Population dynamics of the fast-growing sub-populations of
Pseudomonas
and total bacteria, and their protozoan grazers,
revealed by fenpropimorph treatment
Laila Thirup
a,b,*, Flemming Ekelund
b, Kaare Johnsen
a, Carsten Suhr Jacobsen
aa
Department of Geochemistry, Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark
b
Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen é, Denmark
Accepted 22 March 2000
Abstract
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 ®eld dose (1.3 and 13 mg kgÿ1 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 ®eld.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Predation; Fenpropimorph;Pseudomonas; Protozoa; Bacteria; Soil; Micro CFU
1. Introduction
Though it is generally accepted that protozoa regu-late the size of bacterial populations in soil (Ekelund and Rùnn, 1994), the assumption is mainly based on studies of sterilised gnotobiotic soils (e.g. Rutherford and Juma, 1992; Kuikman and van Veen, 1989; Pus-sard and Rouelle, 1986; Elliott et al., 1980), or on pro-tozoan predation of bacteria introduced to soil (e.g. Heynen et al., 1988; Postma et al., 1990; Recorbet et al., 1992). Only few studies have dealt with the prey-predator dynamics between natural assemblages of bacteria and protozoa in soil. Clarholm (1981, 1989) studied the interactions between indigenous bacterial populations and naked amoebae after rainfall, and
Rùnn et al. (1996) studied the interactions between indigenous bacteria and the total protozoan popu-lation after addition of barley roots to soil. The data from these studies, as well as the short generation times of the involved organisms (Ekelund, 1996), demonstrates that the essential dynamics between the involved populations take place within few days after stimulation. This indicates that frequent sampling is important in such studies. Decomposition of organic matter in many agro-ecosystems seems less dominated by fungi than in non-cultivated ecosystems (Brussaard et al., 1990; Stahl and Parkin, 1996), making the inter-action between bacterial and protozoan populations a very important link in the below-ground food web in agricultural soils.
Besides their eect on the size of the bacterial population, protozoa probably also aect the com-position of the bacterial community in soil by pre-ferential feeding (Singh, 1941); a phenomenon that
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has been demonstrated in experiments on aqueous model systems (Bianchi, 1989; Sinclair and Alexan-der, 1989; Pernthaler et al., 1997). This stresses the importance of studies which involve quanti®cation of particular bacterial groups at the same time as their speci®c protozoan predators.
Organic resources are heterogeneously distributed in soil, which results in patches of substrate with high microbial and microfaunal activity. Such patches are ideal for the study of interactions between bacteria and protozoa. These are isolated transient habitats giving rise to a succession of decomposer organisms (Christensen et al., 1992). For the same reasons, a hotspot is a good model-system for testing the eects of agrochemicals on decomposition. Fenpropimorph is a widely used agricultural fungicide, i.e. the most often used fungi-cide to control fungal diseases on leaves of cereals in Denmark. This fungicide has detrimental eects on plant pathogenic fungi (Loeer and Hayes, 1992), and has been shown to aect non-target soil fungi in a ®eld study, where a delayed inhibition was observed (Bjùrnlund et al., 2000). Furthermore, fenpropimorph has the potential to reduce soil pro-tozoan numbers in a soil environment at a concen-tration of approximately 1 mg kgÿ1 wet wt. (Ekelund et al., 1994; Ekelund, 1999).
Enumeration of the active bacterial population in soil is essential in quantifying the impact of population dynamics within the primary degraders. It is important to consider that only a small part (0.1±5%) of the bac-teria observed by direct microscopy can form colonies on the bacteriological media known today. However, large and culturable cells have in some cases been found to constitute 80±90% of the bacterial biovo-lume, so Olsen and Bakken (1987) speculated that the ecological signi®cance of culturable cells may be large despite the fact that they represent a small fraction of the number of physically intact cells that can be counted in soil by microscopy. This is supported by the ®ndings of Jacobsen and Pedersen (1992a, 1992b) who found good correlation between speci®c metabolic activity and growth as measured by CFU (colony forming units) of speci®c 2,4-D degrading soil bacteria. Furthermore, the number of speci®c CFUs was in good agreement with the numbers of the accompany-ing gene as revealed by quantitative DNA±DNA hy-bridisation (Jacobsen and Rasmussen, 1992).
