Post on 23-Oct-2020
AARHUS UNIVERSITY DEPARTMENT OF ENVIRONMENTAL SCIENCE
VERSITET
Anne Winding, senior scientist Dept. of Environmental Science, Aarhus University, Denmark
AARHUS UNIVERSITY
DEPARTMENT OF ENVIRONMENTAL SCIENCE
July 8th 2014
UNI
Interaction between bacteria and protozoa
in soil
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2
Outline of presentation: bacteria and protozoa
Protozoa
Effect of protozoa on bacteria
Effect of bacteria on protozoa
Interactions in vitro
Interactions in soil
Diversity of soil protozoa
Bacterial pathogens and soil protozoa
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Protozoa
› Protista: unicellular eukaryotic organisms: protozoa, unicellular
algae, and slime molds.
› Proto = first, zoa = animals.
› Aquatic – motile – water films
› 5-500 µm
› Non-monophylogenetic origin
› Ciliates, flagellates, heliozoans (aquatic, with exopodia), and
amoebas.
› Trophozoites: physiological active stage
› Cysts: inactive resting stage, recalcitrant. Water stress; Cyst
bank
3
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Distinct morphological groups
Several phylum
4
Tikhonenkov, 2010 Ekelund, 2002
Foissner and Al-Rasheid, 2007
Smirnov and Brown, 2004
• Flagellates,
1-few flagella, asexual reproduction
• Amoebae,
No flagella, asexual reproduction
(sexual reproduction rare)
• Ciliates,
Many tiny flagella, sexual and asexual
reproduction
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Trophic interactions btw bacteria and predators
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Bacteria
Bacterial-feeding
nematodes
Protozoa
Predatory
nematodes
Omnivorous
nematodes
Enchytraeids
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Protozoa: feeding behaviour
›Suspended bacteria:
–Direct interception: flagellates, individual bacteria in suspension
– Filter feeding: in soil few ciliates
–Diffusion feeding: stationary protozoa, rare in soil
›Attached bacteria
–Raptorial feeding: food searching mobile protozoa (up to
69%)
–Grasping: protozoa feeding on attached bacteria (biofilm), common in soil (up to 30%).
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Predator – prey interaction
7 Jousset 2012
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Food uptake ›Phagocytosis
›Food vacuoles
›Enzymatic digestion
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Protozoa affect abundance of bacteria
(Sinclair and Alexander 1989)
Old news
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Avoidance of predation
(Pernthaler 2005)
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In vitro studies: controlled model systems
Bodo designis
Neocercomonas jutlandica
Bodo caudatus Cercomonas longicauda
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Microbial Pest Control Agents:
• antagonistic effects on fungi and insects • effects on predatory protozoa?
Pseudomonas spp. against root pathogenic fungi Means of microbial pest control:
- Secondary metabolites
- Competition of ressources
- Degradation of pathogenicity factors
- Production of enzymes
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Growth of amoebae and bacteria in vitro
(Andersen and Winding 2004)
Amoebae
Bacteria
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(Pedersen et al. 2010)
pseudomonads
E. aerogenes
protozoa
B. caudatus
Days
0 2 4 6 8 10
C. longicauda
Days
0 1 2 3 4 5 6
CF
U m
l-1
101
102
103
104
105
106
107
108
109
Control
P. chlororaphis ATCC43928
P. fluorescens DR54
P. fluorescens CHA0
Figure 1: Pedersen et al.
Figure 2: Pedersen et al.
N. jutlantica
Days
0 2 4 6 8 10
Flagellates: different sensitivity
N. jutlandica
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Spent bacterial growth media
show effects on C. longicauda
growth - depending on bacteria
(Pedersen et al. 2010)
CHA0
Time (days)
103
105
107
109
C. longicauda
P. fluorescens
E. aerogenes
DSM50090T
Fla
ge
llate
or
ba
cte
ria (
cells
ml-1
)
103
105
107
109
Time (days)
0 2 4 6 8 10
Washed
Not washed
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Effects of secondary metabolites
Growth of soil protozoa inhibited by DR54 cell extract
(Andersen and Winding 2004)
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Cercomonas longicauda swimming
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9 protozoa grown on 7 bacteria:
- 4 secondary metabolite producing
- 3 non-producing
B. designis 23
Time (days)
Fla
gella
tes (
ce
lls m
l-1)
101
102
103
104
105
106
N. jutlantica
Fla
gella
tes (
ce
lls m
l-1)
101
102
103
104
105
106
107
Spumella sp.
