Plant and Soil 170: 75-86, 1995. (~) 1995 KluwerAcademic Publishers. Printedin the Netherlands.

Soil microbial diversity and the sustainability of agricultural soils A.C. K e n n e d y a n d K . L . S m i t h USDA-ARS, Washington State University, Pullman, WA 99164-6421, USA Key words: agroecosystems, biodiversity, community, indices, soil quality, sustainable

Abstract Many world ecosystems are in various states of decline evidenced by erosion, low productivity, and poor water quality caused by forest clearing, intensive agricultural production, and continued use of land resources for purposes that are not sustainable. The biological diversity of these systems is being altered. Little research has been conducted to quantify the beneficial relationships between microbial diversity, soil and plant quality, and ecosystem sustainability. Ecosystem functioning is governed largely by soil microbial dynamics. Differences in microbial properties and activities of soils have been reported but are restricted to general ecological enumeration methods or activity levels, which are limited in their ability to describe a particular ecosystem. Microbial populations and their responses to stresses have been traditionally studied at the process level, in terms of total numbers of microorganisms, biomass, respiration rates, and enzyme activities, with little attention being paid to responses at the community or the organismal levels. These process level measurements, although critical to understanding the ecosystem, may be insensitive to community level changes due to the redundancy of these functions. As microbial communities comprise complex interactions between diverse organisms, they should be studied as such, and not as a "black box" into which inputs are entered and outputs are received at measured rates. Microbial communities and their processes need to be examined in relation to not only the individuals that comprise the community, but the effect of perturbations or environmental stresses on those communities.

Introduction Diversity is a critical environmental topic that concerned people from all walks of life are rallying around. This issue involves science, economics, religion, aesthetics, and ethics, making it not just a scientific, but also a societal issue (di Castri and Younes, 1990; Hawksworth, 1991a; OTA, 1987). Also, the diversity issue can encompass every level of biological organization from the molecular to global, including landscapes, ecosystems, populations and individuals. The biological diversity of many ecosystems may be in flux after being subjected to various degradative processes. These processes include forest clearing, intensive agricultural production resulting in erosion, and non-sustainable mining of land resources. Biological diversity programs have centered on plants, animals, and insects with little attention given to microorganisms (OTA, 1987; Wilson, 1988). All too often, this vast biological component is ignored completely. It is estimated that only 13% of the earth's microbial pop-

ulations are identified, leaving a vast portion of that biota unknown and therefore unstudied (Hawksworth, 1991a). Little research has been done to quantify the beneficial relationships between microbial diversity, soil functioning and plant quality, and ecosystem sustainability. Soil is a complex, interrelated community of soil organisms, which influence, yet are in part determined by the chemical and physical parameters of the soil. The science of soil microbiology is often inexact, with many of our assumptions gleaned from the information derived from aboveground ecology of plants and animals. Since the spatial and temporal stresses of the microbial system may be quite different from those of plants and animals, these differences must be made evident and applied, so that reasonable hypotheses and the correct interpretations are made. The purpose of this paper is to explore the issue of microbial diversity as a means to assess and identify research areas for future consideration, which subsequently achieve sustainability in agroecosystems.

76

Sustainability Sustainability is not easily defined and interpretations of the concept are often vastly different. There are many definitions of sustainability and many more discussions of its meaning (Douglass, 1984; Edens et al., 1985; Edwards et al., 1990; FACTA, 1990). Simply stated, sustainability is the adoption of practices that allow for the long term maintenance of the productive capacity, the viability and quality of life, and conservation of the environment and resource base. The goal of sustainability is to leave future generations an earth that will sustain them, an earth they will be interested in inheriting. Agroecosystems currently occupy 30% of the earth's surface and include the earth's most productive soils (Altieri, 1991; Coleman and Hendrix, 1988). Effective management of agroecosystems is critical to improving the conservation and viability of our biosphere. Hence, they need to be recognized as resources equal to natural ecosystems. Applied ecological principles form much of the basis of any sustainable agricultural system, and must consider species abundance, distribution and function, in both temporal and spatial terms (Thomas, 1992). Any discussion of ecosystem maintenance must consider the energy flux within that system (Odum, 1983). Sustainable agriculture must work toward identifying and minimizing energy loss throughout the system (Pimental and Hall, 1984). Yet, identification and implementation of agricultural practices that minimize energy loss while maintaining productivity is the challenge. Diversity is a key issue in energy flux. Although diversity and ecological stability may not be concepts that usually coexist in agricultural systems, diversity in crops, cropping systems, and management practices will enhance the stability of agriculture (Altieri, 1991; Dover and Talbot, 1987), affect the microbial portion of the agroecosystem, and thus, influence sustainability.

