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1.2 Biodiversity and biomass of organisms in soil

What are the major groups of soil organisms?

Living organisms in soil belong to one of five Kingdoms:

1. Kingdom Monera or Prokaryotae

(single-celled prokaryotes)

Bacteria are single-celled organisms about 0.5 to 2 mm in length or diameter and they occur in several main shapes. Rod-shaped bacteria are particularly common in soil. Spherical shaped bacteria and spirals are also present. Some species of bacteria form resistant endospores that survive in very severe conditions. The common groups of bacteria are cyanobacteria, eubacteria, gram-negative bacteria, gram-positive bacteria and archaeobacteria.

Common genera of soil bacteria include:

Arthrobacter, Streptomyces, Pseudomonas, Bacillus, Clostridium, Azomonas, Azospirillum, Azotobacter, Beijerinckia, Rhizobium, Bradyrhizobium, Agrobacterium, Nitrosomonas and Nitrobacter

2. Kingdom Protista (single-celled eukaryotes)

Single-celled eukaryotes include:

diatoms, heliozoans, yeasts (fungi), ciliates, amoebae and dinoflagellates

3. Kingdom Fungi

Fungi are an extremely diverse group of organisms [https://www.kew.org/plants-fungi/plant-fungi-groups/fungi-kingdom/index.htm]. The major structural unit of most fungi is a hypha. Fungi also produce spores of various kinds and sizes, and these usually have distinctive features. Fungal spores are often formed within special structures, e.g. mushrooms are formed as spore-bearing structures. There are many shapes and sizes of fungal structures that contain spores, but most are not visible without a microscope.

Some of the major groups of fungi that occur in soil include:

Common name [Genus]

slime molds [e.g. Dictylostelium, Physarum]

zygomycetes [e.g. Mucor, Rhizopus, Glomus]

ascomycetes [e.g. Claviceps, Saccharomyces]

basidiomycetes [e.g. Agaricus, Boletus]

fungi imperfecti [e.g. Aspergillus, Trichoderma, Penicillium, Rhizoctonia]

4. Kingdom Animalia

Many types of animals live in soil. Their sizes range from several micrometres to more than a metre. The list includes:

protozoa (flagellates, amoebae, ciliates), nematodes, mites , collembolans, molluscs, enchytraeid worms, earthworms, millipedes, centipedes, isopods, ants, termites, beetles, dipterous larvae (fly maggots) and spiders

5. Kingdom Plantae

Roots of most plants have a major influence on the living mass of organisms in soil. Roots range considerably in their size and growth habit. Algae are also common inhabitants of soil and represent the microscopic component of the plant kingdom.

Lichens

Lichens, although not a kingdom, form another important biological component of soils. Lichens are highly specific symbiotic associations between:

fungi & algae or between fungi & cyanobacteria.

Cyanobacteria are bacteria that can fix atmospheric nitrogen, and therefore, they supply nitrogen to the fungal partner in the lichen.

A characteristic feature of the association between the two organisms that form a lichen is that the lichen does not resemble either of the partners.

Lichens that grow on the soil surface can be important for soil stabilisation in dry areas. These types of lichens form a delicate crust that binds soil particles and helps to prevent wind erosion. Other organisms become embedded in the crust, forming a community of organisms that live in a thin film on the soil surface. This also occurs with lichens that grow on rock surfaces. Crust communities on lichens most commonly occur in environments that are rarely disturbed physically. Lichens grow very slowly and cannot form stable crusts on soil that is frequently disturbed by agricultural practices or animal trampling.

Lichens reproduce by forming small vegetative structures that include both the fungus and the bacteria or algae. Wind or water distributes these structures.

How are soil organisms identified?

The biology of most soil organisms, except for plants, has not been investigated. The majority cannot be isolated from the soil, grown independently of other organisms and studied because of their small size. In fact, most small soil organisms, such as bacteria, have not even been given a scientific name.

Since few soil organisms have been named and we know little about their biology, an alternative framework is necessary to sort out which organisms are involved in important soil processes. Soil organisms may be very different morphologically (i.e. in what they look like), physiologically (i.e. in what they do) and genetically (i.e. in what they have the potential to do).

