Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue11-12.oct2000:
Soil Biology & Biochemistry 32 (2000) 1615±1623
www.elsevier.com/locate/soilbio
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 a
a
Department of Geochemistry, Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark
Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen é, Denmark
b
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. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Predation; Fenpropimorph; Pseudomonas; Protozoa; Bacteria; Soil; Micro CFU
1. Introduction
Though it is generally accepted that protozoa regulate 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; Pussard and Rouelle, 1986; Elliott et al., 1980), or on protozoan 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 preypredator 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
* Corresponding author. Tel: +45-46-301200; fax: +45-46-301114.
E-mail address: lth@dmu.dk (L. Thirup).
Rùnn et al. (1996) studied the interactions between
indigenous bacteria and the total protozoan population 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 interaction 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 composition of the bacterial community in soil by preferential feeding (Singh, 1941); a phenomenon that
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 7 5 - 4
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L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
has been demonstrated in experiments on aqueous
model systems (Bianchi, 1989; Sinclair and Alexander, 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 modelsystem for testing the eects of agrochemicals on
decomposition. Fenpropimorph is a widely used
agricultural fungicide, i.e. the most often used fungicide 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 protozoan numbers in a soil environment at a concentration 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 bacteria 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 biovolume, 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 accompanying gene as revealed by quantitative DNA±DNA hybridisation (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 pseudomonads (Kersters et al., 1996)). In addition, Pseudomonas
is important in agriculture because some of its members 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 culture 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 conventionally cultivated until 1988. From 1989 onwards,
it was organically cultivated without the use of pesticides. 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-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine. In Denmark, Corbel is used on cereals at a recommended
dose rate of 1 l haÿ1. This corresponds to a concentration 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 microcosms 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 centrifuge tubes, each containing 50 g soil (wet wt.). Two
non-sterile barley seeds (Hordeum vulgare type Digger)
were sown in each tube. Centrifuge tubes were incubated in a transparent plastic bag to prevent desiccation. 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.
After submerging root pieces in fenpropimorph sol-
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
ution, they were transferred to sterile polyethylene litter bags, 4 5 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 containers (three treatments) were then supplemented with
0.010 M phosphate-buer with or without fenpropimorph to give ®nal fenpropimorph concentrations of
0, 1.3 and 13 mg kg ÿ1 dry 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 rootsamples were aseptically transferred to glass tubes containing 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, Connecticut). 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 colonies rapidly (early-colony technique) was modi®ed
after the micro-colony method of Binnerup et al.
(1993). 100 ml of a 10ÿ1 dilution of the soil suspension
was suspended in 5 ml Winogradsky salt solution, and
bacteria were collected on a 0.2 mm black polycarbonate ®lter by ®ltration. Filters for enumeration of
Pseudomonas were placed on the surface of Pseudomonas 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 enumeration 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 2 3 well multidish (Nunc, Life Technologies, 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 incubation time diered because of slower development of
early-colonies on Gould's S1 than on 1/10 TSB, leading 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 subsequent 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 association of more than two cells. However, most earlycolonies observed consisted of more than 10 cells
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(>70% on 1/10 TSB; >95% on Gould's S1). On ®lters 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 medium (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 generation 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 suspensions used for enumeration of bacteria were likewise
used as basis for the protozoan estimates. Mild sonication 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 dilutions 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 protozoa 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 program (Rùnn et al., 1995). Estimates of the total population 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 medium. The number of fast growing protozoa were estimated using only the information from the one week
counts. To enumerate protozoa feeding on Pseudomonas, 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 buffer. Phosphate buer (0.1 ml) with 5:0 108 mlÿ1 bacteria was added to each well as prey.
2.5. Data analysis
Samplings were done on day 0, 3, 6, 9, 13, 21 and
24. On day 21, preparations for early-colonies and
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L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
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 a Pseudomonas selective medium. Graphs represent results from
samples that were not treated with fenpropimorph.
detection of protozoa based on Pseudomonas ¯uorescens DR54 could not be accomplished.
Data were log transformed, and analysed by a twoway ANOVA (analysis of variance) for the eects of
sampling time, fungicide treatment and the interaction
between the two eects. To compare fungicide treatments 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 recommended dose of fenpropimorph. The 10 times recommended dose treatment resulted in an increase in
bacterial numbers until day 6, when numbers were signi®cantly higher than the control a 0:001). Protozoan 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. 2. The number of fast-growing total bacteria (a) and protozoa
(b) around decomposing roots in soil microcosms with dierent concentrations of fenpropimorph: (a) Culturable bacteria on 1/10 TSB
enumerated with the early-colony technique; (b) MPN-counts of protozoa on 1/300 TSB estimated after one week of incubation.
