Protozoa Drive the Dynamics of Culturable Biocontrol
Maren Stella Mu ¨ller, Stefan Scheu, Alexandre Jousset*
Georg August University Go ¨ttingen, J. F. Blumenbach Institute of Zoology and Anthropology, Go ¨ttingen, Germany
Some soil bacteria protect plants against soil-borne diseases by producing toxic secondary metabolites. Such beneficial
biocontrol bacteria can be used in agricultural systems as alternative to agrochemicals. The broad spectrum toxins
responsible for plant protection also inhibit predation by protozoa and nematodes, the main consumers of bacteria in soil.
Therefore, predation pressure may favour biocontrol bacteria and contribute to plant health. We analyzed the effect of
Acanthamoeba castellanii on semi-natural soil bacterial communities in a microcosm experiment. We determined the
frequency of culturable bacteria carrying genes responsible for the production of the antifungal compounds 2,4-
diacetylphloroglucinol (DAPG), pyrrolnitrin (PRN) and hydrogen cyanide (HCN) in presence and absence of A. castellanii. We
then measured if amoebae affected soil suppressiveness in a bioassay with sugar beet seedlings confronted to the fungal
pathogen Rhizoctonia solani. Amoebae increased the frequency of both DAPG and HCN positive bacteria in later plant
growth phases (2 and 3 weeks), as well as the average number of biocontrol genes per bacterium. The abundance of DAPG
positive bacteria correlated with disease suppression, suggesting that their promotion by amoebae may enhance soil
health. However, the net effect of amoebae on soil suppressiveness was neutral to slightly negative, possibly because
amoebae slow down the establishment of biocontrol bacteria on the recently emerged seedlings used in the assay. The
results indicate that microfaunal predators foster biocontrol bacterial communities. Understanding interactions between
biocontrol bacteria and their predators may thus help developing environmentally friendly management practices of
Citation: Mu ¨ller MS, Scheu S, Jousset A (2013) Protozoa Drive the Dynamics of Culturable Biocontrol Bacterial Communities. PLoS ONE 8(6): e66200. doi:10.1371/
Editor: Hauke Smidt, Wageningen University, The Netherlands
Received December 14, 2012; Accepted May 5, 2013; Published June 26, 2013
Copyright: ? 2013 Mu ¨ller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge support by the Open Access Program of the German Science Foundation (DFG) and the Publication Funds of the Georg
Aug st University Go ¨ttingen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.u
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Various soil-dwelling bacteria improve plant health by inhib-
iting pathogens, and have been increasingly investigated during
the last decades as biocontrol agents to replace pesticides in
agriculture. Biocontrol bacteria produce antifungal secondary
metabolites suppressing pathogens and have been shown to reduce
disease severity in agricultural systems  and contribute to the
high productivity of species-rich grasslands . In order to
efficiently use beneficial bacteria to increase plant yield, there is
the need to better understand the factors driving their fitness in
soils. Numerous strains with a high biocontrol potential have been
isolated and tested in vitro, but their commercial application is often
limited by their low persistence in soil in the field . To survive in
soil and in the rhizosphere, bacteria must compete with the
indigenous microflora and resist predation . Especially preda-
tion by protozoa is a major selection pressure that shapes the
structure of bacterial communities in the soil and the rhizosphere
[5,6], as well as the competitiveness of single strains . Many
bacterial secondary metabolites known for their activity against
soil pathogens are also active against protozoa [8,9,10]. In
laboratory systems, biocontrol bacteria producing secondary
metabolites outcompete non-toxic ones when confronted with
protozoa [4,7,11]. Due to this protective effect, we expected
predation by protozoa in soils to promote bacteria producing
biocontrol secondary metabolites by preferentially consuming non-
producer bacteria, thereby improving the potential of soil bacterial
communities to inhibit plant pathogens.
We investigated the impact of predation at the community level,
with a particular focus on fluorescent pseudomonads, one of the
most intensively studied groups of biocontrol bacteria .