The major characteristics used to sort and identify soil organisms are:

• morphological characteristics

• physiological characteristics

• genetic characteristics

• ecological characteristics

• molecular characteristics

Morphological characteristics of soil organisms

The structure (morphology) of organisms is of primary importance for their identification. Morphological characteristics such as leaf shape or bone structure are relatively easy to use to identify multicellular organisms like plants and rabbits. Morphological criteria are also used successfully to identify soil animals as well as many fungi and algae in soil. Detailed identification keys have been prepared for particular groups.

It is much more difficult to distinguish among different types of bacteria and some fungi. A microscope is only useful for distinguishing between bacteria in a very superficial way (i.e. to identify their overall shape as rods, spheres or spiral configurations). Many bacteria have a similar shape but carry out very different functions in the soil.

Physiological characteristics of bacteria

For identification of bacteria, it is generally necessary to use methods based on their physiology – i.e. how they function. Physiological characteristics may be related to the activity and presence of enzymes and other proteins (e.g. those that allow the bacteria to break down molecules containing carbon and nitrogen). Some ecological characteristics may also be used to distinguish among bacteria (e.g. their tolerance to soil acidity or low temperature).

BiologTM is a commercial tool to identify bacteria in growth media. In other words, it is a carbon source metabolic fingerprint technique.

Identification of bacteria and fungi using molecular markers

Advances in molecular biology provide new possibilities for identifying bacteria from soil samples without first isolating them (Ranjard et al. 2000). This is an important breakthrough for determining the species or strains of bacteria and fungi present in a soil.

DNA contains the genetic information of the organism. It is a nucleic acid and it contains highly specific segments that are characteristic of the species that the organism belongs to, as well as segments that are shared by many different species. The sequences of molecules in DNA extracted from soil are compared with sequences for known organisms or for known functions, such as nitrogen fixation.

Molecular techniques are especially useful for identifying bacteria that are difficult to grow in culture media (e.g. nitrifying bacteria). Once a characteristic sequence of DNA is known, there is no further need to isolate the bacteria and grow them in culture media prior to identifying them. Molecular techniques can be applied directly to field soil samples that include other living organisms (e.g. Griffiths et al. 2003). Although there remain technical difficulties in doing this type of work, research to perfect the methodology is progressing very rapidly. The development of methodology for analysing the DNA and RNA of soil organisms is advancing quickly, with new techniques being published every year.

The PCR (Polymerase Chain Reaction) technique enables replicates of specific segments of DNA to be made many times. The segments are then separated using electrophoresis (i.e. applying an electrical charge). The specific segments of DNA used in the PCR technique are located between pairs of ‘primers’. The primers can be made artificially so that they match segments on the DNA molecule. These primers mark the starting point and the end point for replication of DNA fragments during the amplification process. DNA fragments are replicated many times (amplified) by the PCR technique then separated by fingerprinting according to the size of each DNA fragment (i.e. the number of base pairs on the DNA molecule). A range of techniques including Restriction Fragment Length Polymorphism (RFLP), Denaturing Gradient Gel Electrophoresis (DGGE) and Temperature Gradient Gel Electrophoresis (TGGE) provide specific information about the characteristics of the DNA.

DGGE and TGGE (and other molecular techniques) can be used to investigate microbial community structure in soils. Other techniques are used to compare random segments of DNA from different organisms. As the technology advances, distinctions can be made between enzyme activities of organisms as well as other molecular functions. These new technologies are opening a window to examine the diversity and function of organisms in soil which was not previously possible.

Serological techniques

Serological methods have been used to identify some soil organisms, but now they have been generally been superseded by the rapid development of molecular tools. Serological techniques depend on an immune response of animals to make contact with the cell wall or cell contents of a foreign organism that enters their body. When an animal (e.g. a rabbit, mouse, sheep, horse or goat) comes into contact with a foreign organism, it produces antibodies which are often highly specific to the foreign organism against which they were formed. An antibody response of an animal to an unknown bacterium with that of a known bacterium can be used to identify an unknown bacterium.