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
Fig. 3. The number of fast-growing Pseudomonas (a) and fast
responding Pseudomonas-feeding protozoa (b) around decomposing
roots in soil microcosms with dierent concentrations of fenpropimorph: (a) Culturable Pseudomonas on Gould's S1 agar enumerated
with the early-colony technique; (b) MPN-counts of Pseudomonasfeeding protozoa estimated after 1 week of incubation.
(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 ¯agellates were found per amoebae. Fig. 2b shows estimates 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 fastresponding protozoa feeding on Pseudomonas (Fig. 3b)
showed no signi®cant eect of fenpropimorph amend-
1619
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 estimated after 4 weeks of incubation.
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. Bacterial numbers peaked signi®cantly twice, on day 3 and
day 13. Fig. 4b shows counts of protozoa in microtitre plates incubated for 4 weeks. The highest fenpropimorph 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 dierences were found only on days 6 and 9.
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L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
CFUs of Pseudomonas were not aected by the fungicide 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 fenpropimorph 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 as Pseudomonas peaked 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 (Marstorp, 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 traditional plate counts, suggesting that early-colonies
enumerated after a few hours represented only a fastresponding subpopulation of the culturable bacteria.
This can be attributed to the fact that slow-growing
bacteria did not have time to develop, and fast-growing, 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 communication). The dierence between early-CFU and
CFU was more pronounced for TSB than Gould's
because of the dierence in growth rate. Thus the calculated mean generation time of bacteria forming
early-CFUs on TSB was 1±1.5 h, while it was approximately 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 isolates. Kasahara and Hattori (1991) found that fastgrowing isolates generally were able to grow in rich
media, while most slow-growing isolates only grew in
dilute media. They were therefore termed as oligotrophic. de Leij et al. (1993) found that fast-developing
colonies dominated in young rhizosphere compared to
older rhizosphere. In bulk soil, slow-developing colonies were even more prevalent than in rhizosphere.
In the initial stages of this experiment, the root environment 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 population of culturable bacteria.
4.2. Protozoa regulate bacteria
Fig. 5. The number of Pseudomonas (a) and Pseudomonas-feeding
protozoa (b) around decomposing roots in soil microcosms with
dierent concentrations of fenpropimorph: (a) Culturable Pseudomonas on Gould's S1 agar enumerated by plate spreading; (b) MPNcounts of Pseudomonas-feeding protozoa estimated after 4 weeks of
incubation.
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-fungicide 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
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
recommended dose fenpropimorph treatment during
the ®rst 9 days was accompanied by a signi®cantly elevated level of fast-responding bacteria on the 6th day
(Figs. 2 and 4). The reason that only early-colonydetected 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 growing bacterial cells (GonzaÂlez et al., 1993; Pernthaler et
al., 1997).
As fenpropimorph is a fungicide, an initial depression 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). Consequently, we ®nd an earlier reestablishment of the fungal population unlikely in this experiment, when
fenpropimorph was applied in a tenfold higher concentration. 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 Pseudomonas was delayed as compared to the peak of fastgrowing Pseudomonas (Fig. 3), as would be expected in
a prey-predator system. This pattern is still obvious,
though less pronounced when slow-growing Pseudomonas-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 as Pseudomonas CFU (Johnsen et al., 1999).
4.3. Eect of fenpropimorph
In this experiment the lowest concentration of fenpropimorph used (1.3 mg/kg soil) corresponded to the
recommended ®eld dose, if we assume that the fungicide is distributed in the upper 5 cm soil. Fenpropimorph was applied on the soil surface of the
microcosm, but due to the strong absorption properties of the compound we wanted to assure contact
between fenpropimorph and the decomposing roots.
Therefore, the roots were brie¯y submerged in a fenpropimorph solution before being buried.
Fenpropimorph was found to diminish protozoan
numbers, especially the fast-growing protozoa (mainly
1621
¯agellates) detected after one week of incubation. The
signi®cant decrease in protozoan numbers at the highest concentration tested 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 manipulate 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 Rhizobium. 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 with Pseudomonas
¯uorescens DR54. This indicates that the Pseudomonas
strain only was a suitable food for some protozoa. It
is possible that Heteromita globosa was less sensitive to
fenpropimorph, which explains why Pseudomonas-feeding 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 fenpropimorphic acid is more mobile. In a ®eld experiment 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
This study strongly indicates that the interaction
between protozoa and bacteria is an important factor
governing the size of their respective populations.
There was a general correlation between the curve
peaks of high levels of bacteria, succeeded by increasing protozoan populations. A speci®c prey-predator re-
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L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
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 treatment resulted in increased levels of early-colony-forming bacteria on TSB. This shows how toxicants can
aect non-target organisms by aecting the interactions 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 Protection Agency (J. No. 7041-0293) as a part of the project 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 Biological Processes in Contaminated Soil and Sediments.
