Diss. ETH No. 14566
Biotic Factors Affecting
in the
2,4-Diacetylphloroglucinol
Biosynthesis
Model Biocontrol Strain Pseudomonas fluorescens CHAO.
A dissertation submitted to the
Swiss Federal Institute
for the
of
Technology, Zürich
degree
of
Doctor of Natural Sciences
Presented
by
Regina E. Notz
Dipl.
sc.
Nat. ETH
July 6th, 1971
of Dagmersellen
born
citizen
Accepted
on
LU
the recommendation of
Prof. Dr. Geneviève
Défago,
referent
Prof. Dr. Dieter Haas, co-referent
Prof. Dr. Bruce
McDonald, co-referent
2002
The truth is
rarely
pure and
never
simple
Oscar Wilde
Table of Contents
Abbreviations
1
Summary
3
Zusammenfassung
5
Chapter 1
9
General introduction
Chapter 2
31
Biotic factors
phlA
affecting expression
of the
2,4-diacetylphloroglucinol biosynthesis
in Pseudomonas fluorescens biocontrol strain CHAO in the
gene
rhizosphere
Chapter 3
55
Fusaric-acid-producing
strains of Fusarium oxysporum alter
expression
biosynthetic
gene
rhizosphere
of wheat
2,4-diacetylphloroglucinol
in Pseudomonas fluorescens CHAO in vitro and in the
Chapter 4
Differential
75
2,4-diacetylphloroglucinol production by
different biocontrol
Chapter 5
pseudomonads
two
ecologically
and
genetically
in response to fusaric acid
99
General conclusions
Acknowledgements
105
Publications
106
Curriculum vitae
107
Abbreviations
lactone
AHL
jV-acyl-homoserine
Amp
ampicillin
ARDRA
amplified
CFU
colony forming
Cm
chloramphenicol
DAPG
2,4-diacetylphloroglucinol
FA
fusaric acid
FORL22
Fusarium oxysporum f sp.
gacA
global
HPLC
high-performance liquid chromatography
INA
ice nucleation
KB
King's
Km
kanamycin
LB
Luria Bertani
LPS
lipopolysaccharide
LSD
least
MAPG
monoacetylphloroglucinol
MS
mass
OD
optical density
OSG
minimal
PCR
polymerase
Pit
pyoluteorin
RBS
ribosomal
Rif
rifampicin
Tc
tetracycline
Tn
transposon
VBNC
viable-but-nonculturable
ribosomal DNA restriction
units
radicis-ly copersici
activator of antibiotic and
medium B
difference
spectrometry
glucose-ammonium
medium
chain reaction
binding
site
1
strain 22
cyanide biosynthesis
activity
significant
analysis
2
Summary
Introduction of fluorescent
alternative to manage soilborne
varies
the
depending
pseudomonads
plant pathogens.
biotic factors that influence
is
fluorescens
biosynthetic
monitor
commercialization. Production of
of the most
important
mechanisms
study
to
was
identify
in the model biocontrol agent Pseudomonas
CHAO.
Strain CHAO,
DAPG
one
promising
a
of these biocontrol agents
The purpose of this
pseudomonads.
DAPG-biosynthesis
performance
hampered
polyketide 2,4-diacetylphloroglucinol (DAPG)
in biocontrol of many biocontrol
biocontrol agents offers
The
the environment and this has
on
as
harboring
genes,
a
phlA,
to
of the DAPG
expression
translational fusion of the promoter
the reporter gene lacZ
biosynthetic
genes. It
gene of
plasmid pME6259
on
was
proximal
was
used to
shown that this reporter gene
system accurately reflected DAPG production by comparing ß-galactosidase activity with
DAPG extracted from media
DAPG
expression
artificial soil.
or
tested in
was
phlA expression
from the
a
plants (bean
cultivars
and
and
maize
no
clay-
in the
rhizospheres
was
shown that
A
peak
was
was
stimulating
effect
Pythium
on
age had
planting
DAPG gene
expression
ultimum. P. ultimum infection
and
a
two- to
Fusaric acid
(FA),
a
and sand-based
of two
rhizospheres
of two
dicotyledonous
was
pathogenicity
of
throughout
22
observed when the host
increased
gene
was
Within 48 h
days.
plant
expression
Using
A
was
infected
of the phlA
screened for FA
production showing
DAPG
that FA
rhizosphere.
produced by
production
expression
3
'-
measured in the cucumber
many Fusarium
species,
in strain CHAO. A collection of 12 F. oxysporum strains
biosynthesis
on
line.
and
the
expressing
pregerminated seedlings.
significantly
factor
maize
were
'Magister'
significant impact onphlA
fivefold increase in the maize
a
between
near-isogenic parent
three- to sixfold increase
represses DAPG
of these Fusarium strains
a
(e.g.
transgenic
fourth and remained at that level
'lacZ reporter gene in CHAO:
rhizosphere
plant
on
also observed at the level of
programs
and its
(crylAB)
reached 24 h after
to a
breeding
observed between
model, it
expression dropped
with
were
insecticidal gene
expression.
the
such differences
thuringiensis
as a
than in the
wheat)
differences among cultivars from traditional
Bacillus
significantly higher
was
This host genotype effect
cucumber).
a
differences among six U.S. and Swiss maize cultivars showed. While there
as
'Antares'),
of maize. Influence of host genotype
gnotobiotic system containing
of CHAO
monocotyledonous plants (maize
rhizosphere
in CHAO
varied between strains. The
was
assessed
was
impact
using pME6259. By
growing CHA0/pME6259
oxysporum strains it
suppress DAPG
was
in media amended with culture filtrates from different F.
shown that
production, (ii)
correlated with the
degree
(i) only
the FA concentrations in the
of DAPG
whereas
a
and
suppression,
strain CHA638, which lacks the DAPG
rhizosphere,
producing F.
FA
specific
enhancing effect,
the phlF mutant CHA638. PhlF
to
seems
levels in strain CHA638
limit DAPG
production
eight
to
were seven
wheat
both without
expression
in the wheat
times
strongly
of CHAO
neither of the two F. oxysporum strains altered phlA
being pathogenic. Again,
phlA expression
even an
able to
abolished in
was
gnotobiotic
a
repressed phlA expression
oxysporum strain had
non-producing F.
the adverse effect
(iii)
were
culture filtrate
fungal
repressor PhlF. In
F A-producing F. oxysporum strain
a
oxysporum strains
in
rhizosphere
higher than
as
in the wild-
type CHAO.
P. fluorescens CHAO and
differ in
CHAO,
two new
of CHAO
in strain
extent
was
and
ecological
genetical
Q2-87
was
also
radicis-lycopersici
In the tomato-rockwool
or
Q2-87
only
important
well
as
performance
was
and to
a more
sustainable
host
was
as a
a
DAPG
biosynthesis
exponential growth phase
F.
two
activity
agriculture.
