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Wood ash recycling to forests is beneficial because it regains nutrients and prevents acidification, but wood ash application is restricted due to its cadmium (Cd) content. We question if Cd in wood ash represents a problem, since decreases in Cd bioavailability due to ash-induced pH changes may counteract increased total Cd concentration. We studied effects of wood ash (0, 3, 9 and 30 t ha −1 ) and lime (pH increase equivalent to the wood ash treatments) on growth and Cd uptake in Deschampsia flexuosa. After four months, we measured plant biomass and Cd accumulation, and extracted Cd from the soil using three different methods; HNO 3 (total), EDTA (chelator-based) and NH 4 NO 3 (salt-based). Wood ash and lime strongly stimulated plant growth. Cd concentration in the plant tissue decreased with wood ash and lime addition, and correlated positively with the NH 4 NO 3 extractable fraction of Cd in the soil. In contrast, HNO 3 and EDTA extracted more Cd with increased wood ash application. We conclude that wood ash amendment increases soil pH, total Cd concentration, nutrient levels and stimulates plant growth. However, it does not increase Cd accumulation in D. flexuosa, as pH-driven decreases in Cd bioavailability leads to reduced plant Cd uptake. Finally, soil bioavailable Cd is best determined using NH 4 NO 3 -extraction.

Cadmium (Cd) is one of the most toxic heavy metals found in soil. Arbuscular mycorrhiza (AM) is known to reduce Cd translocation in plants by immobilising Cd in the root system. The effect of mycorrhiza on plant Cd uptake is usually studied in simple systems with single strains of mycorrhizal fungi and few levels of Cd. Here we studied how a wide range of soil Cd concentrations affected plant AM colonisation, and how the species-specific differences in AM colonisation affected uptake, translocation, and toxicity of Cd in plants in a system with naturally occurring mycorrhizal fungi. Six plant species were grown in pots in a greenhouse across seven levels of Cd, which made it possible to model dose-response curves, and calculate EC50 for each plant species. We found a remarkable trend where Cd at moderate levels stimulated mycorrhizal colonisation until a certain threshold where the symbiosis breaks down. Our results support the existence of a protective effect of AM fungi against Cd, as the symbiosis reduces Cd translocation to shoots, especially in plants with very high AM colonisation. Thus, we conclude that it is the combination of plant species and AM colonisation that determines Cd uptake in plants. AM is therefore an essential trait to consider when growing plants in Cd-polluted soil.

Small heterotrophic protists (flagellates and naked amoebae) are very abundant in soil and play a key role in maintaining soil services. Hence, knowledge on how xenobiotics affect these organisms is essential in ecosystem management. Cadmium (Cd) is an increasing environmental issue as both industrial deposition and recycling of heavy metal rich waste products have led to Cd enrichment of soils. Evaluation of toxicity of Cd to micro-organisms is often performed using a solution of pure Cd (e.g. CdCl) in liquid culture. This approach may be highly misleading as interactions between Cd and other substances, e.g. various ions or inherent soil components often strongly modify Cd toxicity. Hence, we compared the toxic effect of Cd to small heterotrophic protists in soil microcosms and liquid culture. We also evaluated how zinc (Zn) affects Cd toxicity, as Zn usually accompanies Cd in a ratio of c. 100:1, and is known to impede Cd toxicity. In the soil microcosms, we also monitored the primary food source of the protists, i.e. culturable bacteria, and used soil respiration as a proxy of soil functioning. Finally, we examined to what extent Cd actually sorbs to soil. We found 1) that c. 103 times more Cd was required to obtain the same effect in the soil microcosms compared to the liquid culture, 2) that soil sorption explains why Cd, even though highly toxic in aqueous solutions, has very limited effect when applied to soil, and 3) (very surprisingly) that in our experimental systems Zn was as toxic as Cd. Our study suggests that Cd toxicity to soil protists will be small because most Cd in soil will be sorbed to the soil matrix and because the Zn:Cd ratio of 100:1 in most substances, incl. pollutants, will mean that lethal Zn effects will occur before Cd reaches toxic levels.