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www.elsevier.com/locate/soilbio
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 a
a
Department of Geochemistry, Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark
Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen é, Denmark
b
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. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Predation; Fenpropimorph; Pseudomonas; Protozoa; Bacteria; Soil; Micro CFU
1. Introduction
Though it is generally accepted that protozoa regulate 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; Pussard and Rouelle, 1986; Elliott et al., 1980), or on protozoan 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 preypredator 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
* Corresponding author. Tel: +45-46-301200; fax: +45-46-301114.
E-mail address: lth@dmu.dk (L. Thirup).
Rùnn et al. (1996) studied the interactions between
indigenous bacteria and the total protozoan population 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 interaction 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 composition of the bacterial community in soil by preferential feeding (Singh, 1941); a phenomenon that
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 7 5 - 4
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L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
has been demonstrated in experiments on aqueous
model systems (Bianchi, 1989; Sinclair and Alexander, 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 modelsystem for testing the eects of agrochemicals on
decomposition. Fenpropimorph is a widely used
agricultural fungicide, i.e. the most often used fungicide 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 protozoan numbers in a soil environment at a concentration 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 bacteria 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 biovolume, 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 accompanying gene as revealed by quantitative DNA±DNA hybridisation (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 pseudomonads (Kersters et al., 1996)). In addition, Pseudomonas
is important in agriculture because some of its members 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 culture 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 conventionally cultivated until 1988. From 1989 onwards,
it was organically cultivated without the use of pesticides. 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-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine. In Denmark, Corbel is used on cereals at a recommended
dose rate of 1 l haÿ1. This corresponds to a concentration 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 microcosms 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 centrifuge tubes, each containing 50 g soil (wet wt.). Two
non-sterile barley seeds (Hordeum vulgare type Digger)
were sown in each tube. Centrifuge tubes were incubated in a transparent plastic bag to prevent desiccation. 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.
After submerging root pieces in fenpropimorph sol-
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
ution, they were transferred to sterile polyethylene litter bags, 4 5 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 containers (three treatments) were then supplemented with
0.010 M phosphate-buer with or without fenpropimorph to give ®nal fenpropimorph concentrations of
0, 1.3 and 13 mg kg ÿ1 dry 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 rootsamples were aseptically transferred to glass tubes containing 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, Connecticut). 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 colonies rapidly (early-colony technique) was modi®ed
after the micro-colony method of Binnerup et al.
(1993). 100 ml of a 10ÿ1 dilution of the soil suspension
was suspended in 5 ml Winogradsky salt solution, and
bacteria were collected on a 0.2 mm black polycarbonate ®lter by ®ltration. Filters for enumeration of
Pseudomonas were placed on the surface of Pseudomonas 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 enumeration 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 2 3 well multidish (Nunc, Life Technologies, 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 incubation time diered because of slower development of
early-colonies on Gould's S1 than on 1/10 TSB, leading 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 subsequent 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 association of more than two cells. However, most earlycolonies observed consisted of more than 10 cells
1617
(>70% on 1/10 TSB; >95% on Gould's S1). On ®lters 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 medium (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 generation 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 suspensions used for enumeration of bacteria were likewise
used as basis for the protozoan estimates. Mild sonication 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 dilutions 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 protozoa 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 program (Rùnn et al., 1995). Estimates of the total population 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 medium. The number of fast growing protozoa were estimated using only the information from the one week
counts. To enumerate protozoa feeding on Pseudomonas, 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 buffer. Phosphate buer (0.1 ml) with 5:0 108 mlÿ1 bacteria was added to each well as prey.
2.5. Data analysis
Samplings were done on day 0, 3, 6, 9, 13, 21 and
24. On day 21, preparations for early-colonies and
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L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
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 a Pseudomonas selective medium. Graphs represent results from
samples that were not treated with fenpropimorph.
detection of protozoa based on Pseudomonas ¯uorescens DR54 could not be accomplished.
Data were log transformed, and analysed by a twoway ANOVA (analysis of variance) for the eects of
sampling time, fungicide treatment and the interaction
between the two eects. To compare fungicide treatments 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 recommended dose of fenpropimorph. The 10 times recommended dose treatment resulted in an increase in
bacterial numbers until day 6, when numbers were signi®cantly higher than the control a 0:001). Protozoan 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. 2. The number of fast-growing total bacteria (a) and protozoa
(b) around decomposing roots in soil microcosms with dierent concentrations of fenpropimorph: (a) Culturable bacteria on 1/10 TSB
enumerated with the early-colony technique; (b) MPN-counts of protozoa on 1/300 TSB estimated after one week of incubation.
