the difficulties which must be overcome in order to permit valid interpretation of apparent differences between locations. EFFECT OF AGRICULTURAL MANAGEMENT PRACTICES ON THE BIODIVERSITY OF MICROBIAL COMMUNITIES The biodiversity of plants and animals generally declines as an inverse function of the intensity of human intervention, especially where crops are cultivated using mechanized methods and agrochemicals are applied (Anderson, 1994). The loss of species is generally in response to intense and widespread habitat modification (simplification) which causes local extinction of species because the resources required to persist (largely space and food) are no longer available in sufficient quantity. Although competition is commonly imputed as a cause of local extinction, there is little evidence that interspecific competition for resources is responsible for local extinction of such species. Can the same be said for microorganisms? Will the change from forest to cropping result in the loss of individual microorganisms or merely altered patterns of abundance of those already present? Further, within cropping soils, will different management practices result

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in loss of individual taxa of microorganisms or merely differences in the proportions of taxa that make up microbial communities? What are the consequences of any changes for the functioning of the agricultural system? Will it be more or less resilient in the long-term? These are questions which require investigation. There is a substantial literature which documents the impact of soil and crop management practices on different components of the soil microbiota (see reviews by Brussaard et al., 1990; Doran and Werner, 1990; Hendrix et al., 1990; Bethlenfalvay, 1992: Dick, 1992: Roper, 1993: Pankhurst and Lynch, 1994; Rovira, 1994: Neate, 1994). In all cases, populations of individual soil microorganisms (e.g. Rhizobium, root pathogens) and functional groups of soil microorganisms (e.g. cellulolytic microorganisms, denitrifying bacteria, nonsymbiotic nitrogen-fixing bacteria, mycorrhizal fungi, sulphur-oxidizing microorganisms) change in abundance in response to different management practices. These management-induced changes in abundance are usually a consequence of changes in habitat and substrate availability that discourage or favour the growth of selected microorganisms. For example, fallowing and cultivation reduced populations of VA mycorrhizal fungi in a cropping soil (Thompson, 1987). This could be overcome by reducing the length of the fallow period sufficiently to prevenl a large decline in VA mycorrhizal fungi in the soil (Thompson, 1987). In another example, the decline in cereal root disease caused by the take-all fungus after several years of continuous wheat cropping was correlated with an increase in populations of fluorescent pseudomonads antagonistic towards the growth of the fungus (Rovira and Wildermuth, 1981). Direct evidence of the impact of management practices on the diversity of individual functional groups of soil microorganisms has been found. For example, the diversity of Rhizobium species (root nodule bacteria) has been shown to decline with decreasing soil pH (Harrison et al., 1988) and in the presence of heavy metals (Hirsch et al., 1993). In a comparison of Rhizobium diversity in a cropped soil and soil under native vegetation, Ophel-Keller and Wiebkin (1994) showed that the diversity of Rhizobium species was highest in soil under permanent pasture and lowest in soil under native vegetation. In contrast, the genetic diversity within strains of R. leguminosarum by. trifolii and by. viceae was lower in the pasture soil than in soil under a wheat/volunteer pasture (Ophel-Keller and Wiebkin, 1994). In these same soils, genetic diversity within populations of the root pathogen Pythium irregulare was higher in the cropped soil than in the pasture soil (Mathew et al., in press). Genotypes of this pathogen were also present in the native soil but these were quite distinct from those found in the cropped soil. in another example, the diversity of heterotrophic and autotrophic sulphur-oxidizing microorganisms increased following atmospheric pollution of S or repeated application of sulphur fertilizer to the soil (Germida et al., 1992). An interesting aspect of this study was the fact that the Thiobacillus bacteria could not be detected in the unamended soil. The question remains as to what is the significance of management-induced changes in terms of the functioning of the agricultural system. There is no evidence to suggest that the numerous processes mediated by soil microorganisms do not persist across the spectrum from undisturbed soil under native vegetation to intensively cultivated soil under continuous cropping. Whether the spectrum of microorganisms responsible for these processes under different systems remains the same or changes requires further research. There is little reason to believe that individual species become extinct, even though their abundance may alter with different patterns of organic inputs. The small spatial requirements for persistence of many microorganisms (mm-cm rather than m-km) means


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that the spatial requirements will not be drastically altered by major changes in soil management practices. The major exception to this will be mutualistic/dependent associations between plant species and microorganisms (e.g. mycorrhizal fungi, root pathogens, R h i z o b i u m ) where alterations in the suite of plant species present can cause corresponding changes in the presence of the obligate microorganisms (Bethlenfalvay, 1992; Neate, 1994; Ophel-Keller and Wiekin, 1994). Importance of biodiversity of soil microbial communities to the stability of agricultural systems There has been much written about the relationship between species diversity and ecosystem stability. A common view is that species diversity stabilizes ecosystem functional properties (McNaughton, 1977; Van Voris et al., 1980; Klein et al., 1986, Potter and Meyer, 1990; Huston, 1993; Anderson, 1994; Elliott and Lynch, 1994). As agricultural systems are dynamic, an important issue in this diversity/stability relationship resides in a better understanding of the role microbial communities have in the processes which support these systems. In particular we need more knowledge concerning functional diversity responses to environmental stresses (Klein et al., 1986). The genetic diversity of soil microbial communities generally decreases in response to an environmental stress or disturbance which upsets the ecological balance of population interactions within the community (Atlas et al., 1991). Those microbial populations which survive the imposed stress will have specific characteristics that enable them to persist within the perturbed community. Studies of the impact of chemical pollutants or pesticides on soil microbial communities showed a decline in the taxonomic and genetic diversity of the resident microbial community with the gradual build up of selected (less diverse) populations with the capacity to tolerate the pollutant and/or utilize it as a substrate (Atlas et al., 1991; Holben et al., 1992). Such versatility within a community could be an indicator of the robustness of the ecosystem, i.e. its capacity to respond (and hence its resilience) may be directly related to the diversity of organisms present. Current concerns about the environmental impact of intensive agriculture, in particular the increasing reliance on agrochemicals, is shifting the focus away from high energy input agricultural systems to more ecologically sustainable systems. The management of these systems includes the use of reduced tillage, regular inputs of organic matter and nutrient recycling strategies based on crop rotations (Pankhurst and Lynch, 1994). These practices alter the inputs of carbon and nutrient into the soil and thus have major impacts on the biological activity and the biodiversity of the soil organism communities present. The complexity of these communities, and their capacity for compensatory activities, make it currently very difficult to determine the relative importance of specific functional groups. Ecologically, this raises the question of how much redundancy there may be in the biological composition of these communities and which components must be maintained in order to retain the functional processes (Walker, 1992). This will be a fruitful area of research. As more information becomes available on the patterns of biodiversity displayed by soil organism communities under various management regimes, it may become possible to correlate community composition with system stability. Knowledge of the microbial diversity required to maintain functional processes, such as the various transformations associated with soil organic matter decomposition, or symbiotic associations, is required if

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the importance of biodiversity of soil microbial communities to the m a n a g e m e n t and sustainability of agricultural systems is to be understood.

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