Due to their small size, microorganisms can be widely dispersed and they colonize sterilized soils fast. However, details regarding quickness and successional patterns are poorly understood. We investigated the ability of airborne microorganisms to colonize sterilized soil at different heights above ground, and at different distances to a tree. Soil below the tree was colonized by bacteria already after 1 day and the sites above and away from the tree after only 4 days. The site below the tree was the only one to be colonized by culturable protists during the 15 day experiment. Our results show a pattern of succession when sterile soils are colonized by bacteria and protists. The tree was found to have a substantial impact on the recruitment of microorganisms.

International audience; Promoting diverse and functioning biological communities is an important objective of agroecology, with increasing attention given to the important role of soil biodiversity. In an experimental study conducted under field conditions, we followed over four years the dynamic of soil organisms from various sizes and trophic niches in four variants of a cropping system which are differentiated by soil tillage, residue management and N fertilization rate. Differentiation in overall family diversity, as well as in the abundance and diversity of the multiple trophic groups was evaluated every two years. Our study demonstrated a delayed but effective differentiation in soil biota diversity following implementation of the agricultural practices. Soil biodiversity varied throughout time with some groups responding more readily than others, thereby highlighting differences related to trophic position and body size. The visualization of diversity profiles revealed an increasing impact of agricultural practices on group diversity towards higher trophic levels. While tillage appeared a main factor of influence, surprisingly little impact of residue management and nitrogen fertilization could be observed. Predicting the response of the soil biota to anthropogenic influence calls for an understanding of complex interactions between soil organisms in heterogeneous soil microhabitats. Through its multitaxonomic approach, the present study increases our understanding of the dynamic of soil communities in agricultural cropping systems and helps identify possible consequences for soil functioning.

Yellow River Delta undergoes intensive conversion from natural wetland to agricultural fields and artificial woodland. In this study, we analysed how the conversion affects bacterial community diversity and composition by Illumina Miseq sequencing combined with functional prediction. Compared to natural wetland, arable land and woodland were featured with higher soil organic matter, total nitrogen and bacterial diversity, but lower electrical conductivity. The bacteria Gemmatimonadetes related to soil organic matter and total nitrogen, was enriched in arable land, while salt-resistant bacteria (e.g. phylum Chloroflexi and its class Ardenticatenia) were abundant in natural wetland. Moreover, the relative abundances of the nitrifying bacteria Nitrospira and Nitrosospira were significantly higher in arable land and woodland than in natural wetland, suggesting that land use changes significantly affect the bacterial processes involved in nitrogen cycling. Redundancy analysis (RDA) showed that the differences in bacterial community were attributed to soil nutrient-related properties (i.e., total nitrogen and soil organic matter), soil salinity (i.e., electrical conductivity), and heavy metals (i.e. Cu and Cr). PICRUSt results revealed that land use conversion from natural wetland to arable land increased soil functions, e.g., biosynthesis process and oxidative phosphorylation. The data help us elucidate how land use changes affect terrestrial ecosystem function, and advise local farmers to apply the suitable land-use strategies and keep agricultural sustainable development.

Wood ash, the by-product of biomass combustion to energy, can return important nutrients back to the soil and counteract acidification. However, the application of wood ash may affect the emission of greenhouse gases. Here, the effect of wood ash application on nitrous oxide (N2O) emissions from different soil environments were investigated in a 40 days incubation experiment comprising ten different soil types amended with five different wood ash concentrations (0, 3, 9, 20, and 54 t ash ha−1). The emitted N2O was measured continuously, and initial soil properties without ash application (carbon (C), nitrogen (N), ammonium (NH4+), nitrate (NO3−), and pH) and resulting soil properties (pH, NH4+, and NO3−) were measured prior and after the incubation period, respectively. The Random Forests (RF) model was used to identify which factors (initial and resulting soil properties, vegetation, management, wood ash doze, and respiration rate) were the most important to predict the development of emitted N2O after ash application. Wood ash either increased, decreased, or had no effect on the amount of emitted N2O depending on soil type and ash dose. The RF model identified the final resulting pH as the most important factor for the prediction of emitted N2O. The results suggest that wood ash can mitigate N2O emissions from soil, however, this effect depends on soil type where a mitigating effect of wood ash application was observed mainly in low pH soils with high soil organic matter whereas an increase in N2O emissions was observed in mineral soils that had previously received N fertilization. This study emphasises the importance of pH manipulation in regards to N2O emissions from soil.

