Interactions between bacteria and microfaunal predators...

AARHUS UNIVERSITY 16th March 2012

Interactions between bacteria and microfaunal

predators and implications for biodiversity and

turnover of organic matter

Anne Winding, senior scientist

Environmental Science, Aarhus University, Roskilde, Denmark

Flemming Ekelund, associate professor

Terrestrial Ecology, Biological Sciences, Univ. of Copenhagen, Denmark

AARHUS UNIVERSITY

16th March 2012 Interaction between bacteria and protozoa

Anne Winding 2

Interactions in soil and rhizosphere

M. Bonkowski et al. / Eur. J. Soil Biol. 36 (2000) 135–147

Protozoa

Microflora

Bacteria Fungi

Nematodes

Nutrients and

hormones

Microflora

Bacteria Fungi

Nutrients and

hormones

Protozoa

Earthworms

Nematodes

Nutrients and

hormones Root

exudates

Organic

matter

Microflora

Bacteria Fungi

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Anne Winding

› unicellular euckaryotic organisms

› many are motile

› size range from 10 to 52 µm

3

Protozoa

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Protozoa affect abundance of bacteria

(Sinclair and Alexander 1989)

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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

et

al. 2

00

2)

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Interaction between bacteria and protozoa

- but how?

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Aquatic environment

(Pernthaler 2005)

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Environmental Risk Assessment

Microbial Pest Control Agents:

• antagonistic effects on fungi and insects

• effects on predatory protozoa?

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Pseudomonas spp. against root pathogenic fungi

Means of microbial pest control:

- Secondary metabolites

- Competition of ressources

- Degradation of pathogenicity factors

- Production of enzymes

10

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Pseudomonas fluorescens DR54

- isolated from sugar beet rhizosphere

- producing membrane-bound viscocinamide and

cellulytic enzymes

12

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Soil and rhizosphere

inoculated with P.

fluorescens DR54

Small negative effect on

CFU

13

(Johansen et al. 2005)

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Soil and rhizosphere inoculated with DR54

Positive effect on fast-responding protozoa

(Johansen et al. 2005)

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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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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

16

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Fate of CHA0 gfp and CHA0 pME3424

Pseudomonas fluorescens

Days

0 2 4 6 8 10 12 14

log

CF

U g

-1 d

w

6,0

6,5

7,0

7,5

8,0CHA0/gfp1

CHA0/pME3424

Figure 2. Winding and Oberender

(Winding and Oberender unpubl.)

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Soil respiration

Time (days)

0 2 4 6 8 10 12 14 16

Accum

ula

ted

CO

2 (

mg m

icro

cosm

-1)

-2

0

2

4

6

8

10

control

E. aerogenes

P. fluorescens CHA0 gfp1

P. fluorescens CHA0/pME3424



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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


a

b

a

abc

aaa

a

a

a


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PCR-DGGE of Bacteria E

a 0

E

a 1

4

CH

A0

0

CH

A0

14

C

HA

0+

0

CH

A0

+ 1

4

Co

n 0

Co

n 0

C

on

7

Co

n 1

4

Ea

7

CH

A0

7

CH

A0

14

C

HA

0+

7

CH

A0

+ 1

4


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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


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


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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


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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



Winding and Oberender (Winding and Oberender unpubl.)

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Effects of P. fluorescens MPCA in soil

› 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.

› Effect of CHA0 on abundance of protozoa?

› Effect of CHA0 on genetic diversity of Kinetoplastidae at

day 0, no effect later

› The DAPG and Plt wild-type and the overproducing strain

of CHA0 generally had the same effect

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In vitro studies: controlled model systems

Bodo designis

Neocercomonas jutlandica

Bodo caudatus Cercomonas longicauda

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Bacteria used secondary metabolites

P. fluorescens DR54 viscocinamide*, cellulytic enzymes

P. fluorescens CHA0 DAPG, Plt, Prn, HCN

P. fluorescens CHA0 pME3424 overproduction of Plt and DAPG

P. chlororaphis MA342 2,3-deepoxy-2,3-didehydrorhizoxin

Pseudomonas sp. DSS73 amphisin*, HCN

Bacillus licheniformis

Campylobacter jejuni

* membrane bound

Control strains

P. fluorescens DSM50090 na

P. chlororaphis SC na

Enterobacter aerogenes na

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Growth of amoebae in vitro


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Growth of amoebae in vitro


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Bacterial growth during predation by amoeba


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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.


N. jutlantica

Days

0 2 4 6 8 10

N. jutlandica

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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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Cercomonadidae, Rhizaria Excavata, Bodonidae Chromalveo

lata Amoebozoa

Cercomonas

longicauda

Neocercomonas

jutlandica

Heteromita

globosa

Bodo

caudatus

Bodo

designis 23

Bodo

designis

UJ

Spumella sp. Phalansterium

solitarium

Hartmannella

vermiformis

No bacteria added 0 2.05) A 0.84 F 0 0.73 C 0.90 C 0 0 0

E. aerogenes 1.82 A 1.72 AB 3.76 D 1.97 A 1.31 B

2.13

A

B

1.04 B 1.34 B 1.75 A

P. chlororaphis

ATCC43928 1.96 A 1.61 AB 4.82 B 1.90 AB 2.00 A 2.66 A 0.94 B 0.72 C 1.39 B

P. fluorescens

DSM50090T 1.81 A 1.63 AB 4.31 C 1.76 B 1.89 A 2.61 A 2.22 A 1.10 B 1.69 A

P. fluorescens

DR54 1.96 A 1.73 AB 5.62 A 0 0.60 C 0 0 1.82 A 0.71 DE

P. fluorescens

CHA0 *1.84 A 0 0 0 0 0 0 0 1.26 BC

Pseudomonas sp.

