CHAPTER 2 Soil Microfauna: Diversity and Applications of Protozoans in Soil Stuart S. Bamforth CONTENTS Introduction Role of Soil Protozoa Measuring Protozoan Biodiversity Protozoan Diversity in Agroecosystems Applications Conclusions References INTRODUCTION Agricultural plant production depends upon the decomposition of plant and animal residues, as well as fertilizers, into simpler compounds, many of which are transformed into microbial and animal protoplasm. These organic materials are eventually mineralized into simpler compounds, such as CO 2 , ammonia, and phos- phate, which are absorbed by plant roots. ROLE OF SOIL PROTOZOA Microarthropods and larger fauna, especially earthworms, increase the rate and amount of mineralization by comminution of organic matter and by redistribution of “hot spots” of activity through movements. However, mineralization and return © 1999 by CRC Press LLC. of nutrients to plants occur in the water films covering soil aggregates and filling their pores. Here, bacteria and fungi decompose organic matter and immobilize the extracted nutrients into their bodies, but grazing by the microfauna, protozoa, and nematodes regulates and modifies the size and composition of the microbial com- munity and enhances microbial growth through microfaunal excretions. Nematodes also graze fungi (Chapter 1), but protozoa, especially amoebae, can graze bacteria in tiny pore spaces unavailable to nematodes. The degree of nutrient recycling is influenced by external factors of climate and soil management (e.g., inputs of fertilizers and biocides, compaction by farm machinery) and internally by the com- munity of protozoa and nematodes, reflected in their biodiversity. Most of the microfauna are located in small hot spots scattered through the soil mosaic, which is soil aggregates of 1 mm or smaller, containing bits of organic matter, detritus and the overlying litter, rhizosphere, and the “drillosphere” parts of the soil influenced by earthworm secretions and castings. The microbial-feeding microfauna constitute an essential component of the soil ecosystem; therefore, changes in their community structure can influence mineralization and soil fertility. MEASURING PROTOZOAN BIODIVERSITY Soil protozoa comprise four groups: the “naked” rapidly growing flagellates, amoebae, and ciliates, and the more slowly growing shelled amoebae, or testacea. The small size and flexibility of the first two groups allows them to exploit small pore spaces, and they furnish most of the protozoan numbers. The more diverse and larger ciliates and testacea inhabit the larger pore spaces which are subject to desiccation and other stresses; consequently, both groups show a wide spectrum of species of r/K selection and degree of autochthonism (Wodarz et al., 1992). Ciliates are divided into pioneer r-selected Colpodida, competitive K- selected Polyhymenophora, and intermediate remaining taxa. Dividing the number of species of the first group by the second produces a C/P ratio, where C/P > 1.00 indicates a stressed soil of low productivity, and C/P < 1.00 a more productive soil with microarthropods and macrofauna (Foissner, 1987; Yeates et al., 1991). Among the testacea, certain species indicate soil acidity or alkalinity, and the shell conveys information about moisture fluctuations (Bonnet, 1964). Consequently, these two groups can serve as bioindicators of soil conditions. Ideally, biodiversity studies measure both species and numbers per species. However, the small size and transparency of naked protozoa make them too difficult to find among soil particles; consequently, counting has been traditionally performed by the most probable number (MPN) technique of Singh (1946) or its modification by Darbyshire et al. (1974). There are criticisms of the method (Foissner, 1987), in response to which a second direct count method was developed by Griffiths and Ritz (1988) that separates the protozoa from soil particles by percoll phosphate gradient centrifugation and staining for fluorescent microscopy. This method is employed routinely to measure the protozoan component of the soil fauna in experimental field crop studies by the Technical University of Munich. The larger and more motile ciliates can be counted by examining a watered soil suspension drop-by-drop until © 1999 by CRC Press LLC. at least 0.4 g of fresh soil has been examined (Foissner, 1987). The possession of a shell enables direct counting of testacea by this method, or by staining a soil suspension and mounting small samples on slides, providing a permanent record (Couteaux, 1967; Korgonova and Geltser, 1977). Combining the temporary and permanent methods provides a more complete census than either method alone. Estimating species richness is best done by placing 10 to 50 g of sample in a petri dish and adding water until 5 to 20 ml will drain off when gently pressed with a finger. By placing several coverslips, each underlaid with a piece of lens paper on top of the sample, and examining after 1 day, a variety of flagellate species will be revealed. The culture is examined at 3 to 4 day intervals for a month to determine the succession of species of mainly ciliates and testacea (Foissner, 1987). Most amoebae will be found by streaking soil samples on bacterized water (non-nutrient) agar plates or by placing soil samples in wells cut in such plates. The amoebae migrate out from the soil particles (Bamforth, 1995a). PROTOZOAN DIVERSITY IN AGROECOSYSTEMS Studies on grasslands (McNaughton, 1977; Tilman, 1996) show that biodiversity stabilizes community and ecosystem processes, although individual species within the system may fluctuate considerably. Tilman (1996) found wide variations in the biomass of the 24 most abundant species of plants in an 11-year study. In a 6-month study of soil ciliates under a spruce stand, Lehle (1992) found that the proportions of the three dominant ciliate species fluctuated widely: Cyclidium muscicola ranged from 8 to 75% of the total populations and two colpodid species varied from 4 to 45%. The different responses in these two studies may reflect changes in the realized niches of species; thus, biodiversity furnishes a reservoir of biotic abilities contrib- uting to ecosystem sustainability (Bamforth, 