4
are
discussed.
commonly
can
confront biocontrol
age, and the presence of
modulate the
affect biocontrol
on
reporter genes inaZ and lacZ
tested. Here, statistical differences
cultivar, host
step
rockwool system. At the
reporter gene. Advantages and
These factors
might
provides
of inaZ to phlA
better with Q2-87 than with CHAO.
of the biotic factors that
nonpathogenic fungi.
of these factors
late
tomato roots grown in a
species,
one
(FORL22) significantly repressed phlA
be found with lacZ
some
they
'-lacZ fusion of
Q2-87. Amendments of FA producing
FORL22
biocontrol gene and therewith
understanding
during
system, the suitability of the
inoculants in the environment: host
and
on
parallel.
strains but
much smaller extent than in CHAO. The
of lacZ and inaZ to report phlA promoter
This thesis deals with
as
a
in
production
FORL22 -mediated phlA -repression
between treatments could
pathogenic
but to
strain 22
time, protection of tomato against
disadvantages
of lacZ to phlA of Q2-87, and
28% in
only
transcriptional phlA
a
assessed in vitro
repression
in CHAO but not in
reflect FA-
one
suppressed by FA,
of FA-mediated/?M4
oxysporum f. sp.
to
DAPG-producing biocontrol
traits. In addition to
transcriptional fusions,
58% measured in CHAO but
same
both
are
constructed to evaluate their DAPG
were
expression
Q2-87
the way to
expression
performance.
overcome
of
an
Identification
inconsistent
Zusammenfassung
Ausbringen
Das
Alternative,
jedoch
von
fluoreszierenden Pseudomonaden bietet eine
bodenbürtige Pflanzenpathogene
um
durch ihre je nach Umwelt variierende
Diacetylphloroglucinol (DAPG) spielt
eine
wichtige
fluorescens
Reportergen
lacZ auf Plasmid
Vergleich
Der
Extraktionen
aus
Mais-Rhizosphäre
hat
derjenigen
Wirts-Genotyps
von
dikotylen
zwischen
Unterschied zwischen transgenem
(crylAB) exprimiert,
Modell,
,Magister'
das sich
aus
auf die DAPG-
Quarzsand
(Bohne
monokotylen
und
Gurke)
Pflanzen
wies eine
(Mais
und
auf. Dieser Effekt des
um zu
zeigen,
vorhanden waren, konnte kein
Elternlinie
Gen
festgestellt werden.
Mais
24 Stunden nach der
Pflanzung
von
erreicht. In den nächsten 48 Stunden fiel der Wert auf einen Viertel
beobachtet,
steigerte
mal höhere Werte wurden in der
Mais-Rhizosphäre
isogenischen
Genexpression wurde
und blieb auf dieser Ebene während 22
Infektion mitP. ultimum
,Antares')
traditionellen
dass das Alter der Pflanze einen Einfluss auf phlA
hat. Eine maximale
wurde
und
aus
Mais, der das insektizide Bacillus thuringiensis
und seiner beinahe
vorgekeimten Sämlingen
Tagen.
wenn
die
die phlA
Ein
anregender
Wirtspflanze
'-
'lacZ
mit
Expression
Gurken-Rhizosphäre
Effekt auf die DAPG-
Pythium
ultimum infiziert
in CHAO
signifikant:
war.
3- bis 6-
und 2- bis 5-mal höhere Werte in der
gemessen.
Viele Fusarium Arten
unterdrückt die
von
Während Unterschiede zwischen Sorten
Zuchtprogrammen (z.B.
Genexpression
Reportergen-System
zusammensetzte. Stamm CHAO
Rhizosphäre
Pflanzen
DAPG-
Wirts-Genotypen
gnotobiotischen System getestet,
in der
dass dieses
gezeigt,
in CHAO
DAPG-Biosynthese
konnte auch zwischen sechs verschiedenen U.S. und Schweizer Mais-Sorten
festgestellt werden.
Genexpression
die
um
Der Einfluss des
wiederspiegelt.
Korngrössen und einem Tonmineral
als in
mit dem
promotornahen DAPG-Gen, phlA,
vom
signifikant höhere phlA Expression
diente als
Arbeit wurden biotische
gleichzeitigen ß-Galactosidase-Assays und
von
wurde in einem
verschiedener
Weizen)
Krankheitsunterdrückung
im Modell-Biokontrollstamm Pseudomonas
pME6259 wurde benutzt,
Medium und
die DAPG-Produktion gut
Expression
2,4-
von
CHAO beeinflussen.
Eine translationelle Fusion
zu messen.
Rolle in der
wird
Vermarktung
behindert. Die Produktion
vorliegenden
identifiziert, die die DAPG-Biosynthese
Ihre
bekämpfen.
Wirkung
durch viele Biokontroll-Pseudomonaden. In der
Faktoren
zu
vielversprechende
produzieren
DAPG-Biosynthese
den
Pathogenitätsfaktor Fusarinsäure (FA).
in Stamm CHAO. Eine
5
Sammlung von
FA
12 F. oxysporum
Stämmen wurde auf ihre FA-Produktion untersucht und
es
Produktion
dieser Fusarium Stämme auf die
Stamm
von
DAPG-Expression
CHA0/pME6259
zu
Stamm variiert. Die
wurde mit Hilfe
in einem
Unterdrückung
zu
zeigte, (i)
unterdrücken
dass
nur
FA
Expression
Ein
pilzlichen
keine
pathogène
Stämme
FA-produzi er ender F.oxysporum
in CHAO in der
nichtproduzierender
zeigten
von
oxysporum
oxysporum Stämme die
produzierende F.
vermochten, (ii) dass das
mit der FA-Konzentration im
war.
dass die FA-
pME6259 untersucht. Kultivierung
der unterdrückende Effekt in Stamm CHA638, der den
aufgehoben
gezeigt,
Medium, welches mit Kulturfiltraten verschiedener i7.
Stämme versetzt worden war,
DAPG-Produktion
von
Wirkung
wurde
Ausmass der DAPG-
Kulturfiltrat korrelierte und
(iii)
spezifische Repressor PhlF entbehrt,
Stamm unterdrückte die phlA
wogegen ein
gnotobiotischen Weizen-Rhizosphäre,
F. oxysporum Stamm diese sogar noch erhöhte. Beide Stämme
Interaktionen mit den
Weizenpflanzen.
einen Einfluss auf die phlA
Expression
scheint die DAPG Produktion in der Weizen
achtmal höherer phlA
Expressionsgrad
P. fluorescens CHAO und
zeigten
Keiner der beiden F. oxysporum
in der phlF Mutante CHA638. PhlF
Rhizosphäre
zu
limitieren, wie ein sieben- bis
in Stamm CHA638 andeutet.
Q2-87 sind beide DAPG-produzierende Biokontroll-
Stämme, jedoch unterscheiden sie sich sowohl ökologisch als auch genetisch. Zusätzlich
einer phlA '-lacZ Fusion
lacZ
mitphlA
von
von
CHAO wurden zwei
Q2-87 und eine
von
DAPG-Produktion der beiden Stämme
Biosynthese
inaZ
mitphlA
betrug
unterdrückte die
radicis-lycopersici
Stamm 22
Zugabe
(FORL22)
von
CHAO
28% in
nur
des FA
die phlA
in einem viel
Q2-87.
Tauglichkeit
Unterdrückung
mit dem
Reportergen-Systeme
lacZ
nur
gezeigt werden.
Arbeit
vorliegende
beschäftigt
in Stamm CHAO
vor
zur
oxysporum f. sp.
signifikant
FORL22 als CHAO.
Messung der phlA-
Wirkung
konnte
nur
Die Vor- und Nachteile der verwendeten
Expression
sich mit
einigen
in der Umwelt ausgesetzt sind:
Gegenwart von pathogenen
Faktoren die
inaZ und lacZ
geringeren
wird diskutiert.
Biokontroll-Organismen
die
Reportergene
damit die
um
durch FA in der späten
durch FA und FORL22 wurde untersucht. Die hemmende
Reportergen
Die
der zwei
von
In einem Tomate-
produzierenden F.