There are a number of techniques that use serological methods to identify strains of bacteria present in soil or in plants. The Fluorescent Antibody (FA) technique and the Enzyme-linked Immunosorbent Assay (ELISA) technique are two of the commonest methods.

A fluorescent or enzyme-linked antibody can be selected that is highly specific for a soil organism. When a solution containing this specific labelled antibody is added to a filtered solution from soil, bacteria that are very similar to the original known bacteria can be detected if they are present.

The technique has also been used for fungi, but with less success than for bacteria, this is why the molecular tools are being found to be more useful. One reason for the lack of specificity of fungi (based on serological criteria) is that the structure of the cell wall of the hyphae changes with their age. Thus, the molecular sites for recognition between related antigens and antibodies may become less specific or absent in older microorganisms.

Characterisation of bacteria and fungi using protein and enzyme profiles

Proteomic techniques can be used to extract and identify proteins from soil organisms. The extracts are placed at the top of a gel as for the separation of DNA fragments described above. An electric current is applied. Proteins, (this includes enzymes) vary in their molecular weight and electric charge, and consequently, they travel along the gel in the electric current at different rates. After the current is turned off, the gel is placed in a staining solution until distinct bands appear. Organisms have characteristic proteins and enzymes. This process, called gel electrophoresis, is used to identify similarities and differences between the protein or enzyme profiles of closely related organisms. Proteins represent the expression of genes of the organisms being investigated.

How is the abundance of soil organisms measured?

It is very difficult to determine the number of species of bacteria, fungi or other organisms in soil. As already mentioned, most organisms cannot be isolated from soil individually. Indeed, the estimated number of organisms in a soil depends on which technique is used. Usually there are thousands or millions of organisms in a small quantity of soil and this number decreases with increasing distance below the soil surface. The millions of bacteria present in a gram of soil belong to thousands of different species, but often there are many more individuals of some species than of others.

Some techniques assess specific organisms. Others estimate the mass of a group of similar organisms or the total mass of organisms in the soil. The total mass of living organisms in a soil is termed the 'microbial biomass'.

It is not easy to observe where most organisms actually occur in the soil. Larger organisms such as earthworms and species can be easily seen and counted, but a microscope is required to observe them in sufficient detail for identification. Direct observation of organisms in soil allows their shape and size to be recorded. Some techniques extract living organisms from soil (e.g. soil sieving), but techniques that use chemicals such as wetting agents or heat kill organisms during the extraction process.

Measurement of soil microbial biomass

The simplest way to estimate the abundance of living organisms in the soil is to measure the amount of biological matter in the soil, or biomass (Coleman et al. 2004). This method is useful because it does not require the identification of individual organisms, or even the identification of individual species. The soil microbial biomass is a measure of the amount of carbon, nitrogen or phosphorus (and other elements) present within soil microorganisms, small animals and algae. The proportions of microbial carbon, microbial nitrogen and microbial phosphorus to total soil carbon, nitrogen and phosphorus are not the same in all soils. The dynamics of soil processes and the types of organic matter alter these proportions. Organisms also contain other elements, such as sulfur, and this can also be estimated in microbial biomass.

The earliest approach used was the fumigation-incubation (FI) technique (Jenkinson and Powlson 1976).

This approach involves a series of steps:

• the organisms in the soil are killed by fumigation (chloroform is used),

• living organisms are returned to the soil sample,

• time is allowed for the returned living organisms to breakdown recently killed organisms (incubation period),

• the nitrogen (or phosphorus) extracted, or the CO2 released by respiration is measured for the fumigated soil,

• the nitrogen (or phosphorus) extracted, or the CO2 released by respiration is measured for the unfumigated (control) soil,

• the difference between quantities of nitrogen (or phosphorus) in the fumigated and unfumigated soil is calculated.

The fumigation-incubation technique does not work well under all conditions (Martens 1995). For example, in waterlogged or acidic (low pH) soils, the breakdown of dead organisms by the newly introduced living organisms during the incubation period is reduced, therefore an estimate is required of how different soil conditions affect the degradation of biomass when calculating biomass (see Vance et al. 1987).