1995b). Biodiversity, like the compar- ison of nontillage to conventional agriculture, may not produce noticeable increases in crop production, but maintaining biodiversity can retard the deterioration that has characterized agroecosystems through 4000 years of human history. Protozoa can serve as bioindicators of ecosystem conditions, and warn of soil impoverishment. The appeal of protozoan bioindicators is their environmental sensitivity due to their delicate cell membranes, their rapid growth rate, restricted movement in soil, ubiquity, and wide range of morphologies in ciliates and testacea, providing a multispecies approach enabling community analyses to indicate soil conditions (Foissner, 1994). Difficulties arise in the taxonomy and time needed for identification and enumeration, but, as the following applications illustrate, protozoa convey valu- able information about agroecosystems because of their pivotal position in the nutrient cycling that all terrestrial ecosystems depend upon. APPLICATIONS Conventional agriculture creates a special ecosystem by mixing the topsoil (and compacting it) through tillage, removing plant canopies that protect the soil, adding © 1999 by CRC Press LLC. fertilizers and biocides, and removing harvests. A more sustainable agriculture minimizes topsoil disturbance, reduces inputs, and substitutes organic for mineral fertilizers (Doran and Werner, 1990). Plowing in conventional agriculture incorporates crop residues into the soil profile to produce homogeneous soils that favor the bacteria, protozoa, and bac- tivorous nematode portions of the underground food web; in contrast, minimal tillage leaves organic residues on the surface and a rich organic layer near the surface, enhancing the fungal, Collembola, and earthworm portions of the under- ground food web (Hendrix et al., 1986; Lee and Pankhurst, 1992). The protozoan communities differ between the two systems in the greater prominence of r-selected colpodid ciliates (reflecting less species diversity) in conventional fields (Foissner, 1992; Bamforth, unpublished data). The biomass of amoebae and flagellates, how- ever, is greater in the surface layer of ecofarmed systems (DeRuiter et al., 1993) and is associated with increased nitrogen mineralization (DeRuiter et al., 1993). Using testacea as bioindicators, Wodarz et al. (1992) found organically farmed field and vineyard soils showed improved soil conditions over conventionally farmed counterparts. Organic fertilizers, especially straw and animal manures, are more similar to natural organic substrates than chemical fertilizers. Microbial and protozoan activity is highest in organically enriched soils (Schnurer et al., 1985; Aescht and Foissner, 1991; 1992), and is usually accompanied by increases of most soil fauna, especially earthworms (Doran and Werner, 1990), which enhance protozoan biodiversity. The higher protozoan activity in soils under nontillage and organic fertilizer management is enhanced by other fauna, especially earthworms, which disperse bacteria and their protozoan predators to new locations, through burrowing move- ments and passing ingested cysts through guts, providing new hot spots and releasing greater quantities of nutrients, which have led to increased plant yields in a few cases (Brown, 1995). Ingested active protozoa furnish a highly assimilable food source, sustaining the fauna that enhance microbial and protozoan activities (Brown, 1995). Thus, high protozoan biodiversity usually reflects earthworm abundance. The application of biocides often influences other organisms besides those tar- geted. Herbicides have little effect on protozoa, although they may influence them indirectly by altering bacterial nitrogen activities and by modifying the environment in eliminating the vegetation over the soil. Insecticides and fungicides are more toxic, as shown in the study of Petz and Foissner (1990) on the effects of lindane, an insecticide, and mancozeb, a fungicide, on the soil ciliate and testacean commu- nities of a spruce forest. The insecticide decreased both numbers and species, and altered the community structure of ciliates by increasing the abundance of several colpodids. This result shows the value of multispecies-monitoring studies, and also the value of biodiversity to an ecosystem, allowing response to changing conditions (Bamforth, 1995b). The insecticide exerted less effect on testacea, and the fungicide exerted little influence on both groups. The investigation used a randomized block design and extended the study period to the 90 days needed to ascertain if the biocide caused acute toxicity (Domsch et al., 1983). This type of study shows the precision that protozoan bioindicators can provide to assess agroecosystem conditions. © 1999 by CRC Press LLC. The heavy machinery used in modern farming causes soil compaction, destroying not only the worm and root channels that reduce soil porosity and the larger fauna, but also reducing pore spaces in which bacteria and their protozoan predators live. Compaction reduces testacean species diversity and eliminates large forms (Berger et al., 1985), and a number of studies relating pore space to protozoan activity (e.g., Rutherford and Juma, 1992; England et al., 1993 ) show less activity in smaller spaces. Griffiths and Young (1994) found the same trend and concluded that com- paction influences protozoa indirectly by producing anaerobic conditions that inhibit protozoa and reduce the metabolism and reproduction of their bacterial prey. A vital part of agricultural management is soil conservation and restoration, which can be monitored by analyzing the protozoan community to assess the degree of the comprehensive biological activity to productive farming (Yeates et al., 1991; Wodarz et al., 1992). CONCLUSIONS Protozoa and nematodes are pivotal organisms in agroecosystems because their predation upon bacteria increases mineralization of nutrients necessary for plant growth. Since biodiversity stabilizes community and ecosystem processes (Tilman, 1996), maintaining and increasing protozoan biodiversity will contribute to more sustainable agriculture. Ecofarming and organic fertilizer management enhance pro- tozoan activity. 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