Pseudomonads produce a wealth of antimicrobial secondary
metabolites and extracellular enzymes that inhibit other bacteria,
fungi, protozoa and nematodes . Secondary metabolites
(PRN), two broad spectrum antifungal compounds, and hydrogen
cyanide (HCN), an inhibitor of the respiratory electron transport
system . These compounds efficiently suppress phytopatho-
genic fungi , and also efficiently protect bacteria against
nematodes and protozoa [9,14,15]. We tested if predation by
protozoa increases the numbers of bacteria carrying genes
responsible for the synthesis of these toxins and if this results in
improved soil suppressiveness against phytopathogens.
We set up microcosms with barley seedlings growing in soil
containing semi-natural bacterial communities and the model
protozoan predator Acanthamoeba castellanii. We measured the
abundance of total pseudomonads and the frequency of bacteria
carrying the functional genes responsible for the synthesis of
DAPG, PRN and HCN. Further, we tested if predation induced
functional shifts in bacterial communities resulting in increased
(DAPG) and pyrrolnitrin
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plant protection against pathogens in a biocontrol assay with sugar
beet and Rhizoctonia solani.
Materials and Methods
Preparation of protozoa-free bacterial communities
In order to measure the effect of protozoa on soil microbial
communities, we first established protozoa-free, semi-natural
bacterial communities that were submitted to predation by A.
castellanii. Soil from the Jena Experiment field site, Germany
(Eutric luvisol; for details see Roscher et al. ), was dried at room
temperature for 24 h and one volume of soil was suspended in
three volumes of distilled water of 4uC. Bacteria were extracted on
ice according to Prieme ´ et al.  with few modifications: The soil
slurry was ultrasonicated for 10 s to detach bacteria from soil
particles. The suspension was shaken (300 rpm) for 30 to 60 min
and centrifuged at 150 g for 5 min at 4uC to remove soil debris.
The supernatant was filtered through cotton discs for manual milk
filtration (160 mm), and centrifuged on Percoll (GE Healthcare
Bio-Sciences AB, Uppsala, Sweden; density 1.13 g mL21). The
upper phase containing bacteria was then filtered through 5 and
1.2 mm membranes to remove protozoa . Filters were changed
at regular intervals to avoid contamination by small protozoa
(flagellates). The filtrate with bacteria was decanted in tissue
culture flasks and incubated at 14uC . After one week the
bacterial suspension was checked for contamination by flagellates
under an inverted microscope. Prior to use the biofilm in the tissue
culture flasks was resuspended in an ultrasonic bath (Bandelin
electronic, Berlin, Germany) (10% power, 25 s) at 20uC,
centrifuged (4600 rpm, 15 min) and washed twice in Page
Amoeba Saline (PAS) .
Amoebae (A. castellanii) were isolated from a woodland soil 
and grown axenically on PGY medium (peptone 10 g L21,
glucose 10 g L21, yeast extract 5 g L21) in tissue culture flasks
at 14uC . Prior to the experiment, cultures were washed twice
by gentle centrifugation (3006 g, 30 s). The pellet was resus-
pended in PAS and stored at 14uC. Cell concentration was
determined with a Neubauer counting chamber.
Barley (Hordeum vulgare) seeds were surface-sterilised as described
elsewhere . Briefly, seeds were dehusked in 50% sulphuric acid
for 70 min, washed two times with H2O for 5 min and once with
1% NaHCO3to neutralise acidity. Dehusked seeds were surface
sterilized with 2% AgNO3for 20 min, washed twice for 5 min
with 1% NaCl and H2O, four times with H2O and germinated on
1.5% water agar at 24uC in the dark.
Soil from the Jena experiment field site (see above) was sieved
through 2 mm mesh to remove plant debris, macrofauna and
stones. Microcosms consisted of 206300 mm glass tubes filled with
40 g of a 1:1 mix of soil and quartz sand (grain size 0.1–0.5 mm).
Tubes were closed with a cotton plug and aluminium foil, and
autoclaved (121uC, 20 min).
Sterile microcosms were inoculated with 2 mL of a protozoa
free soil bacteria suspension (4*106bacteria mL21) and soil
moisture was adjusted with sterile water to ensure that the soil was
moist but not wet. Microcosms were incubated for one week at
20uC to permit bacterial growth. Barley seedlings were transferred
into microcosms inoculated with soil bacterial suspension. After
four days microcosms were inoculated with 1 mL amoebae
suspension (2*105amoebae mL21), or 1 mL PAS as control.