Modifications of these techniques for estimating microbial biomass include the fumigation extraction method (FE) and the substrate induced respiration (SIR) methods which have been adapted further for specific situations (e.g. Beare et al. 1992; Martens 1995)

From these general approaches, an estimate can be made of the quantity of carbon, nitrogen, phosphorus, sulfur etc. present in the total mass of microorganisms in the soil. These calculations are based on known estimates of the concentration of these elements in soil organisms. Although different soil organisms have different concentrations of these elements, an average for the whole community is used.

Direct observation of bacteria and fungi in resin-embedded soil

Soil can be embedded in a liquid resin that penetrates soil pores then solidifies (Burges and Nicholas 1961; Foster and Rovera 1973). Once solid, the soil block can be sectioned with a diamond knife. Thin sections are placed on a microscope slide for examination at high magnification. It is very easy to view the position of bacteria and fungi in relation to soil particles using this method but the organisms that are seen cannot be identified, only the number of individuals present can be counted.

Rossi-Cholodny slide technique

The Rossi-Cholodny slide method was an early method used to visualise organisms in soil. It requires thin glass slides to be inserted into the soil for some time (days or weeks). The slides were carefully removed from the soil without disturbing one side of the slide. The other side of the slide is heated gently to 'fix' the organisms onto the glass surface. The slide is then flooded with a stain to make the organisms visible (a stain such as Crystal Violet is commonly used). Algae as well as fungi are often visible on Rossi-Cholodny slides when viewed using a dissecting microscope. Microorganisms observed on Rossi-Cholodny slides are killed when the slide is heated. Hyphae can be measured (length and width) but the species of fungi to which the hyphae belong cannot be identified.

Sieving techniques for extraction of animals from soil

Larger soil animals can be extracted from soil by a variety of sieving techniques or by hand-sorting. Earthworms, beetles and spiders are extracted for identification or counting in this way.

Heating techniques for extraction of animals from soil

Many of the smaller soil animals such as mites and springtails are isolated from soil by placing cores of soil, supported on a mesh, under a suspended light bulb (e.g. Osler and Beattie 1999). The animals migrate to the bottom of the soil sample away from the heat of the light bulb and the drying soil. They fall through a supporting mesh via a funnel (called a Tullgren funnel) into a container. The temperature can be increased gradually during the extraction period to increase the rate of extraction of organisms. It is important that the soil does not dry out too quickly. If it does, some animals will not reach the bottom of the core.

A great variety of soil animals can be extracted from soil using the Tullgren funnel method (Smith et al. 2008)

Flotation techniques for extraction of animals from soil

Soil animals can be recovered from roots or soil samples using the Baermann method (McSorley and Frederická2004). In this technique, animals migrate out of a thin layer of moist soil supported on a wire mesh covered with cheese-cloth into the film of water in which they are suspended. This method is very effective in extracting nematodes.

Direct extraction of bacteria from soil

Bacteria can be extracted from soil and counted, and a small proportion can be grown for further study or identification. However, it is not easy to extract all of the bacteria from a sample of soil because they often occur in inaccessible pores within soil aggregates and inside fragments of organic matter. Repeated extractions do not remove all the bacteria. For example, the total number of bacteria removed after four extractions of soil using a fresh solution of calcium chloride (CaCl2) was still more than a quarter of the number removed by the first extraction (Bottomley and Maggard 1990). Similarly, it was difficult to remove all of one group of bacteria (rhizobia) from the soil sample.

A solution of CaCl2 is usually used to extract bacteria from soil because it has similar ionic properties to water held in soil pores (see Ruzek et al. 2005). This water is called the ‘soil solution’. An appropriate concentration of CaCl2 solution is shaken with a small sample of soil to release as many bacteria as possible. It may be necessary to use a wetting agent to disperse soil particles to release bacteria from inaccessible pores, especially in clayey soils. The solution is coarsely filtered to exclude larger soil particles, and filtered again. The holes in the filter need to be less than 1 Ám in diameter to collect the bacteria. The filter is examined using a microscope under very high magnification (at least 1000 times). The bacteria must be stained with a dye to be seen clearly; a common staining procedure used is the Gram Stain.