Soil quality Soil is a key natural resource interacting with aboveground plant and animal communities and contributing to the success of sustainable agriculture (Pimental et al., 1992; Smith, 1974). Soil is a resource critical to the maintenance of any ecosystem that we need to manage effectively. Thus, soil quality is an issue that needs to be included in discussions of sustainability (Papendick and Parr, 1992). Soil quality can be defined as the soil's capacity to function in a desired manner such as

Fig. 1. The components of soil quality.

to produce healthy crops, animals, and humans, resist erosion, and minimize environmental impacts (Parr et al., 1992). It encompasses not only productivity of a soil, but also environmental quality, food safety, animal and human health, pollutant degradation, and land use.

Soil quality consists of the chemical, physical, and biological components of a soil and their interactions (Fig. 1). The major emphasis in soil quality investigations, until recently, has been on the use of chemical and physical attributes of soil to define soil quality (Arshad and Coen, 1992), since the biological portion is so much more difficult to quantify. However, these two soil features are only part of what may impart to a soil its essence or characteristics. Investigations into microbial parameters involved in soil quality are increasing (Hatfield and Stewart, 1994; Turco et al., 1994). The biological component of the soil is responsible for soil humus formation, cycling of nutrients, soil tilth and structure (Lynch and Bragg, 1985; Tisdall, 1991) and a myriad of other functions. It is imperative that we increase our understanding of soil microbiology as a part of managing sustainable agricultural systems. This biological component largely has been ignored as an important aspect of ecosystem functioning, although microorganisms are highly sensitive to disturbance and perturbation (Atlas, 1984). The concept of soil quality needs to be further defined, and parameters involved in those changes cataloged so that the characteristics or essence of that soil can be determined and tracked. More importantly, the changes in soil with perturbations need to be quantified to assist in the rebuilding or maintenance of an ecosystem. The resilience of a soil system to perturbation may greatly contribute to the understanding of soil quality (Elliott and Lynch, 1994; Thomas and Kevan, 1993).

77 Table 1.

Group

Bacteria Fungi Algae Viruses

Numbers of organisms involved in the major microbial groups Number of species collections Described Estimate

3,000 69,000 40,000 5,000

30,000 1,500,000 60,000 130,000

Species in culture % of species total

Number

% of total estimated species

10% 5% 67% 4%

2,300 11,500 1,600 2,200

7.0% 0.8% 2.5% 2.0%

From D. L. Hawksworth (1991).

Microbial ecology Essential to sustainable agriculture is the maintenance of viable, diverse populations and functioning microbial communities in the soil. Anthropogenic activity can directly or indirectly affect the functioning and diversity of a system. Differences in microbial properties and activities of soils have been reported but are restricted to general ecological enumeration methods or activity levels, which are useful, although limited in their ability to describe a particular ecosystem (Alexander, 1977; Paul and Clark, 1989). The microbial portion of the soil and its response to stress has been traditionally studied at the process level, in terms of total numbers of microorganisms, total respiration rates, and enzyme activities (Parkinson and Coleman, 1991). Little attention has been paid to responses at the community or the multiple levels of organismal structure. Process level measurements, although critical to understanding the ecosystem, may be insensitive to community level changes due to the redundancy of these functions and the complexity of relationships within particular communities. Although process level assessment may describe a situation, it does not indicate diversity and location of organisms responsible. Investigations of populations usually focus on a single organism only, and may not have any resemblance to the reality of interactions. Since communities comprise diverse individuals in complex interactions, they should be studied as such, and not as a "black box" into which substrates are entered and products received at measured rates. Microorganisms, along with the processes they are responsible for, need to be examined

~ [

GLOBAL CHANGES

>

O < i: Z

""=========I~

> , / Biological ~ ~ > .~ ~ ~ <

f Ecosystem"-'(Functioning~)

SUSTAINABLE SYSTEMS

I

Fig. 2. The interrelationship among biodiversity, ecosystem function, and global changes (from diCastri and Younes, 1990; Barbault and Hochberg, 1992).

to evaluate response to environmental stress and any resulting changes in biological diversity.