One group of bacteria, which has been found to be associated with decomposing organic matter, is the genus Pseudomonas (Rovira and Sands, 1971; Johnsen et al., 1999) (formerly known as ¯uorescent pseudomo-nads (Kersters et al., 1996)). In addition, Pseudomonas
is important in agriculture because some of its mem-bers are plant growth promoters (O'Sullivan and O'Gara, 1992) or plant pathogens (Schroth et al.,
1991). It is therefore relevant to examine pesticide eects on this group of bacteria and its predators.
The objective of this study was to use dierent cul-ture techniques to investigate the interaction between bacterial and protozoan sub-populations in soil, and the possible eects of fenpropimorph on them.
2. Materials and methods
2.1. Soil and fungicide
The soil used was a sandy loam soil from the Royal Veterinary and Agricultural University experimental ®elds in Hùje Taastrup, Denmark. The ®eld was con-ventionally cultivated until 1988. From 1989 onwards, it was organically cultivated without the use of pesti-cides. Soil characteristics are: coarse sand 23.7%, ®ne sand 26.2%, coarse silt 12.8%, ®ne silt 19%, clay 16%, and organic matter 2.3% (dry wt.) and water holding capacity 28.3% (dry wt.). Soil was sampled in March 1997 and stored at 58C for ®ve days. It was sieved (2 mm), adjusted to 20% water content (dry wt.), and incubated at 108C.
The fungicide formulation used was Corbel (BASF, Copenhagen, Denmark), which contains the active ingredient fenpropimorph (2)-cis-4-[3-(4-tert -butylphe-nyl)-2-methylpropyl]-2,6-dimethylmorpholine. In Den-mark, Corbel is used on cereals at a recommended dose rate of 1 l haÿ1. This corresponds to a concen-tration of approximately 1.3 mg kgÿ1 dry wt. soil, if distributed evenly in the upper 5 cm of the soil. In this experiment, fungicide concentrations in the micro-cosms were chosen to be 0, 1.3 and 13 mg kgÿ1 dry wt.
2.2. Set-up of microcosms and sampling
Barley plants were grown in transparent 50 ml cen-trifuge tubes, each containing 50 g soil (wet wt.). Two non-sterile barley seeds (Hordeum vulgaretype Digger) were sown in each tube. Centrifuge tubes were incu-bated in a transparent plastic bag to prevent desicca-tion. Plants were incubated at 108C with light from a 15 W plant growth light in 12 h light, 12 h dark cycles. From 12-day old barley seedlings, roots with adhering rhizosphere soil were cut in 2 cm pieces (1.5 cm from the root base). These root pieces were submerged in fenpropimorph solution in 0.010 M phosphate buer, pH = 7.4, with a concentration of 0, 5 or 50 mg lÿ1 fenpropimorph for approximately 5 s. 5 and 50 mg lÿ1 fenpropimorph corresponds to the recommended dose and the 10 times recommended dose, assuming that all added fenpropimorph is dissolved in the soil water in the upper 5 cm soil.
sol-ution, they were transferred to sterile polyethylene lit-ter bags, 45 cm, 1 mm mesh size) six root pieces per bag. From each treatment litter bags were placed in a non-transparent plastic container, with 870 g soil dry weight, 20% water content. The litter bags were buried in a depth of 2.5±3 cm. The three plastic con-tainers (three treatments) were then supplemented with 0.010 M phosphate-buer with or without fenpropi-morph to give ®nal fenpropifenpropi-morph concentrations of 0, 1.3 and 13 mg kgÿ1dry wt., and a water content of 25%. Microcosms were incubated at 108C. Water loss during incubation was negligible.
Sampling was done on day 0, 3, 6, 9, 13, 21 and 24 after the microcosms were established. On each sampling occasion three replicate litter bags from each of the three treatments were sampled. The root-samples were aseptically transferred to glass tubes con-taining 2 ml 0.010 M phosphate buer. The tubes were mildly sonicated in an ultrasonic water bath for 30 s (Bransonic 2210, Branson Ultrasonics, Danbury, Con-necticut). These suspensions were used for all bacterial and protozoan enumerations.