0 2 4 6 8 10
Fla
gella
tes (
ce
lls m
l-1)
101
102
103
104
105
106
no bacteria added
E. aerogenes
P. chlororaphis ATCC 43928
P. fluorescens DSM 50090
P. fluorescens DR54
P. fluorescens CHA0
Pseudomonas sp. DSS73
P. chlororaphis MA342
(Pedersen et al. 2011)
N. jutlandica
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Resulting average growth ratewhen fed to nine different protozoa
Food bacterium
P. f
luor
esce
ns D
SM
5009
0
P. c
hlor
orap
his ATC
C43
928
Ent
erob
acte
r aer
ogen
es
P. f
luor
esce
ns D
R54
P. c
hlor
orap
his M
a342
Pse
udom
onas
sp.
DSS73
Pho
spha
te b
uffe
r, no
bac
teria
P. f
luor
esce
ns C
HA0
ave
rag
e g
row
th r
ate
(d
ay
-1)
0.5
1.0
1.5
2.0
2.5
a
e
a
b
c
d
g
f
Food quality
(Pedersen et al. 2011)
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Ratio between growth rate on 4 metaboliteproducing and 3 non producing bacteria
Cer
com
onas
long
icau
da
Neo
cerc
omon
as ju
tland
ica
Het
erom
ita g
lobo
sa
Pha
lans
teriu
m soilitar
ium
Har
tman
ella v
erm
iform
is
Bod
o ca
udat
us
Bod
o de
sign
is 2
3
Bod
o de
sign
is U
J
Spu
mella sp.
Ratio
0.2
0.4
0.6
0.8
1.0
_________ ___
__
Rhizaria
(Cercomonadidae)
Amoebozoa
Excavata
(Bodonidae)
Chromalveolata
b b
bc bc
cdd
de
e
a
Dependence on type
of protozoa
(Pedersen et al. 2011)
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Flow cytometry for counting
(Pedersen et al 2009)
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Difference in food
selectivity by protozoa
and nematode:
protozoa select,
nematodes don’t
(Pedersen et al 2009)
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Interactions in vitro
›Difference in food quality of bacteria
– Correlating with secondary metabolite production
– Importance of membrane bound vs unbound metabolites
– Unknown compounds?
› Feeding behaviour
– Difference between protozoa in growth on the same bacteria
– Difference in selectivity between protozoa and nematode
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Location in soil
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Ciliates
Flagellates
Bacteria
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AARHUS UNIVERSITY DEPARTMENT OF ENVIRONMENTAL SCIENCE
Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Location in soil
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1. Plenty of water: trophozoites and viable bacteria
2. Less water: cysts and viable bacteria
3. Little water: cysts and spores
1 2 3
soil pore
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Biochar as protection against bacterial predators?
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Tetrahymena Pseudomonas
Valentina Imparato
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Soil food web
M. B
onkow
ski e
t al. /
Eur.
J. S
oil
Bio
l. 3
6 (
2000)
135–147
Protozoa
Microflora
Bacteria Fungi
Earthworms Nematodes
Nutrients and
hormones Root
exudates
Organic
matter
Up to 100,000 individuals/gram soil
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Interactions in rhizosphere
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Prashar et al. 2014
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Interaction btw prey and predator
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fenpropimorph
Thirup et al 2000
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Soil and rhizosphere inoculated with P. fluorescens DR54: Positive effect on fast-responding protozoa
(Johansen et al. 2005)
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Rhizosphere of Arabidopsis thaliana: Amoebae decrease total abundance
and change diversity of bacteria
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Rosenberg et al 2009
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Susana Santos unpubl.
AARHUS UNIVERSITY DEPARTMENT OF ENVIRONMENTAL SCIENCE
Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
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P. fluorescens CHA0 and CHA0 pME3424 in soil
› Isolated from tobacco rhizosphere
› P. fluorescens CHA0: DAPG, Plt, Prn, HCN
› P. fluorescens CHA0 pME3424: ++ prod. of Plt and DAPG
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Total culturable bacteria
harvest after 1 day
CF
U g
-1 d
w
107
108
109
control
E. aerogenes
CHA0/gfp1
CHA0/pME3424
harvest after 7 days
CF
U g
-1 d
w
107
108
109
harvest after 14 days
Time (days)
0 10 20 30 40
CF
U g
-1 d
w
106
107
108
109
Figure 3. Winding and Oberender
a
b
a
abc
aaa
a
a
a
(Winding and Oberender 2012)
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
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Soil Protozoa
control
Enterobacter aerogenes
P. fluorescens CHA0 gfp
P. fluorescens CHA0 pME3424
harvest time (days)1 7 14
Fa
st-
gro
win
g p
roto
zo
a g
-1 d
ry s
oil
102
103
104
105
control
E. aerogenes
P. fluorescens CHA0/gfp1
P. fluorescens CHA0/pME3424
harvest time (days)
1 7 14
To
tal p
roto
zo
a g
-1 d
ry s
oil
102
103
104
105
bd
bd
bd
Y
h
h
h
Figure 4 Winding and Oberender
a
b
e
d
cc
c
c
f
X
Y Y
X
Y gnd
nd
nd
nd
nd
nd
(Winding and Oberender 2012)
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
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Effects of P. fluorescens MPCA on soil protozoa
› Slight negative effect of DR54 and CHA0 on CFU, no effect
on soil respiration and bacterial diversity
› Positive effect of DR54 on the abundance of fast growing
and total soil protozoa.