Eight microcosms were set up for each treatment and sampling
date. Plantswere grownat
(250 mmol s21m22).
21uC with12 hoflight
Enumeration of soil bacteria and protozoa
Microcosms were destructively sampled 0, 7, 14 and 21 days
after inoculation with protozoa. For each sampling date and
treatment, eight microcosms were harvested. Barley roots were
shaken in 10 mL 0.16 phosphate buffer saline (PBS) for 1 h to
extract rhizosphere bacteria, dried (50uC, 24 h) and weighed.
Total aerobic bacteria and fluorescent pseudomonads were
enumerated by serial dilution plating on TSA (tryptic soy broth
3 g L21, agar 15 g L21) and Gould’s S1 agar plates ,
respectively. Colonies were enumerated after incubation at 24uC
for at least 48 h and density expressed as CFUs per plant.
Amoebae were enumerated by Most Probable Number (MPN)
using Pseudomonas fluorescens CHA19 as food source as described
For each sample, nine colonies were picked from the Gould’s S1
plates, heat lysed , and the presence of the biocontrol genes
phlD, hcnAB and prnD were assessed by PCR as previously
described [23,24,25]. PCR was carried out in 20 mL reaction
mixtures containing 16KAPA2G Buffer B, 16KAPA Enhancer
1, 0.2 mM dNTP mix, 0.325 mM of each primer and 0.5 U of
KAPA2G Robust DNA Polymerase (PEQLAB, Erlangen, Ger-
many). Amplifications were performed with the following cycling
program: initial denaturation at 95uC for 10 min, followed by 40
cycles of denaturation at 95uC for 30 s, primer annealing at 67uC
(phlD, hcnAB) or 68uC (prnD) for 45 s and extension at 72uC for
1 min, and a 10 min final extension step at 72uC. The presence of
the corresponding amplicons was assessed on 1.5% agarose gel
stained with ethidium bromide.
Biocontrol assay on soil suppressiveness against
The influence of amoebae on the antagonistic potential of the
bacterial community against phytopathogens was tested in a
biocontrol assay including sugar beet seeds (Beta vulgaris L. cv.
Belinda) and the pathogen Rhizoctonia solani Ku ¨hn (AG 2–2 IIIB) as
described in Latz et al. . The sugar beet - Rhizoctonia
combination allows efficient screening of pathogen development.
Especially the Belinda cultivar is sensitive to a broad range of
pathogens and is used as bait plant to isolate generalist soil-borne
pathogens; it rapidly develops infection symptoms and dies in
absence of protective bacteria . The use of this plant-pathogen
system is an extension of a bait plant system: a plant vulnerable to
infection by various pathogens may allow estimating pathogen
virulence and as a reverse function soil suppressiveness, while
avoiding potential biases due to plant-driven accumulation of
protective bacteria that might only be able to protect the host plant
while being of marginal relevance for other plant species .
Briefly, 40 g root free soil from the last harvest at day 21 (8
microcosms without amoebae, 8 microcosms with amoebae) were
transfered into autoclaved Magenta boxes (7.467.469.7 cm) and
rewetted with 500 mL sterile H2O. Eight sugar beet seeds (99.9%
germination rate) were added to each box below the soil surface.
Subsequently, half of a barley seed infested with R. solani was
placed in the center of the box. The boxes were incubated at 21uC
with 12 h light (250 mmol s21m22). Infection was characterized
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by counting brown roots, stems, leaves and snapped stems of the
sugar beet germ buds over a period of 19 days.
The effect of amoebae and time on the frequency of phlD, hcnAB
and prnD positive isolates was analyzed with a Poisson GLM. The
effect of amoebae as well as the abundance of biocontrol genes on
disease development (brown roots, stems, leaves and snapped
stems of sugar beet seedlings by R. solani) was assessed with a
random intercept mixed effect model with Poisson distribution
investigating the effect of the amoebae and the abundance of
bacteria carrying phlD, hcnAB or prnD genes (as measured at the
end of the first experiment) on disease development over time.
Each symptom was analyzed separately. All analyses were carried
out with R 2.12 (R core development Team, Vienna, Austria).