Expression
eine
zu
können. Die DAPG-
messen zu
aber nicht in Q2-87. Dabei schützte Q2-87 die Tomaten besser
Die
angefertigt,
Expressions-Hemmung
58% in CHAO und
Steinwolle-System
transkriptioneile Fusionen,
unterdrückt, aber
Ausmass als in CHAO. Das Ausmass der phlA
Phase
neue
gleichzeitig
wurde auch in Q2-87 durch FA
exponentiellen
dass
eines
oder
nicht-pathogenen
wichtigen
biotischen
Faktoren, denen
Wirtsart, Wirtssorte, Wirtsalter sowie
Pilzen. Es wurde
gezeigt,
Biokontroll-Genes beeinflussen
6
dass diese
können,
was
sich
auf die
Wirkung
der
Biokontroll-Organismen
auswirken kann. Die Identifikation und das
Verständnis solcher Faktoren stellen einen Schritt auf dem
von
Biokontroll-Produkten und damit
zu
einer
Weg
nachhaltigeren
7
zu
einer besserer
Landwirtschaft dar.
Wirkung
8
Chapter 1
General Introduction
10
Chapter 1
General introduction
Biocontrol of soilborne
Roots
rot
can
be attacked
and vascular wilt.
farmers
are
the environment and
offers
promising
a
many biocontrol
soilborne
alternative
one
damage
as
to crops
or
In
general,
but have
a
biocontrol agent
(3, 97).
colonize the entire root system
cause a
efficiently,
microorganisms (3).
It has been shown that
in colonization traits
only,
longer
was no
oxysporum f. sp.
enzymes,
(40).
play
compounds
that induce
None of these mechanisms
action
are
exhibited
by
a
single
and nutrients is believed to be
plant
are
produce
high affinity
iron
supply
a
mutant
may
a
of P.
an
defense
mutually
are
on
not
are
specific
the modes of action
responsible
prerequisite
for
a
for the disease
strain to be
a
on
rhizosphere,
indigenous
chlororaphis PCL1391, impaired
suppression: siderophores,
range of iron-chelating
factors, which
extracellular
mechanisms, and antimicrobial metabolites
exclusive and
frequently
control
fungal plant pathogens
compounds
Under
or
for ferric iron. These bacterial iron chelators
11
(33).
In the
for space
past years,
by rhizosphere inhabiting
(18, 40, 69).
rhizosphere making
several modes of
(33).
biocontrol mechanism
biological
of iron
successfully
several extracellular
between bacteria and
important
focussing
available in the
root-colonizing
pseudomonads
Once the root is
produce
biocontrol agent
pseudomonads through competition
bacteria
a
role in disease
Siderophores: Competition
many studies have been
as
certain
to
and be able to compete with
radicis-lycopersici (11).
important
an
the past 20 years,
able to protect tomato from foot and root rot caused
colonized, plant beneficial pseudomonads
to
biocontrol agents
beneficial effect. It has to survive in the
proliferate,
suggested
for
The biocontrol bacteria must be able to establish itself at
densities sufficient to
are
concern
pseudomonads:
population
by Fusarium
raise
wide host range and suppress several
Root colonization: Efficient root-colonization is
good
fungal
past years, many research groups have focused
of these
(reviewed by 33,
reasons
naturally suppressive
biocontrol
root
by phytopathogenic
biocontrol isolates have been identified
(33).
as
steam-treatment
(13, 40). During
of biocontrol and several mechanisms have been described that
suppressive capacity
rotation,
(e.g. methylbromide)
have been isolated from soils
plant species only,
In the
diseases such
cause
crop
health. Introduction of bacterial and
plant pathogens. Many
pathogens (88).
such
alternative to manage soilborne diseases
organisms
pathogen
techniques
effects of these chemicals
public
which
plant-pathogens
chemical agents for economical
use
bacteria of the genus Pseudomonas
for
microorganisms
available to reduce
generally
40). However, non-target
soil
soilborne
by
Although
and solarization of soils
organisms,
pathogens by
iron-limiting conditions,
siderophores,
are
fluorescent
which have
a
very
thought to sequester the
it unavailable to
pathogenic fungi
and
limited
thereby
Chapter 1
General introduction
their
restricting
soilborne
pathogens. Pyoverdin
(9). However, dynamics
production
of iron
may act
necessarily
example,
reported
are
the
rhizosphere
are
often
associated with disease
production
of the
of fluorescent
siderophores
(10).
elicitors for
as
associated with biocontrol of
are
to
play
role in the
a
complex
7NSK2
and
suppression (10).
siderophore pyoverdin
In
was
of black root rot of tobacco under iron rich conditions
that
in biocontrol in iron rich soils
in the
competition
suppression
generally suggested
for
pyochelin,
fluorescens CHAO,
of importance in the
siderophores
damping-off of tomato by Pseudomonas aeruginosa
of iron chelators is not
biocontrol strain P.
salicylate
and
of Pythium-mduced
suppression
and it is
Several bacterial
growth (40).
The bacterial
pseudomonads
do not
(42)
play
resistance
against pathogens
in
role
a
and
iron-chelating compounds pyoverdine
inducing systemic
not
some
plants (60, 63, 93).
produced by
Extracellular enzymes: Some enzymes
pathogenicity
factors such
degrading
enzymes
and
that
lipase
toxins
as
might play
a
reactions,
or
mechanisms
this
ß-1,3 glucanase)
role in disease
Induced resistance: All
attacks. A virulent
Some Pseudomonas strains
(90).
chitinase and
(e.g.
plants
avoids
pathogen
as
triggered by
phenomenon
is
triggering
known
as
these
mechanisms,
pathogens
can
cell wall-
against pathogen
suppresses resistance
be reduced if these defense
rhizobacteria
or
systemic acquired
as
prior to
infection and
resistance and induced
resistance, respectively. Various non-pathogenic rhizobacteria have been shown
systemic
resistance in
plants
phytopathogenic fungi,
are
claimed to
antigenic
and
by
(reviewed by 93).
resistance and include
side chain of the bacterial outer membrane
Colonization of tobacco roots
caused
thereby providing protection against
bacteria and viruses
produce systemic
increase in
salicylic
CHAO could
acid and PR
only partially
(TNV)
proteins
fluorescens
WCS374
resistance induction
(21).
or
in the
induce resistance
essential for induction of resistance to
in very small amounts
and induces
In the
Botrytis
A
TNV
systemic protection
Several bacterial determinants
acid and the O-
CHAO reduces leaf necrosis
in the
pyoverdine-negative
(60). Salicylic
by P. aeruginosa
of radish
WCS417, the bacterial LPS appeared
12
induce
broad spectrum of
physiological changes
cinerea
(49).
to
systemic
protein lipopolysaccharide (LPS).
leaves).
against
a
siderophores, salicylic
by Pseudomonas fluorescens
tobacco necrosis virus
degrade
protease, phospholipase C,
have natural defense mechanisms
leaf-necrosis
commonly
well
produce
can
suppression (40, 76, 98).
evades the effects of active defenses. Disease
are
biocontrol agents
acid
mutant
of
production
7NSK2 in bean
against Fusarium
to
plant (e.g.
be the trait
wilt
is
even
by P.
responsible
for
Chapter 1
General introduction
H,C
CH,
HO
Fig.