We describe two novel flagellates isolated from soil, Regin rotiferus and Otto terricolus, genera et species novae, which we cultivated and characterized by light and transmission electron microscopy and by 18S rDNA sequence analysis. Both strains exhibit the key characteristic structural feature of Bicosoecida; i.e. the L-shaped cytostomal root system with an x-fiber, used for feeding. Otto terricolus displays unique novel morphological traits; thus, it has a basal swelling on each flagellum, a root 3/root 2 distribution of 10 + 1 microtubules, and an amoeboid stage in its life cycle. Regin rotiferus has flagella without swellings and a root 3/root 2 distribution of 7 + 3 microtubules, a pattern commonly observed in the Bicosoecida. We present an updated exhaustive maximum likelihood phylogeny of 48 cultured, complete or nearly complete (+1600 bp) 18S rDNA Bicosoecida sequences. Both new species fall into a well-supported freshwater Siluaniidae clade, without being particularly closely related. The morphology and phylogeny do not conclusively support Rictus as a member of Bicosoecida.

Isotope fractionation is a ubiquitous phenomenon in natural ecosystems. When chemical elements move through food chains, natural isotope ratios change because biological processes tend to discriminate against heavier isotopes. This effect can be used to trace flows of matter, estimate process-rates and determine the trophic level of organisms in biological systems. While it is widely accepted that 15N-accumulates in natural food-chains, it is disputed to which extent this is the case for 13C. We constructed sand-microcosms inoculated with a dilution series of soil organisms and amended with glucose as the source of organic carbon. We demonstrated that the proportion of 13C in respiratory CO2 correlated inversely with community complexity. Our results therefore suggest that increasing community complexity, with increasing synergy, competition and predation, facilitates increasing 12C–13C isotopic fractionation.

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance. While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (

We grew plants in soil amended with either finely ground ( 2 ) of maize leaves. Finely ground material will be mineralized faster; hence nitrogen is released faster in the soil. We inoculated the soil with the plant growth inhibiting bacterium Arthrobacter BEM463, a relatively poor food source for bacterial feeders, and the plant growth promoter Sphingopyxis BEM760 which is a good food source. Protozoa and nematodes were introduced separately or in combination. In soil with larger maize leaf pieces (low turnover, slow nitrogen release), bacterial feeders generally increased plant nitrogen uptake and growth. In systems with finely ground material (high turnover, fast nitrogen release), plants hardly benefited from presence of grazers; bacterial mineralization activity alone probably provided enough N for the plants. The harmful Arthrobacter thrived better when grazed by protozoa than when grazed by nematodes, probably because the selective protozoa could avoid it, which resulted in reduced plant performance. We suggest that bacterial feeders generally increase plant performance when nutrients are limited. At high nutrient availability, however, bacterial feeders promote grazer resistant bacteria, and, if these bacteria are harmful to plants, the net effect of bacterial feeders on plant growth can be negative.

We know little about the ability of protozoa to colonize soils, including their successional patterns. To elucidate this issue, we investigated in which order different protozoan morpho-types colonize sterile soil. We used sterilized soils with different carbon content, and exposed them to the atmosphere for a period of 14 months. Bacteria and flagellates occurred in high numbers after 25 days. Ciliates reached high abundance levels after 137 days, followed by amoebae after 245 days. Numbers of distinguishable protozoan morpho-types increased until 245 days after exposure and declined thereafter. DGGE bands showed a remarkable shift in bacterial diversity with time and a significant increase of detectable gene-types until day 137.