2.3. Enumeration of bacteria by early-colony and plate spreading techniques
A technique to determine bacteria able to form colo-nies rapidly (early-colony technique) was modi®ed after the micro-colony method of Binnerup et al. (1993). 100ml of a 10ÿ1dilution of the soil suspension was suspended in 5 ml Winogradsky salt solution, and bacteria were collected on a 0.2 mm black polycarbo-nate ®lter by ®ltration. Filters for enumeration of
Pseudomonas were placed on the surface of Pseudomo-nas selective Gould's S1 agar plates (Gould et al., 1985; Johnsen and Nielsen, 1999) amended with 50 mg lÿ1 nystatin to inhibit fungal growth. Filters for enu-meration of total bacteria early-colonies were placed on the surface of liquid 1/10 TSB (Difco, Detroit, MI, USA, 3 g lÿ1), which had been pipetted (5 ml) into each well of a 23 well multidish (Nunc, Life Tech-nologies, Roskilde, Denmark). Optimal incubation time was 20 h on Gould's S1 agar, and 12 h on 1/10 TSB with gentle shaking (40 rpm) at 208C. The incu-bation time diered because of slower development of early-colonies on Gould's S1 than on 1/10 TSB, lead-ing to average generation time of approximately 3 and 1±1.5 h, respectively, when correlating for a lag phase of approximately 6 h (data not shown).
Acridine orange was applied on the back side of the ®lter as described by Binnerup et al. (1993), with sub-sequent washing twice for 3 min. Filters were mounted using immersion oil, and counted at 400 magni®-cation. An early-colony was de®ned as a tight associ-ation of more than two cells. However, most early-colonies observed consisted of more than 10 cells
(>70% on 1/10 TSB; >95% on Gould's S1). On ®l-ters incubated on 1/10 TSB the aim was to count 100 early-colonies per ®lter. If this number was not reached after inspection of 200 microscopic ®elds, counting was ended. On Gould's S1, the aim was to count 20 early-colonies per ®lter and 400 microscopic ®elds were inspected, if fewer than 20 early-colonies were found. One ®lter per root sample was counted.
Dilution series of the basic soil suspensions were spread on 1/10 TSA agar plates used as a general med-ium (Difco), and on Gould's S1 agar plates. Both media were supplemented with 50 mg lÿ1 nystatin. Plates were incubated at 208C in the dark. After three days, CFU were enumerated. Thus the average gener-ation time for CFUs was approximately 3.5 h.
2.4. Enumeration of protozoa
The most abundant protozoa in agricultural soil are ¯agellates and naked amoebae. At present, these organisms can only be enumerated in soil by using a culture-dependent MPN (most probable number) method (Ekelund and Rùnn, 1994). The soil suspen-sions used for enumeration of bacteria were likewise used as basis for the protozoan estimates. Mild soni-cation of the soil suspension resulted in liberation of protozoa from soil particles which was not signi®cantly dierent from the shaking method normally used (data not shown). The number of protozoa was determined by the MPN method of Darbyshire et al. (1974), as modi®ed by Rùnn et al. (1995). Three-fold soil di-lutions were prepared in 96 well micro-titre plates; one plate per root sample. The plates were incubated at 108C in the dark, and examined for occurrence of pro-tozoa after one and four weeks, respectively, using an inverted microscope (Olympus IMT 2, 300 magni®-cation, phase contrast). Micro-titre plate patterns were converted to the MPN of protozoa by a computer pro-gram (Rùnn et al., 1995). Estimates of the total popu-lation of omnivorous protozoa were obtained by using 1/300 TSB (0.1 g/l tryptic soy broth, Difco) suspended in Ne's amoeba saline (Page, 1988) as culture med-ium. The number of fast growing protozoa were esti-mated using only the information from the one week counts. To enumerate protozoa feeding on Pseudomo-nas, a Pseudomonas ¯uorescens DR54 (Nielsen et al., 1998) was used as food source for the protozoa. The bacterium was grown in Luria Broth overnight, and the cells were washed twice in 0.010 M phosphate buf-fer. Phosphate buer (0.1 ml) with 5:010
8 mlÿ1 bac-teria was added to each well as prey.
2.5. Data analysis
detection of protozoa based on Pseudomonas ¯uores-censDR54 could not be accomplished.