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
Fig. 3. The number of fast-growing Pseudomonas (a) and fast
responding Pseudomonas-feeding protozoa (b) around decomposing
roots in soil microcosms with dierent concentrations of fenpropimorph: (a) Culturable Pseudomonas on Gould's S1 agar enumerated
with the early-colony technique; (b) MPN-counts of Pseudomonasfeeding protozoa estimated after 1 week of incubation.
(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 ¯agellates were found per amoebae. Fig. 2b shows estimates 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 fastresponding protozoa feeding on Pseudomonas (Fig. 3b)
showed no signi®cant eect of fenpropimorph amend-
1619
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 estimated after 4 weeks of incubation.
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. Bacterial numbers peaked signi®cantly twice, on day 3 and
day 13. Fig. 4b shows counts of protozoa in microtitre plates incubated for 4 weeks. The highest fenpropimorph 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 dierences were found only on days 6 and 9.
1620
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
CFUs of Pseudomonas were not aected by the fungicide 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 fenpropimorph 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 as Pseudomonas peaked 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 (Marstorp, 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 traditional plate counts, suggesting that early-colonies
enumerated after a few hours represented only a fastresponding subpopulation of the culturable bacteria.
This can be attributed to the fact that slow-growing
bacteria did not have time to develop, and fast-growing, 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 communication). The dierence between early-CFU and
CFU was more pronounced for TSB than Gould's
because of the dierence in growth rate. Thus the calculated mean generation time of bacteria forming
early-CFUs on TSB was 1±1.5 h, while it was approximately 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 isolates. Kasahara and Hattori (1991) found that fastgrowing isolates generally were able to grow in rich
media, while most slow-growing isolates only grew in
dilute media. They were therefore termed as oligotrophic. de Leij et al. (1993) found that fast-developing
colonies dominated in young rhizosphere compared to
older rhizosphere. In bulk soil, slow-developing colonies were even more prevalent than in rhizosphere.
In the initial stages of this experiment, the root environment 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 population of culturable bacteria.
4.2. Protozoa regulate bacteria
Fig. 5. The number of Pseudomonas (a) and Pseudomonas-feeding
protozoa (b) around decomposing roots in soil microcosms with
dierent concentrations of fenpropimorph: (a) Culturable Pseudomonas on Gould's S1 agar enumerated by plate spreading; (b) MPNcounts of Pseudomonas-feeding protozoa estimated after 4 weeks of
incubation.
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-fungicide 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
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
recommended dose fenpropimorph treatment during
the ®rst 9 days was accompanied by a signi®cantly elevated level of fast-responding bacteria on the 6th day
(Figs. 2 and 4). The reason that only early-colonydetected 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 growing bacterial cells (GonzaÂlez et al., 1993; Pernthaler et
al., 1997).
As fenpropimorph is a fungicide, an initial depression 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). Consequently, we ®nd an earlier reestablishment of the fungal population unlikely in this experiment, when
fenpropimorph was applied in a tenfold higher concentration. 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 Pseudomonas was delayed as compared to the peak of fastgrowing Pseudomonas (Fig. 3), as would be expected in
a prey-predator system. This pattern is still obvious,
though less pronounced when slow-growing Pseudomonas-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 as Pseudomonas CFU (Johnsen et al., 1999).
4.3. Eect of fenpropimorph
In this experiment the lowest concentration of fenpropimorph used (1.3 mg/kg soil) corresponded to the
recommended ®eld dose, if we assume that the fungicide is distributed in the upper 5 cm soil. Fenpropimorph was applied on the soil surface of the
microcosm, but due to the strong absorption properties of the compound we wanted to assure contact
between fenpropimorph and the decomposing roots.
Therefore, the roots were brie¯y submerged in a fenpropimorph solution before being buried.
Fenpropimorph was found to diminish protozoan
numbers, especially the fast-growing protozoa (mainly
1621
¯agellates) detected after one week of incubation. The
signi®cant decrease in protozoan numbers at the highest concentration tested 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 manipulate 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 Rhizobium. 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 with Pseudomonas
¯uorescens DR54. This indicates that the Pseudomonas
strain only was a suitable food for some protozoa. It
is possible that Heteromita globosa was less sensitive to
fenpropimorph, which explains why Pseudomonas-feeding 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 fenpropimorphic acid is more mobile. In a ®eld experiment 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
This study strongly indicates that the interaction
between protozoa and bacteria is an important factor
governing the size of their respective populations.
There was a general correlation between the curve
peaks of high levels of bacteria, succeeded by increasing protozoan populations. A speci®c prey-predator re-
1622
L. Thirup et al. / Soil Biology & Biochemistry 32 (2000) 1615±1623
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 treatment resulted in increased levels of early-colony-forming bacteria on TSB. This shows how toxicants can
aect non-target organisms by aecting the interactions 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 Protection Agency (J. No. 7041-0293) as a part of the project 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 Biological Processes in Contaminated Soil and Sediments.
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