DSS73 1.73 A 0.46 C 2.70 E 0.72 D 1.25 B 0 0 0 0.61 E

P. chlororaphis

MA342 1.55 A 1.36 B 1.30 E 1.52 C 0 1.79 B 0 0.24 D 0.95 CD

Growth rates


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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


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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


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DSM50090T

Fla

ge

llate

or

bacte

ria (

cells

ml-1

)

103

105

107

109

C. longicauda

P. fluorescens

E. aerogenes

DR54

103

105

107

109

CHA0

103

105

107

109

Control

Time (days)

0 2 4 6 8 10

101

103

105

107

109


*

Spent bacterial growth

media show effects on

C. longicauda growth

depending on bacteria


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Flow cytometry for counting

(Pedersen et al 2009)

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Protozoa:

Cercomonas longicauda

Nematode:

Caenorhabditis elegans


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DSM50090

DSS73

Withstanding grazing of protozoa

Cercomonas longicauda and

nematode Caenorhabditis

elegans


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Difference in food

selectivity by

protozoa and

nematode:

protozoa select,

nematodes don’t


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Protozoa as Trojan horses

- bacterial lysis of protozoa

- bacterial multiplication inside

amoebae

- bacterial survival

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Campylobacter jejuni and Acanthamoeba castellanii

- poor survival of bacteria inside amoebae

(Xuan et al. Publ. Online 2011 Env Microb)

0 h

24 h

5 h

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A. castellanii increases growth of C. jejuni


+ amoebae, separated

+ amoebae

- amoebae, micro O2

- amoebae

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Oxygen concentration matters

Legends:

blind

C. jejuni

C. jejuni + amoebae

amoebae

C. jejuni + amoebae, contact

C. jejuni + amoebae, - contact


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Anne Winding

› 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

44

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Anne Winding 45

Protozoan growth on spore-forming

Bacillus licheniformis

N. jytlantica H. globosa

Pro

tozo

an a

bund

ance

(cel

ls m

l-1)

1e+2

1e+3

1e+4

1e+5

1e+6

No added Bacillus cells

Vegetative cells

Spores

Inact. spores

dd d

e

c

cb

a

(Pedersen et al. unpubl.)

N. jutlandica

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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?

› Oxygen level matters

› Feeding behaviour › Difference between protozoa in growth on the same bacteria

› Difference in selectivity between protozoa and nematode

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Anne Winding

Interactions between bacteria and microfaunal

predators and implications for biodiversity and

turnover of organic matter

› What is the diversity of protozoa?

› How to measure it?

47

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Anne Winding

Diversity of soil protozoa

› Determination

› Isolate and identify in microscope

› Isolate and extract DNA and use bar coding

› Extract DNA and use bar coding or DNA primers

› Amoebae (Heger et al. 2011, Nassonova et al 2010)

› SSU 18S rDNA: Small SubUnit 18S rDNA

› ITS rDNA: Internal Transcribed Spacer rDNA

› COI: cytochrome c oxidase subunit I

On isolated species

› Kinetoplastida (Rasmussen et al. 2001)

› 18S rDNA DGGE primers

On extracted DNA from soil

48

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Anne Winding

Implications for biodiversity

49

› How does protozoan predation affect microbial diversity? › Hypothesis: selective predation

1. relative abundance of bacteria producing secondary metabolites

› How do bacterial communities affect protozoan diversity? › Hypothesis: secondary metabolites

1. Total abundance of protozoa

2. Relative abundance of protozoa tolerable to secondary metabolites

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Anne Winding

› Individual bacteria fed to protozoan communities

› Bacterial communities exposed to individual protozoan

predation

› Increasing complexity Soil!!!

To come:

Microcosm experiments

50

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Anne Winding

Transect across EU with 90 sites

51

To come:

Determine protozoan diversity across Europe

7 sites across EU, 2

treatments, 3 replicates,

sampled 3 times (2012-2013)

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Anne Winding

To come:

Determine protozoan diversity across Europe

› Test techniques › Determine diversity by newly designed primers

› Assess effect of site (soil type, climate) and land-use on

diversity

1. Inventory of diversity (also microbes and mesofauna)

2. Input to: Interactions in soil food web

3. Input to: Implications for turnover of organic matter

› EU FP7 EcoFINDERS, EU ITN Trainbiodiverse

(2 PhD positions to be filled)

52

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Anne Winding 53

Interactions in soil and rhizosphere

M.

Bo

nko

wski e

t a

l. /

Eu

r. J

. S

oil

Bio

l. 3

6 (

20

00

) 1

35–1

47

Protozoa

Microflora

Bacteria Fungi

Earthworms

Nematodes

Nutrients and

hormones Root

exudates

Organic

matter

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Anne Winding 54

Acknowledgement Annette Pedersen

Karen S Andersen

Karen S Jensen

Jana Oberender

Anne-Grethe Holm-Jensen

Danish Research Councils, EU FP7 EcoFINDERS

Chr. Keel for providing P. fluorescens CHA0 strains

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Anne Winding

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Interactions between bacteria and microfaunal predators...

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