Barley root growth
In this microcosm assay we investigated whether predation by
amoebae affects biocontrol bacterial communities associated with
the production of antifungal secondary metabolites. Barley roots
were weighed to examine the influence of amoebae on root
growth. The fresh weight (F3,54=46.1, P=,0.001) and the dry
weight (F3,54=4.3, P=0.009) significantly increased with time.
Amoebae did not affect barley root growth (F1,54=2.6, P=0.114
and F1,54=0.3, P=0.60 for fresh weight and dry weight,
Dynamics of bacterial and protozoan density
The total number of cultivable bacteria varied with time
(F3,54=30.5, P,0.001); it increased until day 14 and then
decreased (Table S1). By contrast, the number of pseudomonads
was not affected by time (F3,54=1.0, P=0.38). The presence of
amoebae did not affect the abundance of cultivable bacteria and
pseudomonads (F1,54=1.2, P=0.27 and F1,54=0.9, P=0.36,
respectively; Table S1).
The density of amoebae remained constant during the
experiment, with a density of of 2–4*105amoebae per microcosm
at the end of the experiment.
Frequency of bacteria carrying biocontrol genes
Cultivable pseudomonads carrying the phlD, hcnAB or prnD
genes increased in frequency over time in presence of amoebae,
but tended to decline in the control treatment (Table 1, Figure 1)
The proportion of bacteria harboring biocontrol genes was higher
in presence of amoebae with the exception of day 7 (Table 2). This
suggests that the tested biocontrol genes provided a selective
advantage in presence of predators, but did not improve
competitiveness against other bacteria. The effect of amoebae
was more marked at later growth stages (Amoebae6Time
interaction; Table 1) and varied with the tested genes. Amoebae
significantly increased the frequency of bacteria carrying the hcnAB
and phlD genes (Figure 1), in agreement with the importance of
HCN and DAPG as antipredator defense compounds . In
contrast, the frequency of prnD positive bacteria was not
significantly influenced by amoebae.
Amoebae also increased the average number of biocontrol genes
in each isolate, and this effect increased with time (Table 1),
suggesting that the ability to produce a combination of different
secondary metabolites increases bacterial resistance against
Soil suppressiveness against Rhizoctonia solani
In the biocontrol assay with sugar beet we investigated if
amoebae-induced shifts in biocontrol communities resulted in
differences in soil suppressiveness. We followed the infection of
sugar beet by Rhizoctonia solani in microcosms from the microcosm
experiment, containing soil bacterial communities previously
incubated for 21 days with or without amoebae. Plant infection
by R. solani caused different symptoms, including brown roots,
stems, leaves and snapped stems of the sugar beet germ buds
(Table S2). Disease development was negatively correlated with
the abundance of bacteria carrying the genes phlD (z=22.445,
P=0.014) and prnD (z=22.056, P=0.039) at the beginning of the
biocontrol assay, confirming the importance of these two genes for
biocontrol activity. However, the presence of amoebae did not
affect plant disease and even tended to increase the number of
plants with brown roots (z=0.167, P=0.09).
Effects of protozoa on bacterial communities
Predation by protozoa is a major driver of the density and
functioning of bacterial communities [5,6]. Unexpectedly, in this
study amoebae did not affect the abundance of total cultivable
bacteria and pseudomonads, but increased the frequency of
bacteria carrying the genes responsible for the production of
DAPG and HCN. These two compounds function as broad
spectrum antifungal and antihelminthic metabolites, and are
involved in the suppression of various root diseases [26,27,28,29].
The same secondary metabolites also improve bacterial fitness in
presence of protozoa [8,9,10]. Manipulating ecological forces
favoring one function of the tested genes (bacterial fitness) can be
used to obtain another, desired service provided by the same genes
(inhibition of phytopathogens). Bacteria carrying more than one
biocontrol gene were more abundant in presence of amoebae,
suggesting that the production of multiple toxic secondary
metabolites increases protection against predation. This effect of
predation likely also affects the biocontrol function of the bacteria.