Diversity Biological diversity is the variety of species in ecosystems as well as the genetic variability within each species (Conservation Foundation, 1987). It includes all forms and all levels of life, including genetic, taxonomic, and ecosystem levels. In the past, diversity was determined based on taxonomic species, which may limit the scope of information and relationships obtained. The diversity of species units or even of communities or tropic levels may give us a better estimation of the functioning of a system. Biodiversity can also be defined as a richness of life as indicated by the variety

78 Table 2. Richness, evenness and diversity indices and equations Richness

R"'ar-~'e ¢~v~ • = S- 1 RMenhinick = @n Evenness

HI Epiel°u =Eg

ZSheldon = eH_.~ s' EHeip = er~ - 1

Z H i l l -- ~eH EHillmod = ~eIf - 1 Diversity DSimpson = )~ = ~ s ~ n i - l DShannon = H' = Zs [ ( ~ )

DHill-1 = ilk =

In ( ~ ) ]

1

DHilI-2 eH~ =

of biota and interrelationships of biochemical processes in the soil. Species diversity has been used in many investigations and indices have been used frequently (Peet, 1984). Greater attention now is being paid to diversity issues due to the increased awareness of issues of conservation, sustainability, and preservation, and the apparent loss of diversity by anthropogenic influences (Wilson, 1988). Approaches to studies of diversity vary widely and diversity can be studied at a variety of levels: globally, at the community level and at the population level (one or two species). Biodiversity and functioning can be linked to global issues and human activity (Fig. 2; di Castri and Younes, 1990). Redundancy in function within soil microbial popuiations may lessen the importance of species diversity (Walker, 1992). If we base functioning and its importance on simply our knowledge of the populations, then redundancy may be an issue. We may only understand a fraction of the characteristics of a known microbe,

thus we cannot speculate as to whether redundancy is an issue. In microbial systems, there may be less redundancy than in other systems because high specialization may exist. We are unaware of the true extent or dimension of the diversity of soil microbes, although molecular investigations indicate that the diversity in soil is much more vast than we can discern with cultural techniques (Holben and Tiedje, 1988; Torsvik et al., 1990). Also, the actual contribution of diversity to system functioning is unknown. It is necessary for us to increase our knowledge of biotic and functional diversity to resolve this issue. It is estimated that the total number of species inhabiting the earth approaches 10 million, but less conservative estimates put this number at 15 to 25 million. Of these species, only 1.4 million have been identified, leaving many potentially beneficial biological organisms undiscovered (Hawksworth, 199 l b; Wilson, 1988; Wolf, 1987). Until recently, biological diversity programs have centered on aboveground plants, animals, and insect species, with little attention given to microorganisms. The number of known microbial species totals over 110,000, but only a fraction of the species are identified and even fewer are being studied or in culture collections (Hawksworth, 1991b, Table 1). A vast number of microbial species and their genetic traits are unknown. Microbial communities are highly diverse and thought to exhibit even greater diversity than originally thought or seen in higher orders of organisms (Torsvik et al., 1990; Ward et al., 1992). Obviously, we are aware of only a miniscule portion of the total potential of the system. Within the soil microbial population, there is a wealth of genetic information waiting to be discovered. The ability of an ecosystem to withstand extreme disturbance may depend in part on the diversity of the system. Diversity investigations are needed to: 1) increase our knowledge of the diversity of genetic resources and understand the distribution of diversity on this earth, 2) increase our knowledge of the functional role of diversity, and 3) identify changes in diversity associated with disturbance or management. It may be important to monitor diversity as an indicator of change or in response to a stress. We need to determine the extinction rate that still can maintain a stable sustainable ecosystem with intact integrity. The extinction rate within a system may be an important indicator of the status of the system and critical in determining the level of diversity necessary to maintain a sustainable system. The actual numbers of species and