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).

We compared the abundance and diversity of cultivable protozoa (flagellates and amoebae) in a polycyclic aromatic hydrocarbon (PAH) polluted soil and an unpolluted control, by isolating and cultivating clonal strains. The number of cultivable protozoa was higher in the polluted soil; however, the polluted soil displayed an impoverished community, dominated by certain taxa, such as Acanthamoeba sp. We isolated a total of 31 protozoan strains to characterize them morphologically and by 18S rRNA gene sequence analysis. This approach, i.e. combining morphological and molecular information had the advantage of providing quantitative data, information on morphology but also an accurate positioning of the isolates in 18S rRNA trees.

Pseudomonas bacteria isolated during 52 days on Gould's S1 agar from soil spiked with 0, 3.5 and 15 mg Hg(II) kg soil−1 were characterised to reveal whether mercury affected them differently. Isolates from the treatments with 0 and 15 mg Hg kg−1 were characterised using FT-IR characterisation and subsequent 16S rDNA partial sequencing of representative isolates. To verify the selectivity of Gould's S1 agar and the FT-IR characterisation, all 450 isolates were subjected to the following tests: Gram-determination, catalase and oxidase activity, pigment production on PDA and growth at different temperatures. Furthermore, the isolates were tested for their ability to grow on agar amended with 10 mg Hg kg−1 as an indication of mercury resistance. We found that up to 80% of the isolates in soil amended with 15 mg Hg kg−1 were mercury-resistant, whereas only up to 20% were resistant in the treatments with 0 and 3.5 mg Hg kg−1. We found two groups of Pseudomonas, which probably represent non-described species since they did not group closely with any known species of Pseudomonas in the dendrogram. Hg-enhanced isolates were closely related to P. frederiksbergensis. Furthermore, Hg resistance was almost exclusively restricted to P. frederiksbergensis and P. migulae groups. We conclude that Hg caused a shift in the dominating species of culturable Pseudomonas.

The morpho species Bodo designis is widespread and abundant globally in highly contrasting terrestrial and aquatic ecosystems. Whether the forms of Bodo designis from contrasting environments are conspecific, i.e. largely genetically identical, or whether they merely share the external morphology is presently not known. We examined the ability of different strains of Bodo designis isolated from different environments at different geographical sites to survive and grow at a salinity range of 0.5-45%. The Bodo designis strains from marine, freshwater, and terrestrial environments showed a different ability to cope with altered physiological conditions. Most of the tested strains were only able to tolerate a small salinity range, whereas others were able to withstand all tested salinity levels. We further examined the phylogenetic relationship between the different strains by sequencing the small subunit (SSU) rRNA gene. The resulting phylogenetic analyses suggest a huge genetic variation within Bodo designis, and also imply that Dimastigella and Rhyncomonas are developed inside Bodo designis. If the biological species concept is used, the genetic differences as well as the physiological barriers between the different strains of Bodo designis, would suggest that they should be assigned to different species.

We present six new small subunit (SSU) ribosomal DNA sequences from the heterotrophic flagellate genera Cercomonas , Heteromita , and Thaumatomonas . We aligned the sequences against 42 SSU rDNA sequences from GenBank, including organisms which previously have been demonstrated to group with Cercomonas , Heteromita , and Thaumatomonas and some additional outgroup taxa. The ribosomal secondary structure was taken into account when aligning the sequences. To compensate for unequal evolutionary rates, we subjected 1959 homologue positions (including gaps) to the process of “substitution rate calibration”, resulting in a reduced dataset of evolutionarily informative positions (979 bp). We analysed the data by three different algorithms: neighbour-joining, parsimony, and maximum likelihood. The three algorithms all supported monophyly of a clade consisting of Cercomonas , Heteromita , and Thaumatomonas , chlorarachniophytes, the haplosporids, the plasmodiophorids, the euglyphids, Massisteria and Cryothecomonas. Likewise, the three algorithms strongly supported the separation of Cercomonas into two clades. Hence we suggest that some of the members of the present genus Cercomonas should be placed in a new genus Neocercomonas gen. nov.