Bacteria can also be stained with a fluorescent dye (e.g. Roszak and Colwell 1987) such as acridine orange that stains the nucleic acid in living cells. Under a fluorescent microscope, bacteria stained in this way have bright orange nuclei.

It is not possible to identify bacteria seen with any of these direct methods of observation unless a technique such as the fluorescent antibody technique or molecular markers are also used.

Direct extraction of fungi from soil

The structure of fungi is more complex than that of bacteria. Fungi can exist as spores, fragments or strands of hyphae, hyphal mats or structures such as mushrooms or sclerotia. Several methods are used to extract fungi from soil.

Extraction of hyphae from soil

To extract hyphae from soil, a slurry is first prepared by adding water to a soil sample (usually one or two grams of soil). The slurry may be mixed in a blender and washed through a filter. The filter may need to be flushed with a staining solution to highlight the hyphae when viewed under a microscope because many hyphae are colourless and difficult to see. The diameters of the hyphae are measured against a calibrated scale in the eyepiece of the microscope.

Hyphal length is estimated using the line- or grid-intercept method. The length of hyphae is calculated according to the probability of hyphae touching a fine line visible in the microscope eyepiece or the lines of a grid marked a filter holding the hyphae. Hyphae are dispersed at random on the filter and the number of hyphae that intercept the line in the microscope eyepiece is recorded. The length of hyphae is calculated from the area of the field of view of the microscope, the area of the filter, the amount of soil solution added to the filter and the number of intercepts between hyphae and the line in the eyepiece or the lines in the grid (Giovannetti and Mosse 1980).

Hyphae extracted from soil and collected on a filter as described above may be alive, and can be distinguished from dead hyphae using specific stains that indicate the presence of active enzymes in living tissue. Normally there is a high proportion of dead hyphae in soil and many hyphae may have been damaged by the action of the blender. It is not easy to identify the species of fungus to which these segments of dead hyphae belong.

Extraction of fungal spores from soil

The spores of some fungi can be isolated from soil with relative ease. For example, spores of arbuscular mycorrhizal fungi are large (30 - 500 Ám in diameter) and can be removed from the soil by sieving. A soil suspension in water is washed through a set of sieves of with different mesh diameters (40 to 500 Ám diameters). After sieving, the organic matter collected on each sieve is hand sorted under a dissecting microscope and spores are identified if possible.

If a large quantity of organic matter is also present, another step can be included to separate the spores from the particulate organic matter. First, the material collected on the sieve is washed into a tube and centrifuged for a few minutes. The heavy particles sink with the spores and the particulate organic matter floats and is discarded. The remainder is mixed with a solution of sucrose (which is denser than water). The spores float in the sucrose after another short centrifugation. For clayey soils, it may be necessary to use a soil wetting agent to separate the spores from the soil particles. Spores extracted with a wetting agent are likely to be killed.

Isolation of bacteria and fungi from soil and growth in artificial media

When soil solutions are placed on agar plates or artificial media, many different types of bacteria and fungi appear on the plates after 1 to 5 days. Groups of similar species of bacteria or fungi can be selected by changing the composition of the growth medium. Therefore, the composition can be selected to suit the growth of some organisms and discourage others. In addition, antibiotics can be added to the growth media to preferentially grow particular groups. This procedure is used to isolate bacteria from soil without interference from fungi.

However, there are many difficulties in isolating bacteria and fungi from soil and growing them on agar plates. It is important to remember that only about 1 to 5% of all soil bacteria or fungi will grow on the types of artificial laboratory media currently available. Therefore, organisms that are isolated from soil and grown in this way only represent a very small proportion of the total number and types present in the soil.

Bacteria grow at different rates; some grow very slowly. When a soil solution is added to an agar plate, the fast-growing bacteria multiply first, inhibiting the slower-growing bacteria because they are better at competing for nutrients or space. Furthermore, some bacteria, irrespective of their growth rate, produce antibiotics that inhibit the growth of other bacteria. These examples illustrate why artificial culturing media cannot be used to accurately assess the diversity of bacteria or fungi in soil.