79 species makeup may not be as important as the flux within a system. Diversity indices may be useful to further understand the status of soil microbial communities; however, we need to proceed with caution. Diversity is a function of two components: 1) the total number of species present, species richness or species abundance, and 2) the distribution of individuals among those species, evenness, or species equitability (Margalef, 1958). It is the incorporation of these two components into diversity indices that has led to much debate and has caused confusion even of the most knowledgeable (MacArthur, 1960). Data may be interpreted differently and interpretation confounded depending on the calculations used to derive the index (James and Rathburn, 1981). Furthermore, the indices developed may mask the actual changes that may be occurring within a community or ecosystem.

Species richness Species richness, one component of diversity, can be viewed in several ways. Species richness is simply the total number of species (S). The Margalef (1958) and Menhinick (1964) equations are based on the assumption that a relationship between S and n, the total number of individuals, exists (Table 2). One way to avoid deviating from these assumptions is to use samples of equal size. The data can also undergo rarefaction (Hurlbert, 1971), which assumes that the sample size bias can be reduced by calculation (James and Rathburn, 1981).

Species evenness The other component of species diversity indices is species evenness, which indicates the distribution of the individuals within species designations (Table 2). The evenness index that is most widely used is the ratio of the Shannon index (Shannon and Weaver, 1949), and the maximum value for H' occurs when only one individual occupies each species designation (Pielou, 1977). Other equations for evenness have been proposed (Heip, 1974; Sheldon, 1969); however, these values for evenness are strongly influenced by the richness of the sample. The Hill evenness equation (Hill, 1973) is the ratio of the two Hill diversity indices and represents the comparison of the very abundant to abundant species. This value will approach the value of one as one species dominates. The modified Hill equation differs from the above in that as fewer species

dominate the community, the value approaches zero. Neither of the evenness equations as proposed by Hill is influenced by richness as much as the other equations.

Diversity indices Indices of diversity, which combine species richness and evenness together, also have been called heterogeneity indices (Peet, 1984). Since diversity indices are numerous and varied, it is critical to realize their limitations. A diversity index is a single value; thus, it cannot indicate the total makeup of a community. For example, two communities may have the same diversity index value, but one may comprise low evenness and high richness, while the other may comprise high evenness and low richness. Scientists, therefore, need to look at evenness, richness, and diversity. Although there are several diversity indices in the literature (MacArthur, 1960; Peet, 1984), the following equations demonstrate the breadth of differences among the schools of thought (Table 2). The Simpson (1949) diversity index (Table 2) was the first index to be used in ecological studies, and deals with the probability that two individuals are of the same species. In other words, if the probability that two individuals are in the same species is low, then the diversity is high. The Shannon diversity index (Shannon and Weaver, 1949) deals with the uncertainty predicting to which species an individual will belong. This diversity index equation is the most widely used; however, it is related to the number of species in the sample and does not consider rare species (DeJong, 1975). Hill (1973) devised a series of indices with less emphasis on the rarer species. The first Hill equation uses the natural log of the Shannon index and reflects the number of abundant species. The second Hill equation for diversity is the inverse of the Simpson equation (Table 2), which considers the most dominant species. There are many different equations to use to calculate diversity and the values generated can be diverse in themselves depending on the equations. As with any index, these diversity indices must be used with caution to best describe the situation, and to answer the questions posed.