The distribution of heterotrophic flagellates, naked amoebae, testate amoebae and ciliates was investigated in habitats created by Scots pine-Paxillus involutus and -Suillus bovinus ectomycorrhizospheres. The protozoa living on plant and fungal surfaces preferred the non-mycorrhizal pine roots over mycorrhizal roots or external mycelium. The testate amoebae were more abundant on external mycelium than on mycorrhizae regardless of the mycorrhizal fungal species. Numbers of protozoa were higher in the different habitats provided by S. bovinus mycorrhizospheres when compared with P. involutus mycorrhizospheres. Interestingly, the quality of the bacterial flora as food for the protozoa was affected by the mycorrhizal fungi even in the soils adjacent to non-mycorrhizal root tips of pine. These results demonstrate that mycorrhizal fungi create habitats differently suitable for protozoa living in boreal forest soil.

We studied microbial and protozoan activity, diversity and abundance as affected by Cu 2þ amendments ranging from 0 to 1000 m gg 21 over a 70-day period. At the end of the experiment the microbial population size, as indicated by substrate-induced respiration, had normalized for all Cu 2þ concentrations, but 1000 m gg 21 . Protozoan abundance was negatively affected by Cu 2þ , although, only in the first few weeks. A more detailed analysis of the individual components that make up the microbial and micro-faunal populations (phospholipid fatty acid (PLFA) profile and protozoan morphotypes), however, yielded a somewhat more complex picture. For the three highest Cu 2þ amendments (160, 400 and 1000 m gg 21 ), there still was a significant reduction in number of differentiable protozoan morphotypes. The bacterial PLFA pattern suggested a shift from Gram-negative towards Gram-positive bacteria for the high amendments, a process where protozoan grazing most likely played a significant role. The ratio of the trans/cis isomers of the 16:1v7 fatty acid indicated that Cu 2þ , even at low and medium concentrations, induced physiological changes in the microbial population. The relatively slight changes in total microbial and micro-faunal abundance and activity, also at the highest Cu 2þ concentrations, probably reflected the ability of the community to

We investigated whether the prey-predator dynamics of bacteria and protozoa were affected by inorganic mercury at concentrations of 0, 3.5 and 15 mg Hg(II) kg soil 21 . The amount of bioavailable Hg was estimated using a biosensor-assay based on the mer – lux gene fusion. The numbers of bacterial CFUs on the general medium 1/100 tryptic soy agar (TSA) were significantly decreased when the soil had been amended with Hg. In contrast, no effect was seen on the number of CFUs on the Pseudomonas-specific medium Gould’s S1 agar. Protozoan numbers estimated by the most probable number (MPN) method with 1/100 TSB as growth medium were also negatively affected by Hg. The different fractions of protozoa were affected to different degrees suggesting that amoebae were less sensitive than slow-growing flagellates, which again were less sensitive than the fast-growing flagellates. In contrast, Hg did not induce any detectable changes in the diversity of flagellate morphotypes. In the treatment with 15 mg Hg kg 21 a transiently increased number of bacteria was seen at day 6 probably concomitant with a decrease in the numbers of protozoa. This might indicate that Hg affected the prey-predator dynamics in communities of culturable bacteria and protozoa in soil. Furthermore, we showed that the number of Pseudomonas spp. was not affected by Hg whereas the number of bacteria growing on a general medium was. q 2003 Elsevier Ltd. All rights reserved.

Summary Nematodes infested 13 of 100 earthworm cocoons from a compost pile and 17 of 197 cocoons from a permanent pasture soil. Between one and 2000 nematodes were found within the infested cocoons. All nematodes found in cocoons from the compost pile belonged to the genus Rhabditis , while Rhabditi s spp. as well as members of Cephalobidae infested earthworm cocoons in the pasture soil. In cultures established from cocoons found in the pasture soil, at least five different types of nematodes belonging to the family Cephalobidae were found. Acrobeloides nanus was found in six cocoons, Cephalobus persegnis was found in four and Chiloplacus minimus was found in one cocoon. We suggest that earthworm – nematode interactions may be an important pathway for the transfer of carbon in terrestrial ecosystems, and that the inclusion of these pathways may lead to a better understanding of soil food web functioning.