Quantification of soil organisms using the most probable number [MPN] technique

The Most Probable Number (MPN) technique is used to assess the number of some kinds of bacteria, fungi or soil animals (such as protozoa) in water, soil or plants without isolating them (e.g. Adams and Welham 1995). The technique uses a soil dilution procedure. It dilutes a sample of soil until it is highly probable (according to a statistical test) that the organisms under investigation no longer occur in the sample. There are published tables of MPN probabilities available for estimating the number of organisms present based on the number of dilutions required to exclude them from the soil sample. The greater the number of dilutions required the higher the number of organisms in the original soil sample.

The MPN method has been used to estimate the number of rhizobia in soil (Somasegaran and Hoben 1985). To do this, the diluted field samples are added to sterile tubes containing a legume seedling growing either on agar or in a medium such as vermiculite. If rhizobia, which can colonise the particular legume used, are present in the diluted sample, a nodule will form on the seedling (see later for an explanation of the nodulation process). The dilution level at which no nodules are formed is used to calculate the probability that there were a certain number of root nodule bacteria in the original soil sample.

The MPN method can be used to estimate numbers of other soil bacteria, such as nitrifying bacteria that convert ammonium to nitrite and nitrite to nitrate. In this case, calculation of the most probable number of bacteria is based on a physiological process - the conversion of ammonium to nitrate by nitrifying bacteria. Diluted soil samples are introduced into sterile media containing ammonium without an organic carbon source. After many weeks an assessment is made of the presence or absence of ammonium in the media. The long incubation time is necessary because these bacteria grown very slowly and the rate of conversion of ammonium to nitrate is very slow under these conditions. The soil dilution at which no ammonium is converted to nitrate is identified. This dilution level is used to estimate the probability that a certain number of nitrifying bacteria was present in the original soil sample.

The MPN technique is also used to assess populations of protozoa in the soil. Protozoa are cultured on a special nutrient medium, with a bacterial culture as a food source. A dilution series of soil extracts is used to estimate the population size of the protozoa, as explained above for bacteria.

Use of baiting techniques to isolate fungi and bacteria

Baiting techniques use a living plant, seed or other plant parts to attract and encourage the growth of a certain organism so that it can be isolated from the soil and/or quantified. For example, pieces of potato can be used as baits to isolate pathogens of potato that belong to the bacterial genus, Erwinia (de Boer et al. 1979).

Baiting techniques are used to isolate populations of plant pathogenic fungi and mycorrhizal fungi in the soil. In a bioassay, plants are grown for several weeks in field soil and assessed for the presence of root pathogens or other fungi. For some fungi, a bioassay can be used to indicate the abundance of the fungi. However, bioassays usually provide only rough estimates of the number of fungi in the soil unless they are calibrated using field soils where the number of fungi present is known. This calibration is easier to do for some organisms than for others.

Straw and seeds can be used as baits to isolate fungi and bacteria that colonise various forms of organic matter in soil. Similarly, appropriate plants can be grown in soil to bait soil animals such as plant parasitic nematodes.

Estimation of soil microbial biomass from ATP and enzyme assays

The activity of a number of biochemical components of living cells can be used to estimate the abundance or activity of microorganisms in the soil. Enzymes which are suitable for this method are those which are not excreted by soil organisms, whereas enzymes that are excreted by bacteria and fungi through their cell walls are not suitable (Higashida and Takao 1985). Excreted enzymes, called extracellular enzymes, are involved in the breakdown of organic matter or transformation of other molecules in soil. Since extracellular enzymes may remain active after the organisms that produced them have died, they are not necessarily directly related to microbial biomass or microbial activity.

In this study, one enzyme (fructase) has a different level of activity in the soil to another (urease). The activity of fructase was more closely related to soil microbial biomass than the activity of urease. Fructase is not released into the soil and is associated only with living soil microbial biomass. In contract, urease is an extracellular enzyme.

Characterisation of communities of fungi and bacteria using fatty acid methyl ester (FAME) assays

Fatty acids can be extracted from bacteria or from their cell membranes and separated using a gas chromatograph (Olsson 1995). The pattern produced by the gas chromatograph has peaks representing the presence of certain types of fatty acid. Similar techniques have been developed for distinguishing between groups of bacteria and groups of fungi in soil without first having to isolate the organisms.