Functional diversity In the past, functional groups of microorganisms have been given very broad designations. Microbes had been classified as to their principle means of obtain-

80 ing carbon and energy, i.e., phototrophs, chemotrophs, autotrophs, heterotrophs, or lithotrophs. The use of such categories is limited in information as many situations overlap (Alexander, 1977). In present studies of functional groups, the divisions need to be more precise. (Walker, 1992). Ecosystem functioning before and after disturbance is largely governed by soil microbial population dynamics. Microbial biomass and activity can provide advance evidence of subtle changes in content long before it can be accurately measured against background organic matter levels already present in the soil (Powlson et al., 1987). The forces driving the ecosystem of interest will ultimately determine the functional groups of importance. For example, in an agroecosystem, nutrient cycling, residue decomposition, soil structure, and pest balance will be of paramount importance to the productivity and sustainability of that system. If the soil is to be used as a waste repository, then infiltration, porosity, structure, and degradation capacity are of greatest importance. Soil microorganisms constitute a large dynamic source and sink of nutrients in all ecosystems and play a major role in plant litter decomposition and nutrient cycling (Cambardella and Elliott, 1992; Collins et a1.,1992; Smith and Paul, 1990), soil structure (Lynch and Bragg, 1985), dinitrogen fixation (Sprent, 1979), mycorrhizal associations (Barea, 1991), reduction in plant pathogens (Cook and Baker, 1983), and other alterations in soil properties influencing plant growth. Differences in microbial properties have been reported, but they are restricted to general ecological enumeration methods and activity levels or single species identification, which are limited in their ability to adequately describe a particular ecosystem. Soil organisms are one of the most sensitive biological markers available, and the most useful for classifying disturbed or contaminated systems, since diversity can be affected by minute changes in the ecosystem. The use of microorganisms and their functioning for examination of environmental stress and declining biological diversity needs to be investigated more fully (OTA, 1987). As early as 1927, Waksman (1927) investigated the use of microorganisms coupled with physical and chemical parameters and concluded that these parameters could be used to indicate the fertility of a soil. The criteria identified then were numbers of microbes, nitrification, C02 evolution, cellulose decomposition, nitrogen fixation, catabolic activity and oxidation/reduction power. Domsch et al. (1983) indicated other microbial parameters to study, such as

those populations of greater sensitivity to perturbation, which include the nitrifying population, Rhizobium, actinomycetes, and those responsible for organic matter degradation. Those of moderate sensitivity were populations of algae, bacteria, fungi, and the processes of soil respiration, denitrification, and ammonification.

Community status Microbial diversity indices have been used only minimally to describe the status of microbial communities and their response to natural or human disturbances. Microbial diversity indices can function as bioindicators of the stability of a community and can be used to describe the ecological dynamics of a community and the impact of stress on that community (Atlas, 1984; Mills and Wassel, 1980). An important limiting factor to greater use of the indices is the absence of detailed information on the microbial species composition of soil environments (Torsvik et al., 1990).

Keystone or indicator species It is possible that individual species can function as indicators of the status of an ecosystem. These keystone species need to be common and reflect the response of the community they represent. There is concern over the use of single populations to identify stress responses of a system (Cairns, 1983; Kimball and Levin, 1985). The effect of stress on single species cannot predict what will happen throughout the community and cannot identify all the interactions that may be altered (Cairns et al., 1985). Ecosystem based analyses are critical, with identification of interactions having greatest utility (Odum, 1959). An example of the use of the potential of soil diversity include the area of biological control of plant pathogens (Cook and Baker, 1983). Another example is the investigation of deleterious rhizobacteria, which were discovered in the early 1980s. These investigations have led to changes in management practices for many crops and may eventually lead to biological control of weed species (Fredrickson and Elliott, 1985; Kennedy et al., 1991; Kremer et al., 1990). There is a wealth of genetic material in the soil that may have potential in biotechnology programs; thus, diversity investigations will benefit more than one area of this science (Bull et al., 1992; Malik and Claus, 1987).