Summary 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.

Hydrophobic contaminants sorb to sludge in wastewater treatment plants and enter the soil environment when the sludge is applied to agricultural fields. The mineralization of pyrene was examined in soil, in sludge mixed homogeneously into soil, and in sludge-soil systems containing a lump of sludge. Sludge-amendment enhanced the mineralization of pyrene in the soil compared to soil without sludge, and the most extensive mineralization was observed when the sludge was kept in a lump. The number of protozoa, heterotrophic bacteria and pyrene-mineralizing bacteria was much higher in the sludge compared to the soil. The amendment of sludge did not affect the number of protozoa and bacteria in the surrounding soil, which indicated that organic contaminants in the sludge had a little effect on the number of protozoa and bacteria in the surrounding soil.

Microbial communities differing in biodiversity were established by inoculating sterile agricultural soil with serially diluted soil suspensions prepared from the parent soil. Three replicate communities of each dilution were allowed to establish an equivalent microbial biomass by incubation for 9 months at 15°C, after which the biodiversity–ecosystem function relationship was examined for a range of soil processes. Biodiversity was determined by monitoring cultivable bacterial and fungal morphotypes, directly extracted eubacterial DNA and protozoan taxa. In the context of this study biodiversity relates to the numbers and proportions of different microbial species. Biodiversity decreased by ca. 15, 40 and 60% at each successive dilution step. There was no consistent effect of biodiversity on a range of soil processes measured (incorporation of thymidine and leucine, potential nitrification, nitrate accumulation, respiratory growth response, community level physiological profile and decomposition). Neither was there a direct effect of biodiversity on the variability of the processes, nor on the stability of decomposition when the soils were perturbed by heat or copper. The biodiversity of, and inter-relationships within, the microbial communities was such that the experimental reductions had no direct effects on soil function.

We found that five tested pesticides (the insecticide Dimethoat 40 EC, the herbicide Tolkan, and the fungicides Tilt 250 EC, Tilt Top, and Corbel) decreased the oxidation of atmospheric methane in slurries from a Danish forest soil. Dimethoat 40 EC was the most toxic with an EC50 value (i.e. the concentration which caused a 50% inhibition of the methane oxidation) of 10 mg active ingredient (AI) l 21 , followed by Tilt 250 EC (EC50a 56 mg AI l 21 ). EC50 of Tilt Top was 350 AI mg l 21 , the value of Tolkan was 410 mg AI l 21 , while Corbel had a value of 1600 mg AI l 21 . Dimethoat 40 EC and Tolkan inhibited the oxidation of atmospheric methane at concentrations expected in natural soil after application of the pesticides. Pesticides, therefore, may be partly responsible for the lowered methane oxidation rates in arable soils compared to forest soils. q 2001 Elsevier Science Ltd. All rights reserved.

The numbers and biomass of protozoa, bacteria and fungi were measured at various depths (1.5–122.5 cm) in the unsaturated zone of three contrasting pristine Danish forest site profiles: a dry beech (Fagus silvatica) forest on mor, a wet peaty spruce (Picea abies)/birch (Betula pubescens) forest and a dry spruce (P. abies) forest on mor. All sites were situated on a Weichel moraine. Except for a bacterial peak at 42.5 cm in the peat profile, the general tendency was a decrease in biomass with increasing depth for all groups examined. Protozoa decreased more rapidly with increasing depth than the other two groups of organisms examined. An evaluation of the bacterial–protozoan relationship by a simple mathematical model indicated that the subsurface protozoan populations are active and not accidental percolated cysts. The low protozoan numbers found in shallow subsurface sites contrast markedly with the results from contaminated sites where much larger protozoan populations have been reported even at considerable depths. Consequently, the results suggest that protozoa are good indicators of organic pollution in subsurface soils; however, more work involving the comparison of polluted and unpolluted soils is needed to confirm this suggestion.

