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The success of biocontrol bacteria in soil depends in part on their ability to escape predation. We explored the interactions between Pseudomonas strain DSS73 and two predators, the nematode Caenorhabditis elegans and the flagellate Cercomonas sp. Growth of the nematode in liquid culture was arrested when it was feeding on DSS73 or a DSS73 mutant (DSS73-15C2) unable to produce the biosurfactant amphisin, whereas a regulatory gacS mutant (DSS73-12H8) that produces no exoproducts supported fast growth of the nematode. The flagellate Cercomonas sp. was able to grow on all three strains. The biosurfactant-deficient DSS73 mutant caused severe dilation of the nematode gut. In three-species systems (DSS73, Cercomonas and C. elegans), the nematodes fed on the flagellates, which in turn grazed the bacteria and the number of C. elegans increased. The flagellates Cercomonas sp. usually kill C. elegans. However, DSS73 protected the nematodes from flagellate killing. Soil microcosms inoculated with six rhizobacteria and grazed by nematodes were colonized more efficiently by DSS73 than similar systems grazed by flagellates or without grazers. In conclusion, our results suggest that C. elegans and DSS73 mutually increase the survival of one another in complex multispecies systems and that this interaction depends on the GacS regulator.

Flagellates are very important predators on bacteria in soil. Because of their high growth rates, flagellate populations respond rapidly to changes in bacterial numbers. Previous results indicate that actinobacteria are generally less suitable than proteobacteria as food for flagellates. In this study, we investigated the growth of the flagellate Cercomonas sp. (ATCC 50334) on each of the two bacteria Sphingopyxis witflariensis (Alphaproteobacteria) and Rhodococcus fascians (actinobacteria) separately and in combination. The growth rate of the flagellate was lower and the lag phase was longer when fed with R. fascians than when fed with S. witflariensis. This supports our initial hypothesis that the actinobacterium is less suitable as food than the alphaproteobacterium. However, after longer periods of growth the peak abundance of flagellates was higher on R. fascians, indicating that the food quality of bacterial prey depends on the time perspective of the flagellate-bacterial interaction. There was no evidence that the flagellates selected against the actinobacterium when feeding in mixed cultures of the two bacteria. Experiments where flagellates were fed with washed bacterial cells or with bacteria growing with different substrate concentrations suggested that the low food quality of R. fascians is related both to the intrinsic cell properties and to the extracellular metabolites.

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.

Heterotrophic flagellates occur in nearly all soils and, in most cases, many different species are present. Nevertheless, quantitative data on their community structure and diversity are sparse, possibly due to a lack of suitable techniques. Previous studies have tended to focus on either total flagellate numbers and biomass, or the identification and description of flagellate species present. With the increased awareness of the role of biodiversity and of food web interactions, the quantification of species within the community and their response to environmental change is likely to become more important. The present paper describes a modification of the most probable number method that allows such a quantification of individual flagellate morphotypes in soil samples. Observations were also made on the biomass of flagellate morphotypes in soil. 20 to 25 morphotypes of heterotrophic flagellates were detectable per gram of two different arable soils, which were treated experimentally to test the technique. One of the soils was fumigated with chloroform vapour for different lengths of time (0, 0.5, 2 or 24 hours); this led to a reduction in the number of morphotypes, in the Shannon diversity index and in the evenness. The other soil was planted with wheat, and while rhizosphere soils contained the same morphotypes as bulk soil, the abundance of individual morphotypes was significantly different and the Shannon diversity index in rhizosphere soils was significantly higher. Soil influenced by an elevated CO2 level likewise differed significantly in morphotype abundance when compared to soil exposed to ambient levels of CO2. The technique recovered more than 80% of the discernible morphotypes and could also be used to quantify amoebal and ciliate communities in a similar way.

An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.

Heterotrophic flagellates and naked amoebae are usually very numerous in agricultural soils; with numbers in the magnitude of 10,000 to 100,000 (active+encysted) cells per gram of soil. In 'hotspots' influenced by living roots or by dead organic material, the number may occasionally be as high as several millions per gram of soil. An exact enumeration of these organisms is virtually impossible. As they most often adhere closely to the soil particles, direct counting will underestimate numbers since the organisms will be masked. The method usually applied for enumeration of these organisms, the 'most probable number (MPN) method', is based on the ability of the organisms to grow on particular culture media. This method will in many cases underestimate the total protozoan number (active+encysted). It is uncertain how many of the heterotrophic flagellates and naked amoebae are actively moving and how many are encysted at a particular time; the 'HCl-method' which has usually been used to discriminate between active and encysted has proven to be highly unreliable. Despite the methodological difficulties many investigations of these organisms indicate that they play an important role in agricultural soils as bacterial consumers, and to a minor extent as consumers of fungi. Because of their small size and their flexible body they are able to graze bacteria in small pores in the soil in which larger organisms are precluded from coming. Key factors restricting the number and activity of heterotrophic flagellates and naked amoebae in soils seem to be water potential and soil structure and texture. In micro-cosm experiments, small heterotrophic flagellates and naked amoebae regulate the size and composition of the bacterial community. Bacterial activity seems to be stimulated by these organisms in most cases as well as the mineralization of carbon and nitrogen and possibly other mineral nutrients. In the rhizosphere of living plants the activity of protozoa has proven to stimulate uptake of nitrogen in pot experiments, and it has been hypothesized that organic matter liberated by plants in the root zone will stimulate bacterial and protozoan activity, leading to mineralization of organic soil nitrogen which is subsequently taken up by the plants.