Data were log transformed, and analysed by a two-way ANOVA (analysis of variance) for the eects of sampling time, fungicide treatment and the interaction between the two eects. To compare fungicide treat-ments with the control, and the eect of time on the population development, appropriate LSD values (least signi®cant dierence) were used. A 95% con®-dence level was used, unless otherwise stated.
3. Results
3.1. Bacteria enumerated by the early-colony and plate spreading technique
The short-term early-colony technique was used to count the fast-responding sub-population of culturable bacteria. These numbers, early-CFU, were consistently below the corresponding CFU data for both media, 1/10 TSB and Gould's S1 (Fig. 1).
3.2. Fenpropimorph eects on interactions between subgroups of bacteria and protozoa
The fast-responding bacteria enumerated, using the early-colony technique on 1/10 TSB (Fig. 2a) peaked on day 3 in samples with and without the ommended dose of fenpropimorph. The 10 times rec-ommended dose treatment resulted in an increase in bacterial numbers until day 6, when numbers were sig-ni®cantly higher than the control a0:001).
Proto-zoan numbers estimated after 1 week of incubation of micro-titre plates re¯ect mainly growth of small soil ¯agellates, which are fast-responding and have high growth rates compared to other types of soil protozoa
Fig. 1. The number of culturable bacteria around decomposing roots in soil microcosms during the experimental period. Bacterial numbers were estimated by two methods: a short-term incubation early-colony technique, and traditional plate counts. Two dierent media were used for both methods. 1/10 TSB/TSA was used as an unselective medium and Gould's S1 as aPseudomonasselective medium. Graphs represent results from samples that were not treated with fenpropimorph.
(Ekelund, 1996; Ekelund and Rùnn, 1994). After 1 week of incubation there were around 15 ¯agellates per amoebea in both types of micro-titre plates, but after four weeks of incubation only three to four ¯a-gellates were found per amoebae. Fig. 2b shows esti-mates of protozoa preying on indigenous soil bacteria enumerated after 1 week of incubation of micro-titre plates. The highest fenpropimorph concentration reduced numbers of protozoa signi®cantly on days 3, 6, 9 and 24. There were no signi®cant dierences between the control and the recommended dose.
The fast-responding Pseudomonas (early-CFU) (Fig. 3a) peaked on the third day for all treatments with signi®cantly higher numbers than on all other sampling days. The fenpropimorph treatment had no signi®cant eect on Pseudomonas. Estimates of fast-responding protozoa feeding onPseudomonas (Fig. 3b) showed no signi®cant eect of fenpropimorph
amend-ments. A signi®cant peak in protozoan numbers was observed on day 6. The number of protozoa which feed on Pseudomonas ¯uorescens DR54 constituted only 5±10% of the total protozoan number. One species, the ¯agellate Heteromita globosa, was observed to dominate the protozoan population detected in micro-titre plates with Pseudomonas ¯uorescens DR54 as food source.
The number of bacterial CFUs on 1/10 TSA (Fig. 4a) did not dier signi®cantly in samples from fungicide-treated soil compared to untreated soil. Bac-terial numbers peaked signi®cantly twice, on day 3 and day 13. Fig. 4b shows counts of protozoa in micro-titre plates incubated for 4 weeks. The highest fenpro-pimorph concentration reduced numbers of protozoa, but the decrease in protozoan numbers in the 10 times recommended dose treatment was less pronounced than on protozoa estimated using micro-titre plates incubated for 1 week (Fig. 2b). Here signi®cant dier-ences were found only on days 6 and 9.
Fig. 4. The number of total bacteria (a) and protozoa (b) around decomposing roots in soil microcosms with dierent concentrations of fenpropimorph: (a) Culturable bacteria on 1/10 TSA enumerated by plate spreading; (b) MPN-counts of protozoa on 1/300 TSB esti-mated after 4 weeks of incubation.
CFUs ofPseudomonas were not aected by the fun-gicide treatments either (Fig. 5a). A signi®cant peak was observed during the 3±6 day, and again on day 13. Estimates of protozoa feeding on Pseudomonas
(Fig. 5b) showed no signi®cant eect of fenpropi-morph amendments. A signi®cant peak in protozoan numbers was observed on days 6 and 13.