'
1. Chemical structure of
Antibiosis and
the
biocontrol. In several
promoting activity
(41),
kanosamine
(83), oligomycin
well documented
DAPG
by comparing
parental
strain.
protection
DAPG to
play
This
of plants
metabolite in the
Organization
In strains P.
organized
as an
use
antimicrobial and therefore
plant growth
compounds including
(96),
A
DAPG
(39), phenazines (87), pyoluteorin
xanthobaccin
(Fig. 1),
HCN
has
(67),
and zwittermicin A
emerged
(84).
biocontrol strains of Pseudomonas
Indirect evidence for the in situ
of the mutant with
various
role in
genetic
and helminths is
production
suppression
the addition of
when
regulation
is
of
usually
(Fig. 2)
synthetic
of the DAPG
and
are
monoacetylphloroglucinol (MAPG),
methods
proved
the
DAPG to soil
strains
are
importance
Further indications for
a
biosynthetic
for the
precursor
13
(43)
or
severity
in the
the detection of the
operon
and CHAO, the four
which is
of DAPG in
present (6, 41, 61, 74).
biosynthetic
indispensable
a
DNA sequences restores
control is the reduction of disease
producing
fluorescens Q2-87
wild-type
pathogens (15, 16, 41, 43, 94).
biological
The
of the most
as one
activity against bacteria, plants, fungi
of molecular
against
through
operon
mechanisms in
important
spp. and evaluation of its role in disease
rhizosphere
and
of the most
well established that
now
the biocontrol activities of DAPG deficient mutants with that of
primary
absence of bacteria
an
compounds produced by
Complementation
a
(89),
(23, 30, 40, 62, 68, 82).
ability.
one
(45), oomycin
DAPG
Its broad toxic
obtained
the
metabolite,
by Pseudomonas
biocontrol
A
viscosinamide
antimicrobial
fluorescens (41, 88).
is
compounds
found for many different
was
antimicrobial
important
It is
2,4-diacetylphloroglucinol (DAPG):
plant pathogen systems,
(60), pyrrolnitrin (51),
polyketide
OH
2,4-diacetylphloroglucinol (DAPG)
of antimicrobial
production
'
or
production
genes,
phlACBD
of DAPG
degradation product
as
well
are
as
of DAPG.
Chapter 1
The
General introduction
operon is flanked
biosynthetic
repressor
of the DAPG
protein
(Fig. 2) (2, 80).
\-
synthesis
phiG
transcribed phlF gene
divergently
and
addition, Schnider-Keel
In
frames downstream of phlF,
phtH
the
by
a
et
coding
gene
al.
(80)
have
for
putative
a
sequenced
encoding
efflux
a
protein (phlE)
reading
two new open
designated/»/z/G andphlH (Fig. 2).
>.pWF|
y
Chalcone
Repressor
Acetoacetyl-CoA
synthesis (?)
1 kb
synthase
Permease
DAPG
export (?)
PhlF
Salicylate
Pyoluteorin
Carbon
2. Genetic
At
a
organization
of DAPG genes and factors
transcriptional level,
repressor PhlF
m
P. fluorescens
DAPG
Q2-87,
turn-helix motif typical for DNA
Fl
influenced
by
a
range of
Schnider-Keel et al.
through
PhlF. In
salicylate
as
well
as
on
and FA
a
requires
the
(80)
is
DAPG
regulated by
of PhlF resulted in
metabolites
a
pathway specific
a
helix-
and has been shown to bind to the phlA-
repression
biosynthesis (19, 80). Repression
have shown
the
13, and CHAO (2, 19, 80). PhlF exhibits
binding proteins
secondary
production.
mediated
and inactivation of
by
PhlF
can
be
produced by rhizosphere microorganisms.
positive autoregulation
of DAPG in CHAO mediated
addition, the microorganism's extracellular metabolites pyoluteorin and
negative impact
genes at the
of DAPG
RpoD/RpoS
influencing
biosynthesis
phlF intergenic region (19). Overproduction
phlF'm derepression
\
GacS/GacA
source
Fig.
©
©
t
Fusaric acid
fusaric acid
DAPG
(FA),
stationary phase
pathogenicity
production (24, 80).
functional PhlF
transcriptional
a
level is
factor gs
protein (80).
proposed
The
factor of Fusarium oxysporum, exert
negative regulation
mediated
Additional control of DAPG
for the
housekeeping sigma
(rpoS) (79, 77). Amplification
14
of the
rpoD
a
by salicylate
biosynthesis
factor
gene
gd (rpoD)
and
encoding gd
in
Chapter 1
General introduction
P. fluorescens CHAO resulted in
ultimum
against Pythium
DAPG in strain Pf-5
Although
was
(99).
on
secondary
prokaryotic two-component systems,
an
environmental
phosphorylation.
thought
block
to
lead
to
reduced in
required
of
including
the membrane bound
regulator undergoes
mutant
suppression
of P.
mutant
overproduced
against P.
(gh)
has
DAPG
(77).
been
sensor
(14, 48, 76).
kinase
by
CHAO
(48).
(85).
(GacS)
functions
as
change,
which is
secondary metabolites (14, 26,
In this strain
of
Like in other
Mutations in either gene
dicotyledons only
dependence
of
regulator (GacA) by
conformational
a
of target genes
of soilborne diseases of
It has been demonstrated that GacA
ultimum
never
basicola induced black root rot of tobacco is
fluorescens
of cucumber
in Pf-5 has been shown
of extracellular enzymes, DAPG, and other
Thielaviopsis
protection
gacS/gacA globally regulates production
metabolites
transcriptional regulation
agacA
for
pyoluteorin production
and activates the cognate response
The response
biosynthesis
48). Biocontrol
(78).
sensor
and
RpoS"
an
function for the heatshock factor
The two-component system
extracellular enzymes and
production
in enhanced biocontrol of cucumber
demonstrated, its negative impact
Whistler et al.
enhanced DAPG
improved (79). Conversely,
resulting
DAPG-regulatory
a
an
drastically
however, gacA is
and not of Gramineae
cyanide synthase
genes
(hcnABC)
^Signal(s)?
.
GacS
(sensor kinase)
Peri
plasmic space
Cytoplasmic membrane
GacA
(response regulator)
Transcription
of
RsmA
regulatory RNA(s)
(•)
(translational repressor)
S/\
s
mRNA
(hen, apt, phi,..)
5'
3'
RsmZ
5'
3
Translation
Fig.
3.
Posttranscriptional regulation
(35).
mechanism
involving
P. fluorescens CHAO
15
the
two-component system GacS/GacA in
Chapter 1
General introduction
and the protease gene
the repressor
site
protein
RsmA
(Fig. 3) (5).
RsmA is
(Fig. 3) (5).
the ribosomal
overlapping
translation
in CHAO operates via
(aprA)
binding
site
suggested
rsmZ
suppressed
the
a
a
distinct
recognition
mimicked
partially suppressed
regulatory RNA,
effect of gacS and
negative
bind to
overexpression
RsmA
of GacA function and mutational inactivation of rsmA
Moreover, GacA positively regulates
to
involving
of the target RNA and thus blocks its
(RBS)
fluorescens CHAO,
In P.
mechanism
posttranscriptional
a
called RsmZ
a
partial
gacS defect (5).
(36). Overexpression
expression
RsmZ is
sequester the repressor protein thus allowing the translation of the
respective
to
of
mutations and inactivation of rsmZ
gacA
resulted in reduced
thought
loss
of GacS/GacA target genes
(36). According
to
the
model,
mRNA's.
Limitations of biocontrol
Fewer than ten Pseudomonas
made it to the market
(97).
An
bioproducts
biocontrol
ability (81).
An
fungi
issue is the need for suitable inoculant formulations
important
that allow the Pseudomonas cells to survive
loosing
for the control of phytopathogenic
long-time storage
even more
important
in
high
concentrations without
obstacle to commercial
development,
however, is the inconsistency of performance of many biocontrol agents. Although their
ability
to
reduce the
and
laboratory
50)
greenhouse
inconsistent
of diseases caused
severity
conditions has been
performance
in commercial
obstacle. Because
antibiosis
(88),
production
a
it is
primary
thought
of antimicrobial
reported
part of variability in DAPG production
in several studies
of variability is
suppression
performance might
such
compounds
can
as
fungal pathogens
under
(reviewed by 18,
and field trials tends to be the
sources
mechanism of disease
that variable
soilborne
settings
the
disappointing reality (88). Understanding
by
key
to
overcoming
available to these bacteria is
result from variation in
DAPG. Recent evidence indicates that at least
be attributed to variations in environmental
conditions, abiotic and biotic, that might confront bacterial metabolite production
rhizosphere.
fluorescens
For
example, glucose
Pf-5 and CHAO
but not
(25, 68),
this
glycerol
whereas in P.
enhanced DAPG
fluorescens
Fl 13
production
in the
in P.
production
of DAPG
Q_i_
and MAPG is stimulated
(28).
