deciphering belowground microbe-microbe and plant-microbe...
TRANSCRIPT
Paolina [email protected]
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