First, bacteria producing multiple antifungal compounds are more
likely to protect plants against a broader range of soil-borne
pathogens than bacteria producing one single antifungal com-
pound. Second, bacteria carrying multiple antifungal genes might
be able to better persist in soil, a property that may help
developing effective biocontrol inocula. The promotion of
biocontrol bacteria may be further enhanced by using other
microfaunal predators. The amoebae used in this study are less
prey-selective than other protozoa such as flagellates . More
selective protozoan species may consume only non-toxic bacteria
and thus promote biocontrol bacteria by eliminating competitors.
Further studies are needed to identify which protozoan taxa are
responsible for promoting plant beneficial soil microbial commu-
nities in field soil.
Soil suppressiveness against Rhizoctonia solani
In contrast to our hypotheses, plants growing in soil previously
inoculated with bacteria and amoebae developed the same levels
or slightly more disease symptoms than plants growing in the
control soil containing bacteria only. Nonetheless, disease devel-
opment was negatively correlated with the abundance of prnD and
phlD positive isolates at the end of the first microcosm experiment.
This underpins the role of DAPG and PRN for inhibiting R. solani,
but appears contradictory to the net effect of amoebae: parallel to
the amoebae-mediated increase in the abundance of biocontrol
bacteria we expected reduced disease development. Potentially,
the lack of disease reduction resulted from the interaction between
Protozoa and Rhizosphere Bacteria
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protozoa and biocontrol bacteria at early plant growth stages. In
the barley microcosm experiment, protozoa reduced the estab-
lishment of biocontrol bacteria in very young seedlings, but they
fostered them later. A similar effect may also have occurred in the
biocontrol assay with sugar beet. Despite higher abundance of
biocontrol bacteria at the beginning of the experiment, amoebae
may have reduced their numbers during the early growth phase of
the sugar beet seedlings, affecting plant protection. Notably,
DAPG producers need to reach a threshold density of 105CFU g
root21to effectively suppress pathogens [31,32], and predation
may have transiently reduced their number under this critical
threshold thereby reducing protection against the pathogen in
young seedlings. Further studies on the dynamics of bacteria and
predators during the full plant growth cycle are needed to allow
predicting plant protection by rhizosphere bacterial communities
in presence of predators.
Overall, the study highlights the importance of predation as
driver of the functionality of soil microbial communities with
biocontrol potential. We propose that manipulating predation
pressure may allow developing new strategies improving pathogen
Figure 1. Effect of Acanthoeba castellanii on the frequency of phlD, hcnAB and prnD positive Pseudomonas (A–C) and on the average
number of genes per bacterium (D) during the microcosm experiment with barley (means ± SE). Presence of each gene was tested by
colony–PCR on isolates growing on the Pseudomonas specific Gould’s S1 medium. closed symbols: bacterial communities co-cultivated with
Acanthamoeba castellanii, open symbols: control treatment without protozoa.
Table 1. Results of Poisson General Linear Models on the effects of Amoebae, Time and interaction between Amoebae and Time
on the frequency of phlD, hcnAB and prnD positive bacteria, and on the frequency of biocontrol genes per colony in the microcosm
experiment with barley.
positive bacteriaBiocontrol genes/colony
Amoebae1 0.1 0.785.4 0.02
Time3 1.80.61 5.00.186.0 0.12 3.80.43
Significant effects are highlighted in bold (P,0.05).
Protozoa and Rhizosphere Bacteria
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suppression and may be used to develop new biocontrol inocula
with high persistence in soils.
monads at the different time points after incubation
with or without Acanthamoeba castellanii. Bacteria were
enumerated on TSA (total heterotrophic bacteria) or Gould S1
(Pseudomonas), abundances are expressed as CFU per plants.
Abundance of cultivable bacteria and pseudo-
sown in each microcosm) with infection symptoms in
the biocontrol assay.
Number of sugar beet germ buds (out of the 8
Conceived and designed the experiments: AJ. Performed the experiments:
MM. Analyzed the data: MM AJ. Wrote the paper: MM AJ SS.
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Table 2. Number of screened bacterial isolates carrying biocontrol genes in the microcosm experiment with barley.
Time (days)Amoebae Isolates screened
Isolates carrying biocontrol genes forIsolates with…
DAPG HCN PRN
70 5516207 12113
7171 11 175 11110
140 68 13641241
141 65 161882 144
211 63131888 141
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