In strain S272 the
carbon
source
by
Fe
-ions and
highest yield
(102). Furthermore,
enhanced in the presence of
of antimicrobial
as
but is poor in the presence of succinate
of DAPG has been obtained with ethanol
in P. fluorescens CHAO the
Cu2+, Mo2+,
Microbial metabolites
sucrose
signal
and
the sole
of DAPG is
Zn2+ (25).
molecules
compounds produced by
production
as
play
also
an
important
role in the
regulation
biocontrol bacteria. One well-documented class of
16
Chapter 1
General introduction
diffusible
signal
which
are
involved in
When
a
certain
molecules
fluorescent
In
communication between bacterial
phenazines (71).
recent
evidence
be involved
In P.
that
(36).
In
an
addition,
populations
DAPG
even
fungal
inhibitor of DAPG
However,
and root
a
synthesis
have
can
impact
AHL's
is not
CHAO
operon in
play
a
role in
and
signal
but
molecule
might
several extracellular
by
salicylic
acid
(80).
of metabolites
production
on
cross-
of
regulated by AHL's,
different Fusarium
produced by
fluorescens
sensing.
phenazine biosynthetic
in CHAO is affected
an
quorum
thereby influencing production
synthesis
is needed about the
insight
more
the
as
repressed by pyoluteorin
wilt factor
in P.
DAPG
production
metabolites
relevant for biocontrol. FA,
and
as
solvent-extractable extracellular
analog
metabolites. DAPG is autoinduced and
Interestingly,
known
addition, there is evidence that
fluorescens CHAO,
implies
(AHL) (reviewed by 29),
threshold concentration of AHL is
a
of target genes such
pseudomonads (72).
regulation
gene
of bacterial cells is present,
expression
lactones
iV-acyl-homoserine
cell-density-dependent
a
density
reached to stimulate
the
are
strains, is
a
potent
(24, 80).
ecological
interactions
taking place
in soil
environments, which might influence production of antimicrobial metabolites such
DAPG. With this
biocontrol strains could be customized for
knowledge,
use
in
as
particular
environments, inoculum preparations developed for optimal performance, the environments
could be modified to be
independent
Reporter
favorable to
more
of environmental
genes
are an
or
strains could be constructed that
are
signals.
genes used in microbial
Reporter
strains,
ecology
attractive tool to monitor
substrates where detection of its gene
product,
e.g.
expression
of
a
DAPG, is difficult
gene in natural
or
impossible.
A
reporter gene system consists of a gene lacking its natural promoter, which will be transcribed
only
a
if fused downstream from
exogenous promoter
(reviewed by 54).
reporter gene should be conveniently detectable. Ideally,
sensitive,
uncommon
quantification,
and
responsive
transcriptional
containing
its
fusions.
own
in
a
changes
They join
no
in
background levels,
transcriptional activity (54).
ecology to study promoter strength
a
gene of interest with the
translational initiation
chimeric mRNA, but
transcriptional
to
hybrid protein.
and
a
translational
product
Most reporter gene
regulation
are
of such
fusion,
a
a
gene
construct
results in
reporter gene lacking its
and translation start sites is fused in-frame to the gene of interest. This
hybrid protein,
which must retain its
activity (85).
17
In
of
reliable for
promotorless reporter
regions. Transcription
In
The gene
reporter gene system should be
a
in natural substrates in order to avoid
fusions used in microbial
a
an
some
cases, the site of
yields
reporter gene
Chapter 1
insertion
General introduction
can
destabilizing
influence the
sites
expression
of the reporter, e.g.
by disrupting
stabilizing
mRNA
(70).
Several different reporter genes have been used in recent studies of microbial
and
they
can
be
Escherichia coli encodes
provided
their
quantified according to
fluorescent upon
With the
cleavage (31).
help
the carbon metabolism of F. oxysporum f. sp.
substrate to
from the TOL
of the
aggA
plasmid
locus of P.
galactosidase
produce
in
of gusA
nonpathogenic Fusarium
and herewith reduced disease. Catechol
a
on
and
can
effect of biotin
on
the
of
production
used
(38)
an
(luxAB)
or
luciferase
firefly
O2. Many studies
use
lux
as a
a
activity
to asses
few /wx-fusions
(GFP),
the
were
(59).
spp.
been
addition,
bdhA
a
as a
marker, fused
visualization studies. With this
dynamics
gfp,
be
can
a
by
that
conversion
in the
stimulating
a
can
be
easily
light-emitting
reaction that
requires
in natural environments
of /wx-bioluminescence
(57, 73).
In microbial
on
(22,
metabolic
ecology, only
Green fluorescent
a
protein
with shifted
fluorescens spectroscopy
protein
was
recently
or
confocal
isolated from Discosoma
quantify promoter strength. They
tomato roots
detected
and its color variants enhanced cyan,
technique, Bloemberg
on
ß-
the bacterial luciferase
expression (12, 75).
used to
isolated
of the enzyme
strong constitutive promoter for localization
of P. fluorescens WCS365
inaZ is
a
an
polyhydroxybutyrate degradation
phenotype
dependence
red fluorescent
to a
of
study expression
to
by quantifying the
microorganisms
quantified by
widely
activity
originally
'lacZ fusion to show
'-
catalyze
gene
activity
yellow o-nitrophenol
of the bacteria
study
was
yellow (autofluorescent proteins, AFPs),
AFPs have not yet been
applied
the
be measured
jellyfish Aequorea victoria,
excitation and emission maxima
In
of the
status
constructed to
isolated from the
microscopy.
use
physiological
enhanced green, and enhanced
laser
which both
xylE,
successfully
It is either conferred
marker to track
44, 91, 101). Some authors make
used
was
enzyme involved in
samples.
(lue),
reporter gene, Duijff et al. (27)
Its gene,
in Sinorhizobium meliloti. Bioluminescence is another
and measured in environmental
or
is another enzyme that cleaves
(8). Similarly,
of o-nitrophenyl-ß-D-galactopyranoside into the
Hofmann et al.
pigmented
oxysporum Fo47 lowered the
pigmented product.
bean roots
gene of
colorless substrate
as a
2,3-dioxygenase
transcribed from the lacZ gene
spectrophotometer.
a
gusA
ecology,
Uni, the causal agent of Fusarium wilt of flax,
Pseudomonasputida
putida
The
unique phenotypes.
which turns
ß-glucuronidase,
evidence that the
appropriate
or
et
al.
using
(4)
observed the
have often
or
population
three different AFPs. Other than
reporter gene that is widely used for quantitative studies in microbial ecology. In
the absence of ice
nucleating agents,
water
will
supercool
18
to
nearly
-40°C. A few bacterial
Chapter 1
General introduction
species (e.g.
P.
syringae,
herbicola, P. viridiflava, P. fluorescens and Xanthomonas
Erwinia
campestris) have the ability
to
low
ability
-2°C. Ice nucleation
as
protein
be
can
iron
availability
(55)
to
a
a
single
droplet freezing
pyoverdine
assay
(92).
bean, gypsophyla,
It
was
during growth
are
reporter gene is
on
this reporter
(e.g.
are
an
humic
leaves and pear flowers
tomato
much lower than under
important
might
environments.
and
be present in certain
results
experimental
samples
acids)
2,3-dioxygenase, ß-galactosidase,
enzymes
Both,
(31, 52).
synthesis
a
quantifiable
can
(66)
found inaZ to
response of
a
ß-glucuronidase (52).
bacteria
indigenous
these two systems have the
of
population.