4. Discussion
4.1. Bacterial succession during decomposition
Total bacteria as well asPseudomonaspeaked within the ®rst 6 days (Fig. 1). This peak was probably caused by a high level of easily degradable organic matter liberated from the added root material (Mar-storp, 1996).
Bacterial numbers obtained by the traditional
plate-spread method and the micro-colony technique diered (Fig. 1). Early-CFU gave lower numbers than the tra-ditional plate counts, suggesting that early-colonies enumerated after a few hours represented only a fast-responding subpopulation of the culturable bacteria. This can be attributed to the fact that slow-growing bacteria did not have time to develop, and fast-grow-ing, but in the soil inactive bacteria, do not germinate within these few hours. For example, inactive Bacillus
spores from soil are more likely to give normal CFU colonies than early-CFU colonies, because spores from stressed Bacillus start germination after longer and more varied time than spores from Bacillus grown in normal laboratory culture (Binnerup, personal com-munication). The dierence between early-CFU and CFU was more pronounced for TSB than Gould's because of the dierence in growth rate. Thus the cal-culated mean generation time of bacteria forming early-CFUs on TSB was 1±1.5 h, while it was approxi-mately 3 h for Pseudomonas forming early-CFUs on Gould's S1. On both media the mean generation time of bacteria forming regular CFUs was around 3.5 h.
Some eort has been made to describe the ecological dierences between fast- and slow-growing soil iso-lates. Kasahara and Hattori (1991) found that fast-growing isolates generally were able to grow in rich media, while most slow-growing isolates only grew in dilute media. They were therefore termed as oligo-trophic. de Leij et al. (1993) found that fast-developing colonies dominated in young rhizosphere compared to older rhizosphere. In bulk soil, slow-developing colo-nies were even more prevalent than in rhizosphere.
In the initial stages of this experiment, the root en-vironment was undoubtedly rich in easily degradable organic matter (Marstorp, 1996). This environment promoted bacteria with fast growth rates, re¯ected by the subpopulation detected as early-colonies. These bacteria had a lower occurrence in later stages of the decomposition process. Still, the number of CFUs was relatively high in later stages. Possibly, this re¯ects a higher percentage of slower growing bacteria utilising more complex carbon sources among the total popu-lation of culturable bacteria.
4.2. Protozoa regulate bacteria
Interaction between the indigenous fast-responding bacteria and protozoa was indicated by the initial rapid growth of early-CFU on 1/10 TSB, succeeded by an increasing protozoan population in the non-fungi-cide treated root soil (Fig. 2). The persistent high level of total protozoa after day 13 probably re¯ects a high proportion of inactive cysts, which were produced during the vigorous initial activity around the roots (Ekelund and Christensen, 1996).
A signi®cant decrease in protozoa in the 10 times Fig. 5. The number of Pseudomonas (a) and Pseudomonas-feeding
recommended dose fenpropimorph treatment during the ®rst 9 days was accompanied by a signi®cantly el-evated level of fast-responding bacteria on the 6th day (Figs. 2 and 4). The reason that only early-colony-detected bacteria responded in this manner could be that they represent bacteria with a high growth rate in the root environment, which causes them to be more susceptible to protozoan grazing. A marked eect on TSB CFUs (which contrary to the early colonies also includes bacteria that are slow-growing or not active in the root environment) is less expected since other studies con®rm that protozoa prefer feeding on grow-ing bacterial cells (GonzaÂlez et al., 1993; Pernthaler et al., 1997).
As fenpropimorph is a fungicide, an initial de-pression of soil fungi followed by reestablishment of the fungal population between days 6 and 9, could explain the elevated number of early colonies on the 6th day (Fig. 2a), as competitive release of bacteria. However, in a similar experiment we found that the soil fungi population, measured as FDA-active hyphae, was signi®cantly reduced by around 50% for more than 10 days when fenpropimorph was applied in normal ®eld dose (Thirup et al., submitted). Conse-quently, we ®nd an earlier reestablishment of the fun-gal population unlikely in this experiment, when fenpropimorph was applied in a tenfold higher concen-tration. Therefore, such eect on soil fungi can not explain the decline in early colonies on 1/10 TSB.