81 Disturbed areas

Efforts to develop methodologies for the investigation of microbial diversity have been slow, although some attention has been given to chemically stressed systems such as chemical spills and hazardous waste sites (Atlas, 1984). In these systems, much of the work has focused on specific organisms for potential degradation of toxic substances. Spent oil shale soils are commonly characterized by low microbial numbers, fewer types of bacteria, and low rates of cellulose decomposition, compared to nondisturbed soils (Segal and Mancinelli, 1987). The addition of top-soil and revegetation in soil reclamation projects were found to increase populations of heterotrophic aerobic bacteria, actinomycetes, ammonium oxidizers, and fungi to levels found in undisturbed soils. Decline in microbial populations soon after soils are reclaimed has been attributed in part to utilization of soil organic matter (Fresquez et al., 1986). Reclamation of degraded lands often begins with nonmycorrhizal plants with the mycorrhizal inoculum increasing with each stage (Cundell, 1977; Waaland and Allen, 1987). Management practices

Management practices influence microbial activities in long term agricultural lands (Bolton et al., 1985; Doran, 1980; Martynuik and Wagner, 1978; Ramsay et al., 1986). The ecology of the root-microbe interactions after minimum tillage practices is vastly different from the extensive plowing to prepare the seedbed. The changes in the soils' physical and chemical properties resulting from tillage greatly alter the matrix supporting growth of the microbial population. Within a given soil, there is considerable variation with depth in the composition of the microbial community. In no-till agricultural systems, microbial activities drastically differed with depth, with the greatest microbial activity occurring near the no-till surface; while in the tilled system, activities were more evenly distributed throughout the plow layer (Doran, 1980). The time frame or outcome of changing management practices are not well understood. Only when diversity studies of the total community are conducted will we be able to understand the type and magnitude of the ecological disturbance and the means to return the soil to its near original state. Microbial biodiversity is key to the integrated functioning of nutrient cycles and decomposition in terrestrial soil systems. These systems range from tropi-

cal forest, savannah, temperate evergreen forests, to agricultural ecosystems. Each system is influenced by parameters which dictate vegetative growth and ecosystem energy flux. Inherent in each system is a unique microbial flora to which a disturbance facilitates shifts in its diversity. One can track changes in microbial diversity, and then use these methodologies to obtain background information on specific undisturbed ecosystems. Once background baseline data are established, changes in ecosystem functioning can be detected and management systems developed to protect these systems from irreversible ruin. Therefore, diversity of the organisms is important. However, it may be of more use to indicate functional groups and their diversity and changes with perturbations. Not only diversity, but the rate of activity may need to be incorporated.

Microbial diversity with an agroecosystems Prairie versus cultivation

Recognizing the lack of information on the functioning of individual species in soil microbial communities, we investigated the differences in soil microbial activities and communities between a prairie grassland and a cultivated wheatland. Such investigation would increase our information about these agroecosystems and shed light on the possible changes occurring within microbial communities associated with management practices.

Material and methods Site

Plow layer soil (0 to 30 cm) was collected in the summer of 1991 from two sites in the Pacific Northwest, near Pullman, WA. The sites had natural prairie and cultivated land plots on the same slope and aspect in close proximity. Samples were collected from the two sites in a grid pattern across the toposequence. The cultivated site was in a wheat-barley-pea rotation using conventional tillage and weed management practices representative of the farming practices of the Palouse region. The prairie had not been disturbed since the family had homesteaded 100 years ago. The soil series was a Palouse silt loam. Soil was collected to a depth of 30 cm and kept on ice or refrigerated until anal-

82 ysis. All analyses were conducted within four weeks of sampling. Any plant patches were avoided in the prairie and the interrow areas were sampled in the cultivated system. Nine samples were collected at each of three toposequences. Soil was analyzed for gravimetric water content, pH, inorganic N, soluble C (McGill et al., 1986), biomass carbon (Jenkinson and Powlson, 1976), denitrification potential (Tiedje, 1982), nitrification potential (Schmidt and Belser, 1982), dehydrogenase (Casida et al., 1964), and phosphatase enzyme activities (Tabatabai and Bremner, 1969). For those methods that relied on colorimetric analyses, the methods were modified for use with microtiter plates, which allowed rapid reading of numerous samples. All experiments were analyzed by analysis of variance. Treatment means were compared using a Fishers (protected) least significant difference atp = 0.05 (Steel and Torrie, 1980). Soil was plated on various media selected to represent broad nutritional groups microbial groups found in soil.The media used were tryptic soy agar (bacteria), Sands and Rovira medium (Gram-bacteria), Martin's minimal medium (fungi), potato dextrose agar (fungi), and starch casein (actinomycetes; Wollum, 1982). Plate counts were taken and morphological differences noted. Bacterial populations were log transformed before statistical analysis.