A nice
of this is shown in Miller et al.
promoter
to
gfp
and showed
a
or
cell and do not
large spatial
only
In
inactive cells because it
To the present
demonstrate
(39)
time, reporter
production
studied the role of
Hv37aR2 in the inhibition
using
a
lacZ
phenazine
transcriptional
locus in P.
levels of pigments
by
or
who fused
(66),
requires
on
falsify
they
can
in
expression
a sucrose
a
regulated
the surface of bean leaves.
expressed by
been used
rhizosphere.
a
(64).
few times to
Howie and Suslow
produced by Pseudomonas fluorescens
ultimum in the cotton
spermosphere
fusion. The influence of seed exudates
PGS12
that
the flavin mononucleotide FMNH2
only
two
oxygen for the emission of
striking advantage
variation of sugars
metabolites
aureofaciens
assessment
in anaerobic
expressed
estimate average
gene systems have
of'Pythium
nucleus per cell
material and thus
require
of antimicrobial metabolites in the
antifungal
one
quantitative
Unlike with GFP and AFPs, the bacterial bioluminescence will not be
metabolically
up
sucrose-regulated
addition, the latter
plant
Not all reporter genes will be
single
for
require
which interfere with the colorimetric assays of catechol
expression
use
high
provide high background
detect gene
a
of the
the
laboratory conditions,
issue. Miller et al.
GFP and bacterial bioluminescence
light (37, 54). Nevertheless,
biosensor to
useful under all circumstances. For studies in natural
Environmental
material
particulate
inaZ
as a
genes that control the
that strong promoters transcribe at levels too
(54).
studies,
also used to estimate
promoter. However, maximum detectable level of transcription of inaZ is
means
of its
its host.
6100-fold fewer cells than lacZ to obtain
which
transcription
In many
gene has been used
(27, 53, 58).
biosynthetic
and
(inaZ)
gene
as
coronatine of P. syringae pv. tomato and showed that their
habitats, where populations sizes
of
on
fused inaZ to the
Not all reporter genes
efficiency
by
in environmental habitats
chlorosis-inducing phytotoxin
is induced
a
of the
in Erwinia herbicola
Ma et al.
expression
is conferred
regulated promoter
IAA-production
(7, 56).
the formation of ice-nuclei in water at temperatures
quantified conveniently by
fused to the iron
assess
catalyze
was
examined with the
19
on
the
help
and
rhizosphere
expression
of
of the
aphz '- 'inaZ fusion
Chapter 1
General introduction
(32). Pyoluterorin expression
cotton
and cucumber
of P. fluorescens Pf-5
rhizosphere (46). Chin-A-Woeng
chromosomal reporter to show in situ
tomato roots. In a recent
study,
a
studied with
was
al.
et
of P.
phenazine expression
chit33
'-
'gfp
fusion
was
(12)
pit'- 'inaZ fusion
a
used
in the
aphz '- 'luxAB
chlororophis PCL1391
made to
study regulation
on
of chitinase
33, which is involved in the biocontrol ability of Trichoderma harzianum (20). However, little
is known about
influence
positive-
expression
or
negative-acting signals
in the
environment that
rhizosphere
of antimicrobial metabolites and thus may determine the
success
of
biocontrol in the field.
The
present study
In the
of DAPG
from
biosynthetic
soil
a
of tobacco
such
as
present study, P. fluorescens CHAO
chambers
or
under
many of these
biosynthetic
acid
(65),
the
conditions
and HCN
phospholipase (76).
of DAPG
biosynthetic
host health when attacked
were
pathogenic F.
by
root
Q2-87
were
well
as
(96),
oxysporum strains
several
by
to FA was
and
evaluated.
used to reflect actual
tritici
different
Ggt
to
on
be
the
isolated
a
root-pathogenic fungi
(Ggt), Pythium
grown in
plants
wheat in the field
key
factor in the
growth
(100).
indole-3-acetic-acid
CHAO has been
a
(34),
model
study regulation
biological
and extracellular enzymes
organism
thereof
to
identify
(5, 36, 47, 48, 76, 80).
genes has been discussed in detail
rhizosphere.
on
the
the DAPG
P. ultimum
A second part
expression
rhizosphere
genetically
of DAPG
previously.
DAPG gene
on
investigates
biosynthetic
in relation to their FA
pathway-specific
expression
impact
of
of non¬
genes of strain
producing ability.
repressor
a
the
age, and
PhlF,
was
The
used to
third part, the FA
sensitivity
different biocontrol P. fluorescens strains CHAO and
Throughout the
production
In
siderophores pyoverdine (1), pyochelin (95)
the mode of action of the microbial interaction. In
ecologically
was
expression
DAPG, the strain produces the antimicrobial
pathogenic fungi
evaluated in the
phlF mutant CHA638, lacking
of the two
as
on
the
study
work, the influence of host species and host cultivar, host
CHAO in vitro and in the wheat
investigate
(17)
In addition to
phytohormone
first part of this
strain CHAO
Fusarium oxysporum
genes of HCN and DAPG and
regulation
a
(41).
pyoluteorin (17)
salicylic
In
greenhouse
of CHAO
like protease and
The
solani, and
studies, production of DAPG has emerged
activity
metabolites
and
CHAO reduces the extent of disease caused
Rhizoctonia
model to
the causal agent of black root rot
Thielaviopsis basicola,
to
Thielaviopsis basicola, Gaeumannomyces graminis var.
ultimum,
control
as a
genes under various environmental conditions. CHAO
naturally suppressive
(86).
used
was
whole
study,
lacZ
or
inaZ reporter gene fusions
of DAPG in vitro and in the
20
rhizosphere
of different
Chapter 1
General introduction
The
plant species.
monitoring
DAPG
advantages
and
biosynthetic
disadvantages
genes
of lacZ and inaZ
as
reporter genes for
discussed.
are
Literature cited
1.
Ahl, P., Voisard, C, and Défago, G. 1986. Iron-bound siderophores, cyanic acid, and antibiotics
involved in
suppression
Phytopathol.
2.
Bangera,
for
synthesis
biocontrol
4.
of the
polyketide
by
a
Pseudomonas fluorescens strain. J.
antibiotic
Lugtenberg,
rhizobacteria. Curr.
G. V.,
2,4-diacetylphloroglucinol
gene cluster
from Pseudomonas
J. Bacterid. 181:3155-3163.
G. V., and
by
Bloemberg,
basicola
G., and Thomashow, L. S. 1999. Identification and characterization of a
M.
Bloemberg,
Thielaviopsis
116:121-134.
fluorescens Q2-87.
3.
of
imaging
different autofluorescent
Opin.
in the
and
Sturman, N., and Lugtenberg, B. J. J.
ofPseudomonas fluorescens WCS365
proteins
growth promotion
Plant Biol. 4:343-350.
A. H. M., Lamers, G. E. M.,
Wijfies,
2000. Simultaneous
B. J. J. 2001. Molecular basis of plant
rhizosphere:
populations expressing three
perspectives
New
for
studying
microbial
communities. Mol. Plant-Microbe Interact. 13:1170-1176.
5.
Blumer, C, Heeb, S., Pessi, G., and Haas, D. 1999. Global GacA-steered control of cyanide and
exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.
Proc. Natl. Acad. Sei. USA 96:14073-14078.
6.