The peak of fast-growing protozoa feeding on Pseu-domonaswas delayed as compared to the peak of fast-growingPseudomonas(Fig. 3), as would be expected in a prey-predator system. This pattern is still obvious, though less pronounced when slow-growing Pseudomo-nas-feeding protozoa and Pseudomonas (Fig. 5) are compared. The decrease in culturable Pseudomonas
could be caused by a loss of culturability, but this is not likely since we showed earlier that the amount of
Pseudomonas-speci®c DNA in a similar experiment declined at the same time asPseudomonas CFU (John-sen et al., 1999).
4.3. Eect of fenpropimorph
In this experiment the lowest concentration of fen-propimorph used (1.3 mg/kg soil) corresponded to the recommended ®eld dose, if we assume that the fungi-cide is distributed in the upper 5 cm soil. Fenpropi-morph was applied on the soil surface of the microcosm, but due to the strong absorption proper-ties of the compound we wanted to assure contact between fenpropimorph and the decomposing roots. Therefore, the roots were brie¯y submerged in a fen-propimorph solution before being buried.
Fenpropimorph was found to diminish protozoan numbers, especially the fast-growing protozoa (mainly
¯agellates) detected after one week of incubation. The signi®cant decrease in protozoan numbers at the high-est concentration thigh-ested is in accordance with previous observations (Ekelund et al., 1994; Ekelund, 1999). Fenpropimorph is a sterol biosynthesis inhibitor, and probably interacts with protozoa by changing their sterol pattern. This was demonstrated for the soil amoeba Acanthamoeba polyphaga, when exposed to the fungicide in vivo (Raederstor and Rohmer, 1987). Thus, fenpropimorph can be used to manipulate groups of soil microorganisms in a selective manner. The fungicide thiram has previously been used to ma-nipulate soil protozoa to elucidate the importance of grazing on inoculated Rhizobium in the rhizosphere of bean (Ramirez and Alexander, 1980). Besides protozoa and Rhizobium, soil bacteria and bacteriophages were also detected, and the results suggested that protozoa were a regulating factor on total bacteria and Rhizo-bium. However, even though thiram is a fungicide, the eect on soil fungi was not studied.
We noticed that the ¯agellate Heteromita globosa
was dominating in micro-titre plates withPseudomonas ¯uorescens DR54. This indicates that thePseudomonas
strain only was a suitable food for some protozoa. It is possible thatHeteromita globosawas less sensitive to fenpropimorph, which explains whyPseudomonas -feed-ing protozoa were not inhibited in this experiment. Fenpropimorph acts on the sterol biosynthesis, which presumably is very variable among protozoa, which indeed is a broad phylogenetic group.
The eect of fenpropimorph on soil organisms is greatly dependent on the behaviour of the compound in the soil habitat. Stockmaier et al. (1996) found, that fenpropimorph absorbs to the upper 0±5 cm soil after surface application, while the degradation product fen-propimorphic acid is more mobile. In a ®eld exper-iment degradation half lives of fenpropimorph were found to be about 36 and 47 days in a clayey silt and a silty sand soil, respectively (Stockmaier et al., 1996). The bio-availability of the compound, however, is probably much lower than suggested by half lives determined on the basis of acetone extraction of the fungicide. Possibly the toxic eect of fenpropimorph on protozoa in this experiment was transient because of low bio-available concentrations after the ®rst 10 days.
5. Conclusions
re-lation was observed between Pseudomonas and their grazers. The coupling between protozoan and bacterial populations was also con®rmed by the observation that the decrease of protozoa in the fungicide treat-ment resulted in increased levels of early-colony-form-ing bacteria on TSB. This shows how toxicants can aect non-target organisms by aecting the inter-actions between organisms.
Acknowledgements
We thank Ole Nybroe for helpful criticism on the manuscript, Svend J. Binnerup for help and comments on the parts concerning the micro-colony technique, and Anita Lùve Nielsen for technical assistance. This work was funded by The Danish Environmental Pro-tection Agency (J. No. 7041-0293) as a part of the pro-ject Pesticide Eects on Agricultural Soil Ecosystems, and two centres under the Danish Environmental Research Programme, Centre for Eects and Risks of Biotechnology in Agriculture, and Centre for Biologi-cal Processes in Contaminated Soil and Sediments.
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