Communitycomposition We examined heterotrophic bacterial communities in the soil from these two sites. To obtain isolates, 60 isolates for each sample treatment were randomly selected and streaked from plates containing the highest number of separate colonies. These isolates were cultured on their initial medium and stored in that medium plus 40% gycerol at -80 ° C. Each isolate was analyzed for its ability to utilize substrates representing various functional groups. Substrate utilization was determined using a basal growth medium containing several substrates including glutamate, glycine, citrate, serine, xylan and various hemicelluloses, cellulose, and mannans as the sole source. These were selected as they represent basic nutritional groups or pathways and functional groups, and had been previously shown to differentiate soil bacteria (A C Kennedy, unpublished). Starch hydrolysis, proteolytic activity, lypolysis, and growth at pH 4, 7, and 9 were also assessed and included in the overall assessment of substrate utilization. Ability to grow in the presence of heavy metals, polyethylene glycol,

or antibiotics (Gerhardt, 1981) was also tested and categorized as stress parameters. All parameters were screened using 96-well microtiter plates and growth was indicated by the redox dye, triphenyl tetrazolium chloride (Kennedy, 1994). Each isolate was ranked and assigned three numerical designations, one based on its ability to utilize substrates, another on its ability to grow in the presence of the select 'stresses' of metals and antibiotics and osmotica. The third ranking was a combination of the two, which we designated as the species unit number. In the first ranking set, the isolates were ranked according to their ability to utilize substrates with those isolates utilizing only a few substrates given a small numerical ranking. Those isolates able to utilize a large number of substrates and substrates of increasing complexity were given higher ranking numbers. The isolates were ranked in order of their ability to flourish in the presence of antibiotics, polyethylene glycol or metals. This ranking was determined by the collection and type of 'stress' an individual could withstand, with those isolates sensitive to the components receiving small numbers and those more resistant were assigned larger numerical rankings. The rankings, thus the characteristics, of each species unit were used to calculate diversity indices (Hill, 1973; Shannon and Weaver, 1949; Simpson, 1949). The community structure also was visualized by graphing the relationship of substrate utilization and stress response. Graphs were constructed using the substrate utilization ranking on the abscissa and the stress response ranking graphed on the ordinate axis.

Results and discussion

The two sites, prairie and cultivated soils, were different in almost all parameters assayed (Table 3) except the plate count data (data not shown). The process level functioning of the two systems was quite different as has been reported in similar comparisons of systems (DeLuca and Keeney, 1993). Biomass C, phosphatase, dehydrogenase, denitrification and nitrification potential all were significantly higher in the prairie system. Inorganic N values were higher in the cultivated soils and pH was not different between the two systems. Although the values for diversity varied depending on the equation used, in general, the cultivated sites had a greater diversity overall (Table 4). The greater diversity in the cultivated systems illustrates the use of diversity indices in community differentiation. Although

83 Table 3. Soil microbial activity parameters from a grass prairie and cultivated wheat field in eastern Washington Parameters

Units

Grass

Cultivated

Prairie

Wheat

380.0b

278.0a

Biomass C

~zg Biomass C/~g s o i l - ~

Phosphatase

/~g g i n - 1 m i n - l

28.0b

6.54a

Dehydrogenase

~ g formazan 24 hr -1 g soil -1

15.5b

1.72a

Denitrification potential

/zg N20- N evolved g soil- l day- 1

18.0b

Nitrification potential

# g NO 3 Ng s o i l - l

N inorganic

# g Ng s o i l - l

pH

8.2a

1.45b

0.82a

3.98a

4.4b

6.01a

5.82a

25

+

+

20

+ ¢.0 15

+44-

CO n" 10 ¢n ¢n

÷

+

0 0

4-

4-

+

~

/ + ,, ~ . ~ 4+

+

+

+ +

+÷ //±-11-..4+ +

++. ~ .

+

+

. .