› Negative effect of CHA0 on abundance of protozoa
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Protozoa increase abundance of MPCA in rhizosphere
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With protozoa
No protozoa
Müller et al. 2013
Protozoa positively increase effect of MPCA in rhizosphere?
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Protozoa change the physiological profile of bacteria in soil
(Rønn et al. 2002)
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Variation in changes of bacterial community depends on protozoan species
(Rø
nn e
t al. 2
002)
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Effects of predation in rhizosphere
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The percentage of Verrucomicrobia and Actinobacteria of eubacterial biomass as revealed by the
FISH method at the last harvest date (day 32). The relative abundance of these bacterial groups
was significantly higher when protozoa were present ( P = 0.033 and 0.005; respectively). Ekelund et al. 2009.
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What is the diversity of protozoa? How to determine it? ›Determination
– Traditional: Isolate and identify in microscope
–Molecular analysis of soil protozoa
− Isolate and extract DNA and use bar coding
− Extract DNA and use bar coding or DNA primers or NGS or??
›Challenges:
–Many phylums – design of primers
– Extraction of DNA – trophozoites and cysts
–Databases
41
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Molecular techniques demonstrate protists diversity is higher
than morphological studies suggest
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Genetic diversity of Kinetoplastidae
1 day 7 days 14 days E. aer. b
CHA0/gfp1 a
CHA0/gfp1 c
CHA0/gfp1 b
CHA0/pME3424 a
CHA0/pME3424 b
CHA0/pME3424 c
Control a
Control b
E. aer. c
Control c
E. aer. a
0.7 0.8 0.9 1.0 SAB
CHA0/gfp1 c
CHA0/gfp1 a
E. aer. b
E. aer. a
CHA0/pME3424 c
Control b
Control a
Control c
E. aer. c
CHA0/gfp1 b
CHA0/pME3424 a
CHA0/pME3424 b
0.7 0.8 0.9 1.0 SAB
0.7 0.8 0.9 1.0 SAB
Control b
Control a
Control c
E. aer. a
E. aer. b
CHA0/gfp1 a
CHA0/gfp1 c
CHA0/pME3424 b
E. aer. c
CHA0/pME3424 a
CHA0/gfp1 b
CHA0/pME3424 c
(Winding and Oberender 2012)
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harvest time (days)
1 7 14
ba
nd
s o
n D
GG
E g
el
6
8
10
12
14control
E. aerogenes
P. fluorescens CHA0/gfp1
P. fluorescens CHA0/pME3424
Figure 6. Winding and Oberender
(Winding and Oberender 2012.)
Genetic diversity of Kinetoplastidae
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45 Letendu et al 2014
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Protozoa as Trojan horses
Potential impacts on survival and spreading of human pathogens
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Protozoa as Trojan horses
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Fate of bacteria after phagocytosis
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- Protozoan lysis of bacteria
- Bacterial lysis of protozoa
- Bacterial multiplication inside
amoebae
- Bacterial survival
- Increased gene transfer
- Increased virulence
- Increased antibiotic resistance
- Transcriptome changes
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Campylobacter jejuni and Acanthamoeba castellanii - poor survival of bacteria inside amoebae
(Bui et al. 2012 Env Microb)
0 h
24 h
5 h
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A. castellanii increases growth of C. jejuni
(Bui et al. 2012 Env Microb)
+ amoebae, separated
+ amoebae
- amoebae, micro O2
- amoebae
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Anne Winding.
July 8th 2014 Interaction between bacteria and protozoa in soil
Bacteria packaging by amoebae Dig. B: digestible bacteria Res. B: resistant to lysosomal degradation
51
Legionella
pneumophila
in Multimellar
bodies (MLB)
Empty MLB
Denoncourt et al 2014
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›Resting stage
›Resistant to draught, low food concentration, adverse temperatures etc.
›Resistant to predation?
›Germinate at high nutrient availability
›Spore germination inside protozoa?
Bacterial spores
52
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Tetrahymena and Bacillus
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Interactions between bacteria and protozoa in soil
›Tight interaction :
– Protozoa affect bacteria
–Bacteria affect protozoa
›Effect on plant growth and N-cycling
›Survival and transport of bacteria
54
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Acknowledgement
Co-authors: Susana Santos, Valentina Imparato,
Annette Pedersen, Karen S Andersen, Karen S
Jensen, Jana Oberender, Flemming Ekelund,
Anders Johansen,
Technicians: Anne-Grethe Holm-Jensen, Tina Thane
Funding: Danish Research Councils, EU FP7
EcoFINDERS, ITN-Trainbiodiverse