Bonsall, R. F., Weller, D. M., and Thomashow, L. S. 1997. Quantification of 2,4-
diacetylphloroglucinol produced by
of wheat.
7.
Appl.
fluorescent Pseudomonas spp. in vitro and in the
rhizosphere
Environ. Microbiol. 63:951-955.
Brandi, M. T., and Lindow, S. E. 1997. Environmental signals modulate the expression of an
indole-3-acetic acid
biosynthesis
gene in Erwinia herbicola. Mol. Plant-Microbe Interact. 10:499-
505.
8.
Buell, C. R., and Anderson, A. J. 1993. Expression of the aggA locus ofPseudomonas putida
vitro
9.
and
m
planta
as
by the reporter
gene,
Buysens, S., Heungens, K., Poppe, J., and Höfte,
pyoverdin
in
suppression
aeruginosa 7NSK2.
10.
detected
of Pythium-induced
Appl.
biocontrol agents,
Pages
New
179-188 in:
Mol. Plant-Microbe Interact. 6:331-340.
M. 1996. Involvement of pyochelin and
damping-off of tomato by Pseudomonas
Environ. Microbiol. 62:865-871.
Campbell, R., Renwick, A.,
by
xylE.
m
and
Coe, S. K. A. M. 1986. Antagonism and siderophore production
plant growth promoting organisms,
and the
general rhizosphere population.
Iron, siderophores, and plant diseases. T. R. Swinburne, ed. Plenum Press,
York, London.
21
Chapter 1
11.
General introduction
Chin-A-Woeng,
T. F.
C, Bloemberg, G. V., Mulders, I. H. M., Dekkers, L. C, and Lugtenberg,
B. J. J. 2000. Root colonization
chlororaphis
by phenazine-1-carboxamide-producing
bacterium Pseudomonas
PCL 1391 is essential for biocontrol of tomato foot and root rot. Mol. Plant-Microbe
Interact. 13:1340-1345.
12.
Chin-A-Woeng,
T. F.
C, Bloemberg, G. V.,
Schripsema, J., Kroon, B., Scheffer,
F.
Bij,
A. J.,
van
der
Drift, K. M. G. M.,
J., Keel, C, Bakker, P. A. H. M., Tichy, H. V., de Bruijn,
R.
Pseudomonas
Fusarium oxysporum f. sp.
chlororaphis
radicis-lycopersici.
PCL 1391 of tomato root rot caused
by
Mol. Plant-Microbe Interact. 11:1069-1077.
Cook, R. J. 1993. Making greater use of introduced microorganisms for biological control of
plant pathogens.
14.
der
J., Thomas-Oates, J. E., and Lugtenberg, B. J. J. 1998. Biocontrol by phenazine-1-
carboxamide-producing
13.
van
Annu. Rev.
Phytopathol.
31:53-80.
Corbell, N., and Loper, J. E. 1995. A global regulator of secondary metabolite production in
Pseudomonas fluorescens Pf-5. J. Bacteriol. 177:6230-6236.
15.
Cronin, D., Moenne-Loccoz, Y., Fenton, A., Dunne, C, Dowling, D. N., and O'Gara, F. 1997.
Ecological
interaction of a biocontrol Pseudomonas fluorescens strain
diacetylphloroglucinol
with the soft rot potato
pathogen
producing 2,4-
Erwinia carotovora
subsp. atroseptica
FEMS Microbiol. Ecol. 23:95-106.
16.
Cronin, D., Moënne-Loccoz, Y., Fenton, A., Dunne, C, Dowling, D. N., and O'Gara, F. 1997.
Role of 2,4-diacetylphloroglucinol in the interactions of the biocontrol
with the potato cyst nematode Globodera rostochiensis.
17.
Défago, G., Berling,
and
C. H.,
ofPseudomonas fluorescens:
B.
18.
Schippers,
Défago, G.,
borne
and
Environ. Microbiol. 63:1357-1361.
root rot of tobacco and other root diseases
potential applications
plant pathogens.
and P. R.
strain Fl 13
Burger, U., Haas, D., Kahr, G., Keel, C, Voisard, C, Wirthner, P.,
Wüthrich, B. 1990. Suppression of black
control of soil-borne
Appl.
pseudomonad
D.
Hornby,
and mechanisms.
R. J.
Pages
93-108 in:
by
strains
Biological
Cook, Y. Henis, W. H. Ko, A. D. Rovira,
Scott, eds. CAB international.
Keel, C. 1995. Pseudomonads
pathogens. Pages
as
biocontrol agents of diseases caused
137-148 in: Benefits and risks of introducing biocontrol
by
soil¬
agents. H.M.T.
Hokkanen, and J.M. Lynch, eds. Cambridge University Press, England.
19.
20.
M., Fenton, A., Bardin, S., Aarons, S., and O'Gara, F. 2000. Regulation
Delany, I., Sheehan,
M.
of production of the
antifungal
of phlF
metabolite
2,4-diacetylphloroglucinol in Pseudomonas fluorescens
genetic analysis
de las
Mercedes, D. M., Limon, M. C, Mejias, R., Mach, R. L., Benitez, T., Pintor, T. J. A., and
as a
transcriptional
repressor. Microbiol. UK 146:537-546.
Fl 13:
Kubicek, C. P. 2001. Regulation of chitinase
33
(chit33)
gene
expression
in Trichoderma
harzianum. Curr. Genetics 38:335-342.
21.
de
Meyer, G., Capieau, K., Audenaert, K., Buchala, A., Métraux, J.-P., and Höfte,
Nanogram
amounts of
salicylic
acid
produced by the
22
M. 1999.
rhizobacterium Pseudomonas aeruginosa
Chapter 1
General introduction
7NSK2 activate the
resistance
systemic acquired
pathway
in bean. Molecular Plant-Microbe
Interact. 12:450-458.
22.
23.
de
L.
procedure
to
der
Bij,
A. J., and
visualize root colonisation
rapidly
Lugtenberg,
B. J. J. 1997. Use of a lux-based
by Pseudomonas fluorescens
in the wheat
Anton. Leeuw. Int. J. G. 72:365-372.
Dowling,
N., and O'Gara, F. 1994. Metabolites of Pseudomonas involved in the biocontrol of
D.
disease. Trends Biotechnol. 12:133-141.
K., and Défago, G. 1997. Zinc improves biocontrol of Fusarium
Duffy,
B.
tomato
by Pseudomonas fluorescens
inhibitory to
25.
van
rhizosphere.
plant
24.
A., Kuiper, I.,
Weger,
Duffy,
bacterial antibiotic
and represses the
production
biosynthesis. Phytopathology
crown
and root rot of
of pathogen metabolites
87:1250-1257.
K., and Défago, G. 1999. Environmental factors modulating antibiotic and siderophore
B.
biosynthesis by Pseudomonas fluorescens
biocontrol strains.
Appl.
Environ. Microbiol. 65:2429-
2438.
26.
Duffy,
K., and Défago, G. 2000. Controlling instability in gacS-gacA regulatory
B.
inoculant
ofPseudomonas fluorescens biocontrol strains.
production
genes
during
Environ. Microbiol.
Appl.
66:3142-3150.
27.
Duijff,
B.
J., Recorbet, G., Bakker, P. A. H. M., Loper, J. E., and Lemanceau, P. 1999. Microbial
antagonism
at the root level is involved in the
suppression
of Fusarium wilt
by the
Fusarium oxysporum Fo47 and Pseudomonas putida WCS358.
nonpathogenic
combination of
Phytopathology
89:1073-1079.
28.
Dunne, C, Delany, I., Fenton, A., and O'Gara, F. 1996. Mechanisms involved in biocontrol by
microbial inoculants.
29.
Dunlap,
Pages
69-106 in: Bacteria
multicellular
organisms.