• + • + +

+

:~-,~+

/

+

• +

+

+

+ 4-

4I

J

t

J

I

5

10

15

20

25

30

Substrate Response P r a i r i e Grass

+

Cultivated W h e a t

Fig. 3. The effect of long term prairie or cultivation on the distribution of species units.

these results are somewhat in disagreement with other findings (Lal, 1991), our data indicate changes in the communities occurring. Obviously, diversity values only indicate a portion of the difference between these two management systems. The mixing of the substrate by plowing or other disturbance further increased the diversity of the population. Each isolate tested was ranked by both its ability to grow on the substrates and its ability to withstand stress. The rankings were higher for isolates able to use a greater number or more complex substrates and for isolates which grew under several of the stress conditions. When the isolates from each system were plotted relative to their substrate utilization and stress

rankings, definite patterns of communities could be seen (Fig. 3). The isolates from prairie soil exhibited a tighter or more similar population of organisms with a greater diversity and broader range of substrate utilization and stress resistance in bacteria isolated from the cultivated soil. The prairie soil community can be represented in general terms by the oval in Figure 3. All functional groups, i.e. substrate utilization groups, studied were represented within the community in each management system, but a greater difference between these communities was found in the individual's ability to respond to imposed stress. Differences were seen within the populations; however, these changes were not occurring in functional grouping. Rather a deft-

84 Table 4. Richness,evenness, and diversityvalues for microbial populations from soil from a grass prairie and cultivated wheat field in eastern Washington

Value

Richness Margalef Menhinic

Grass Prairie index

Cultivated Relationship Wheat index

31.18 17.33

37.58 19.42

gp < cw gp < cw

Evenness Pielou Sheldon Heip Hill Hill mod

0.278 0.065 0.042 0.640 0.446

0.450 0.990 0.085 0.291 0.165

gp < cw gp < cw gp < cw gp > cw gp > cw

Diversity Simpson Shannon Hill 1 Hill 2

0.546 1.05 1.83 2.86

0.515 1.89 1.94 6.67

gp > cw gp < cw gp _
nite shift in the communities' ability or adaptation to withstand stress was measured. The communities identified from the cultivated site occupied a larger spread of types of organisms, mainly due to an increase in stress response. These species types were quite different from the tight population found in the prairie soil. These data indicate that substrate utilization and stress response data of isolates can be used to differentiate among soil populations. It must be noted, however, that the isolates used in this study were obtained from soil dilution culture plates, which investigate a subset of the population, not the total population. Nevertheless, differences in the microbial communities were detected and quantified. Further investigations of other management systems and temporal changes will be necessary for greater understanding of the effect of management on the microbial portion of soil quality. The challenge remains to draw these comparisons among several more similar types of systems.

Future considerations Research is needed to increase our understanding of the function of soil microbial communities. Investigations of diversity and ecosystem function may assist in deciphering the meaning and use of functional diversity. A key question to ask is what function is of interest in the investigation. In agroecosystems, the most notable functions are those involved in N and C cycling, soil structure maintenance, antibiosis, and so on. It is often easy to identify functioning, but more difficult to further dissect the function and to recognize the interrelationship with others. It is not only one species involved in function, but rather the species involved can be quite diverse and interrelated. It is impossible at this point in time to fully comprehend total functioning of the soil system. The functioning of a system, however, may not be as important as the resiliency of the community to withstand stress. The research priorities that need to be addressed in the coming years are to: I) clarify the dimensions of diversity of the genetic resource in the soil, 2) increase our knowledge of the functional roles of microbes we recognize as important to those whose importance we have yet to comprehend, 3) assess changes that occur with perturbations, and 4) identify potential indicators of diversity and soil quality. It is the added information on the extent of diversity in microbial systems and integration of this knowledge, which will lead to sustainability.

Acknowledgements Support from the USDA - Agricultural Research Service in cooperation with the College of Agriculture and Home Economics, Washington State University, Pullman, WA 99164 is greatly appreciated. We are grateful to C R Petersen, J Warner, H McCrindle-Zimmerman for excellent technical assistance. Trade names and company names are included for the benefit of the reader and do not imply endorsement or preferential treatment of the product by the U S Department of Agriculture or Washington State University. All programs and services of the U S Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.

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