Paolina Garbevap.garbeva@nioo.knaw.nl

Netherlands Institute of Ecology

Deciphering belowground microbe-microbe and plant-microbe chemical communication

@PaolinaGarbeva

Research line: understanding belowground microbial interactions & communication

Garbeva research group

Soil & rhizosphere

fungi bacteria

protists

Broad-spectrum antibiotics

Bacteriocin

BiosurfactantSiderophores

Volatile Organic

Compounds

Microbial Volatile Organic Compounds (mVOC)

• small molecules (<300Da) • low boiling points• high vapor pressure• able to diffuse through both water- and gas- filled pores in soil

Dimethyldisulfide

Geosmin

Chemical classes:

AlkenesAlchoholsKetones

PyrazinesTerpenes

Sulfur VOCs

Headspace sampling

Thermal Desorption Tubes

(adsorbent: tenax/carbopack)

Polydimethylsiloxane (PDMS) silicone tubes

GC*GC- Q-TOF-MS (Agilent)

Cheatomium sp. Mucor sp.

Rhizoctonia solani

Many soil and rhizobacteria emit volatiles with antifungal activity

Garbeva et. al., (2014) FEMS Microbiology Ecology

Log Dilution0 1 2 3 4 0 1

Myc

elia

l ext

ensi

on (c

m2 )

0

10

20

30

40

50

60

C

Fusarium oxysporum

The loss of soil bacterial species can lead to a decline in the production of antifungal volatiles

Hol et al., Ecology (2015)

2-methylfuran2-furaldehydebenzothiazole

F-Mix1

_t0

F-Mix2

_t0

F-Mix3

_t0

F-Mix1

_tend

F-Mix2

_tend

F-Mix3

_tend

16S

rRNA

gen

e co

py n

o. p

er g

soi

l

1.0e+7

2.0e+7

3.0e+7

4.0e+7

5.0e+7

6.0e+7

5.0e+8

1.0e+9

1.5e+9

2.0e+9

2.5e+9 Burkholderia sp. AD024 Dyella sp. AD056 Janthinobacterium sp. AD080 Pseudomonas sp. AD021Paenibacillus sp. AD087

A

a

B

b

C

c

D D

A

A

B B

C C

D

EE

E

EE

E

a a

b

b

d

c

d

c

d

4-Mix 5-Mix5-Mix

4-Mix

Monocultures

Mixture

Schulz et al., Frontiers in Microbiology (2015)

Volatile production is affected by microbial interactions

2,5-bis(1'-Methylethyl)-pyrazineC10H16N2

Burkholderia sp. Monoculture

Paenibacillus sp. Monoculture

Interaction

2,5-bis(1'-Methylethyl)-pyrazine

Effect of interspecific bacterial interactions on volatiles blend

C10H16N2

Antimicrobial activity of the pyrazine-compound

0

100000

200000

300000

400000

500000

600000

F. culmorum Control F. culmorum

R. solani Control R. solani

Myc

elia

l gro

wth

in

pixe

l^2

Tyc et al., Microbial Biotechnology (2017)

Soil suppressive to Fusarium culmorum

Effect of volatiles emitted by suppressive and conducive soils on disease suppression

1-Octen-3-ol

Isobutyric acid

α-Terpinene

Can bacteria sense the presence fungi and oomycetes based on volatiles and react with specific phenotypic responses?

Bacterial response to fungal volatiles

Bacterial response to fungal volatiles

Screening for Volatile-Mediated Phenotypes

Schmidt et al., (2015) Frontiers in Microbiology

Effect of volatiles emitted by F. culmorum on bacterial motility

Serratia plymuthica

FC Control 0

1

2

3

4

5

swar

min

g di

amet

er (c

m)

Fusa

rium

cul

mor

umm

otili

ty W

A+A

RE

Volatile profiles of fungal and oomycetal isolates(PDA and WA+ARE)