Quantification of functional groups of soil organisms

Different soil organisms are able to carry out the same functions in soil. This applies to common processes such as the release of ammonium from organic matter and the conversion of ammonium to nitrate. This is because many soil organisms perform similar functions, and are collectively called 'functional groups'. These groups can be quantified.

One method for quantifying the number of functional groups of soil organisms is to determine the 'microbial catabolic diversity' of soils. This approach quantifies the microbial population in the soil based on its ability to degrade a wide selection of organic carbon molecules, which are added separately to the soil. The amount of soil respiration is measured after the addition of each carbon molecule and this is used to indicate the level of degradation (see earlier description of the substrate induced respiration method). The pattern of degradation of these substrates corresponds with the expected activities of organisms in soil (Degens and Harris 1996). An alternative method measures enzyme activity in response to the addition of a range of carbon substrates to a solution containing organisms washed out of the soil.

Quantifying the functional groups of organisms in soil involved in specific processes is an important research area as it provides knowledge about the genetic diversity of soil communities. Understanding the diversity of soil organisms may be for important for questions about the sustainability of land use.

Relationships between abundance of different types of organisms

Close links exist between the abundance of some groups of organisms in the soil and in some instances, it is possible to use an assessment of the abundance of one group of organisms to estimate the abundance of another. An example of this is the strong correspondence between the number of fungus-eating amoeba and the quantity of fungal hyphae in soil (Gupta and Germida 1988). This reflects the strong link between the quantity of amoebae and their food source (hyphae).

The relative abundance of fungi and bacteria can also be estimated as an indication of the status of the biological component of soil (see Joergensen and Wichern 2008). Generally, a higher ratio of fungi to bacteria is indicative of more stable soil communities, whereas bacterial dominance is more common in intensively managed agricultural soils.

Why is it useful to use more than one technique to quantify soil organisms

Quite often, different techniques produce quite different estimates of the number of soil organisms for the same soil. For example, direct observation and plate counting can produce widely different estimates of the number of bacteria (Russell 1973). The number of bacteria counted using the plate-count method is usually only about 1% of the number that is counted by directly counting bacteria extracted from soil and collected on a filter. Two reasons for this are (i) that many of the bacteria will not grow on the artificial media used in plate counting, and (ii) that not all bacteria isolated on a filter would have been alive in the soil at the time of sampling.

The difference between the direct and indirect methods for assessing soil organisms is particularly important when making comparisons between different soil management treatments. Adding manure increases the abundance of bacteria in an alkaline soil, but decreases abundance in a more acid soil (Russell 1973). This effect was much greater when plate counts of bacteria were used than when direct observation counts were made.

This example demonstrates why it is necessary to have a good understanding of:

• the reliability of different methods for counting soil organisms,

• the ecology of the organisms being assessed, and

• how this information is to be used.

Molecular tools are now also used for identifying or estimating the abundance of a particular organism in soil (e.g. Griffiths et al. 2003). Detailed knowledge of each technique and the assumptions contained within them is essential for interpreting the results of assessment of the abundance of soil organisms. Furthermore, it also needs to be emphasised that every method of estimating the number of organisms in a soil sample is, at best, only an approximation of the real number that is present.

Summary Points (1.2)

• Soil contains an immense diversity of organisms.

• Most of the organisms that live in the soil have not been identified and cannot be grown in the laboratory.

• Specific techniques are required to estimate the abundance of different types of soil organisms.

• It is not easy to isolate all the bacteria from soil because they can occur in inaccessible pores within soil aggregates.

• Many different techniques can be used to identify bacteria and fungi extracted from soil, including the techniques of molecular biology, serology and plant bioassays, as well as direct observation.

• Soil animals can be extracted using techniques that involve sieving, heating and baiting.

• Microscopic examination or molecular tools can be used to identify soil organisms.

• Different techniques may give different estimates of the abundance of the same group of soil organisms, therefore, care is needed in interpreting this information.

• The complex nature of quantifying and identifying soil organisms has hampered the study of organisms in soil.

• New methods are available to quantify functional diversity of soil organisms.

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