The population dynamics of indigenous soil bacteria and protozoa on decaying barley roots were followed by using litter bags buried in laboratory-incubated soil. The soil was either non-treated or treated with the fungicide fenpropimorph (in the formulation Corbel) at concentrations corresponding to the recommended and at 10 times field dose (1.3 and 13 mg kg ˇ1 dry wt.). Number of total bacteria and number of Pseudomonas were detected, using both traditional plating and short-time incubations of ‘early’ colonies, to determine the fast-responding subpopulation of the culturable bacteria. The number of protozoa corresponding to the two subpopulations was followed. The results strongly indicate a predatory association between the protozoa and bacteria. This was shown by a tight temporal association, and by a stimulation of bacteria following predatory release when protozoa were inhibited by fenpropimorph. Thus, fenpropimorph disturbed population dynamics in concentrations, which can be reached in surface soils after distribution in the field. 7 2000 Elsevier Science Ltd. All rights reserved.

Litterbags containing freshly harvested wheat roots with adhering rhizosphere soil were placed in their native soil. Four levels of the fungicide fenpropimorph were applied: 0, 1, 10 and 100 mg kg −1 soil. The experimental set-up was a randomised block design with five replicates. Litterbags were sampled six times over a 4-month period and microbial numbers determined. Bacteria (colony forming units, CFUs) and protozoa (most probable number) followed a similar pattern: a sudden decrease at the onset of the study period was followed by a fast recovery and increase. This ended at maximum mean values of ≈7.5 × 10 7 bacteria g −1 root litter by day 7 and 3 × 10 6 protozoa g −1 root litter by day 20, respectively. Fenpropimorph had no significant effect on either of these two groups, for which mean population sizes seemed to interact in a prey–predator manner. The fungi (stained by calcofluor white and fluorescein diacetate) were unaffected by the pesticide until day 20. Hereafter, fluorescein diacetate-active (FDA-active) hyphae were significantly inhibited in all fungicide treated plots, compared to the untreated plots 50 and 114 days after the application of fenpropimorph. Fungal colony forming units (fungal CFUs) on fenpropimorph-containing agar demonstrated a selection towards more fenpropimorph tolerant fungi in the 100 mg kg −1 fenpropimorph treated plots. The delayed effect of fenpropimorph on FDA-activity could be explained as a combined effect of the pesticide and the degradation product itself, and the fluctuating water stressing field conditions of this year. Our results, thus, demonstrated long term effects of fenpropimorph on the activity of saprotrophic fungi at recommended field dose, and that high dose fenpropimorph can select for fenpropimorph tolerant fungal populations.

Nutrient availability is a major constraint to plant production and carbon storage in arctic ecosystems, but there are few studies coupling processes in the decomposer and microbial food web and the implications these processes have on the control of nutrient mineralization. We studied the relationship between microbial biomass and the abundance of microbivore and the role of grazing on nutrient turnover after annual addition of carbon (sucrose), fertilizer (NPK), fungicide (benomyl) and bactericides (streptomycin and penicillin) to two dwarf shrub communities, a low and a high altitude heath. After four years of repeated additions, we measured microbial biomass by fumigation-extraction and phospholipid fatty acid (PLFA) analysis, the fungal to bacterial biomass ratio by PLFA analysis and estimated the numbers of protozoa and nematodes, assigned into feeding groups. The fungal to bacterial ratio of nematode feeding groups was around 0.2, indicating a bacterial-based food web in both communities. The size of the microbial biomass did not change after the additions, except when the amount of available carbon was increased (low altitude heath) or when addition of carbon was combined with fertilizer (high altitude heath). In contrast, fertilizer but not carbon increased the number of microbivores. This suggests that the amount of available carbon and not grazing pressure controls the size of the microbial biomass. Furthermore, it suggests that the food quality, e.g. nutrient content of the micro-organisms, had a larger effect on the microbivore than the size of the microbial biomass. The addition of bactericides and fungicide did not significantly change the fungal to bacterial biomass ratio of the micro-organisms. We could not detect any effects of the bactericides. In contrast, the fungicide strongly decreased nematode density, least in the fungal feeders, probably due to increased abundance of the insensitive Aphelenchoides ssp.