MH- Mucor hiemalis; RS- Rhizoctonia solani; PU- Pythium ultimum; VD- Verticillium dahliae; FC- Fusarium culmorum; TH- Trichoderma harzianum

p-Cymene (1)β-Phellandrene (2)cis-p-Menth-2-en-1-ol (3) trans-p-Menth-2-en-1-ol (4) cis-Piperitol (5) trans-Piperitol (6) β-Elemene (7) β-Cubebene (8) β-Longipinene (9) Longifolene (10) β-Ylangene (11) β-Copaene (12) β-Barbatene (13)β-Acoradiene (14) Amorpha-4,11-diene (15) Isochamigrene (16) 5-epi-Aristolochene (17) Cuparene (18) Trichodiene (19) Longiborneol (20) β-Acorenol (21) Gossonorol (22) (E)-β-Farnesene (23)Kaur-15-ene (24) Kaur-16-ene (26)Tetradecanol (28)

Identified terpenes in F. culmorum

Ecological role ???

Effect of individual terpenes on bacterial motility

α-Terpineneβ-Phellandrene,

3-Carene Camphene

What are the molecular responses of bacteria to volatiles emitted by F. culmorum?

Extraction of RNA and proteins (t1=48h, t2=72h)

Transcriptomics and Proteomics

S. plymuthica exposed to F. culmorum VOCs

Fusarium

VOCs

Serratia plymuthica

Fumarate

Succinate

FRD

Glutathione

Glutathione-Conjugate

GST

Ener

gy m

etab

olism

Degradation of β-alanine

Putative 3-oxopropanoate dehydrogenase

α-Acetolactate

Acetoin

α-ALD

Isocytrate

Succinate

ICL

Environmetal stressOmpW

Biosynthesis of lipid A

Fimbria A protein/YfcS

Peptidoglycan biosynthesis

Peptidoglycan recycling

Methionine

MetQ

DacA

GDP-alpha-D-mannose GlucoseManC

Cell envelope

Motility

MCP IV

Attractants

Citrate-specific fermentation genes

CitB

Signal transduction

Fructose

EIIBC Fructose

YhjH

Cellulose synthesis

Cyclic di-GMP

AdrA

HipA

Multidrugresistance

NarL

NO3-/NO2-

N-limitation

Nitrogen regulatory protein PII

NasR

Glutamine synthetase

Glucose

IIA Glucose

Glucose-6-P

QseC

Flagellar regulonFructose-1-P

Mevalonate pathway

Terpene?

Terpene synthase

Biosynthesis of natural products

Red: higher expression in response to fungal volatilesBlue: lower expression in response to fungal volatiles

Terpene cyclase

Induced terpene production in response to F. culmorum volatiles

Sodorifen

S. plymuthica in response to F. culmorum

S. plymuthica alone

Serratia plymuthica PRI-2C

Maize (Zea mays L.)

F. culmorum

Can volatiles emitted by plants into the rhizosphere attract soil bacteria from a distance ?

Is there difference in the bacterial attraction when plants are infected by pathogen?

Glass olfactometer for testing volatile mediated attraction

Carex arenariaBurkholderia sp. AD024

Collimonas pratensis Ter91

Dyella sp. AD056

Janthinobacterium sp. AD080

Paenibacillus sp. AD087

Pseudomonas sp. AD021

Fusarium culmorum

Synthetic Bacterial Community

Volatiles emitted by soil or F. culmorumcould not stimulate bacterial motility

Volatiles emitted by Carex rootsattracted all soil bacteria from a distance

When infected Carex plants attracted only certain soil bacteria

A distinct blend of volatiles was emitted by: - F. culmorum- C. arenaria- C. arenaria infected with F. culmorum

Diffusion assay of pure volatiles

VOCs can diffuse over long distance (≈ 12 cm) but with different diffusion abilities

Volatiles play important role in belowground interactions

Schulz-Bohm et al., (2017) Frontiers in Microbiology

Schmidt et al., 2015 ISME Journal

Thank you for your attention !

Netherlands Science Foundation

Acknowledgment:

Prof. Dr. Jeroen DickschatUniversity of Bonn

Prof Katharina Riedel University Greifswald

Maria Hundscheid

Gera Hol

Hans Zweers

Kees Hordijk

Ruth Schmidt

Kristin Schulz

Olaf Tyc

Adam Ossowicki