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.

Mycophagous protozoa occur in most soils, but data on their quantitative importance are sparse, possibly due to a lack of suitable counting techniques. I describe a modification of the most probable number method for enumeration of mycophagous protozoa, especially flagellates, with indigenous soil fungi and yeasts as their food source in large individual wells in 24-well microtitre plates. About 103 mycophagous flagellates g−1 dry soil were present in all soils examined. Mycophagous flagellates responded by a 4-fold increase in a week after addition of glucose to soil. Their abundance and quick response to environmental changes suggest an important role for mycophagous flagellates in determining the size of populations of yeast cells and fungal conidia in soil.

Summary The flagellates of an Australian garden soil were studied by placing coverslips on wet soil and subsequently examining the coverslips by light microscopy. A number of genera and species were found which have not previously been reported from soil samples. Besides the three new species, Apusomonas australiensis sp. nov., Peltomonas hanelisp. nov., and Sciviamonas terricola gen. nov. sp. nov. they include species from the genera Amastigomonas, Cryptaulax, Paraphysomonas, and Protaspis. Among genera which have been reported from soils, we recorded a number of species previously unreported from soils: Petalomonas pusilla, Bicosoeca epiphytica, Bicosoeca mignotii, and Ancyromonas sigmoides. In addition, we extracted a number of forms which have been found in soil but which are usually not considered as members of the soil flagellate community. They are: Codosiga botrytis, Salpingoeca amphoridium, and Goniomonas truncata. Only a minority of the taxa recorded are thought of as common and widespread in soils, they include: Apusomonas proboscidea, and species of Cercomonas and Spumella. At least part of the difference between communities of flagellates from freshwaters and those of soils are due to different sampling methods and not only to the existence of different communities.

In a sandy loam soil cropped to barley bacterial production in the rhizosphere was compared to the results of a parallel investigation on rhizodeposition. Bacterial production was stimulated in the rhizosphere as revealed by an increased biomass of bacteria (643–883 μg C g −1 soil) and protozoa (7.2–15 × 10 4 cells g −1 soil) as well as elevated thymidine incorporation (9.7–12 pmol g −1 soil) in rhizosphere soil compared to bulk soil. Rhizodeposition, as determined by several pulse labellings with 14 CO 2 , was estimated to be 412 μg C g −1 dry wt soil in the 0–15 cm layer. Bacterial production, as determined by incorporation of 3 H-labelled thymidine converted to bacterial C, revealed a plant-induced formation of 1348 μg bacterial C g −1 soil in the 0–15 cm layer. This is probably the first estimate for bacterial production based on thymidine incorporation which has been compared to an estimate of C availability in a terrestrial ecosystem. The discrepancy between the rhizodeposition data and the estimate of bacterial production suggests that conversion factors between [ 3 H]thymidine incorporation and bacterial production obtained in laboratory incubations are not generally applicable to the field situation. It is proposed that estimates of bacterial production should be based on measurements of actual conversion factors to improve the accuracy of the method.

Different methods of estimating protozoan diversity in soil are discussed in this paper, with the major emphasis on heterotrophic flagellates. Although many species of ciliates and testate amoebae seem to be unique to the soil environment, the communities of heterotrophic flagellates and naked amoebae are probably best considered as restricted versions of their aquatic counterparts. Soil protozoa are difficult to observe directly, hence culture techniques are usually used. These techniques enable us to explore certain functional aspects, but their major drawback is that some soil protozoa cannot be cultured. Molecular methods, however, have the potential to detect such non-culturable forms.