J. A.
Shapiro
Unity
and M.
and
diversity.
Dworkin, eds. Oxford
Fenton, A. M., Stephens, P. M., Crowley, J., O'Callaghan, M., and O'Gara, F. 1992. Exploitation
capability to
32.
as
lactone autoinducers in bacteria:
London.
of gene(s) involved in
31.
16:721-729.
TV-acyl-homoserine
P. V. 1997.
University Press,
30.
Agronomie
Gallagher,
a
2,4-diacetylphloroglucinol biosynthesis to
Pseudomonas strain.
Appl.
S. R. 1992. GUS Protocols:
confer
a new
biocontrol
Environ. Microbiol. 58:3873-3878.
Using
the GUS gene
as a
reporter of gene expression.
Academic Press, St.
Paul, Minn.
Georgakopoulos,
G., Hendson, M., Panopoulos, N. J., and Schroth, M. N. 1994. Analysis of
expression
a
mutant
of a
D.
phenazine biosynthesis
carrying
a
locus of Pseudomonas
phenazine biosynthesis
aureofaciens
PGS12
on
locus-ice nucleation reporter gene fusion.
seeds with
Appl.
Environ. Microbiol. 60:4573-4579.
33.
Glick, B. R., Patten, C. L., Holguin, G., and Penrose, D. M. 1999. Biochemical and genetic
mechanisms used
by plant growth promoting
bacteria.
23
Imperial College Press,
London.
Chapter 1
34.
General introduction
Haas, D., Keel, C, Laville, J., Maurhofer, M., Oberhansli, T., Schnider, U., Voisard, C,
Wüthrich, B., and Défago, G. 1991. Secondary metabolites ofPseudomonas fluorescens strain
CHAO involved in the
genetics
of plant-microbe
Academic
35.
suppression
of root diseases.
Pages
450-556 in: Advances in molecular
interactions, Vol. I. H. Hennecke, and D. P. S. Verma, eds. Kluwer
Publishers, Dordrecht.
Haas, D., Keel, C, and Reimmann, C. 200X. Signaling in plant-beneficial rhizobacteria.
Submitted.
36.
Heeb, S., Blumer, C, and Haas, D. 2002. A regulatory RNA
in
press
Heim, R., Prasher, D. C, and Tsien, R. Y. 1994. Wavelength mutations and posttranslational
autoxidation of green fluorescent
38.
mediator in GacA/RsmA-
control in Pseudomonas fluorescens CHAO. J. Bacteriol.
dependent global
37.
as a
protein.
Proc. Natl. Acad. Sei. USA 91:12501-12594.
Hofmann, K., Heinz, E. B., Charles, T. C, Hoppert, M., Liebl, W., and Streit, W. R. 2000.
Sinorhizobium mehloti strain 1021 bioS and bdhA gene
transcriptions
are
both affected
by
biotin.
FEMS Microbiol. Lett. 182:41-44.
39.
Howie, W. J., and Suslow, T. V. 1991. Role of antibiotic biosynthesis in the inhibition of Pythium
ultimum in the cotton
spermosphere
and
rhizosphere by Pseudomonas fluorescens.
Mol. Plant-
Microbe Interact. 4:393-399.
40.
Keel, C, and Défago, G. 1997. Interactions between beneficial soil bacteria and
Mechanisms and
Systems:
the 36th
University
41.
ecological impact. Pages
Symposium
of London.. A. C.
27-46 in:
of the British
Gange,
V. K.
Multitrophic
root
pathogens:
Interactions in Terrestrial
Ecological Society, Royal Holloway College,
Brown, eds. Blackwell Science, Oxford.
Keel, C, Schnider, U., Maurhofer, M., Voisard, C, Laville, J., Burger, U., Wirthner, P., Haas, D.,
and
G. 1992.
Défago,
Suppression
of root diseases
by Pseudomonas fluorescens
CHAO:
Importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol. Plant-Microbe
Interact. 5:4-13.
42.
Keel, C, Voisard, C, Berling, C. H., Kahr, G., and Défago, G. 1989. Iron sufficiency,
prerequisite
for the
CHAO under
43.
suppression
gnotobiotic
conditions.
as
antagonists
2,4-diacetylphloroglucinol
strain
79:584-589.
in the
of plant
pathogens
suppression
in the
rhizosphere:
of black root rot of tobacco.
Killham, K., and Yeomans, C. 2001. Rhizosphere carbon flow
from
45.
Phytopathology
by Pseudomonas fluorescens
Keel, C, Wirthner, P. H., Oberhansli, T. H., Voisard, C, Burger, U., Haas, D., and Défago, G.
1990. Pseudomonads
44.
of tobacco black root rot
a
isotopes
to
Role of the antibiotic
Symbiosis
measurement and
9:327-341.
implications:
reporter genes. Plant Soil 232:91-96.
Kim, B. S., Moon, S. S., and Hwang, B. K. 1999. Isolation, identification, and antifungal activity
of a macrolide
antibiotic, oligomycin A, produced by Streptomyces hbani. Can. J. Bot. 77:850-
858.
24
Chapter 1
46.
General introduction
Kraus, J., and Loper, J. E. 1995. Characterization of a genomic region required for production of
the antibiotic
pyoluteorin by the biological
control agent Pseudomonas fluorescens Pf-5.
Appl.
Env. Microbiol. 61:849-854.
47.
Laville, J., Blumer, C,
von
Schroetter, C, Gaia, V., Défago, G., Keel, C, and Haas, D. 1998.
Characterization of the hcnABC gene cluster
ANR in the
regulation by
strictly
encoding hydrogen cyanide synthase
and anaerobic
aerobic biocontrol agent Pseudomonas fluorescens CHAO. J.
Bacteriol. 180:3187-3196.
48.
Laville, J., Voisard, C, Keel, C, Maurhofer, M., Défago, G., and Haas, D. 1992. Global control
in Pseudomonas fluorescens
mediating
antibiotic
synthesis
and
suppression
of black root rot of
tobacco. Proc. Natl. Acad. Sei. 89:1562-1566.
49.
Leeman, M.,
Schippers,
van
Pelt, J. A., den Ouden, F. M., Heinsbroek, M., Bakker, P. A. H. M., and
B. 1995. Induction of
lipopolysaccharides
50.
resistance
ofPseudomonas fluorescens.
against
Fusarium wilt of radish
Phytopathology
by
85:1021-1027.
Lemanceau, P., and Alabouvette, C. 1993. Suppression of Fusarium wilts by fluorescent
pseudomonads:
51.
systemic
Ligon,
J.
Mechanisms and
applications.
Biocontrol Sei. Technol. 3:219-234.
M., Hill, D. S., Hammer, P. E., Torkewitz, N. R., Hofmann, D., Kempf, H. J. and
van
Pee, K. H. 2000. Natural products with antifungal activity from Pseudomonas biocontrol bacteria.
Pest.
52.
Manag. Sei.
56:688-695.
Lindow, S. E. 1995. The
use
of reporter genes in the
study
of microbial
Mol. Ecol.
ecology.
4:555-566.
53.
Loper,
J.
E., and Henkels, M. D. 1997. Availability of iron
rhizosphere
and bulk soil evaluated with
an
to Pseudomonas fluorescens in
ice nucleation reporter gene.
Appl.
Environ.
Microbiol. 63:99-105.
54.
Loper,
J.
E., and Lindow, S. E. 1997. Reporter
expression by
soil- and
microbiology.
C. J. Hurst, G. R.
plant-associated
gene
bacteria.
systems useful in evaluating in situ gene
Pages
482-492 in: Manual of environmental
Knudsen, M. J. Mc Inervey, L. D. Stetzenbach, and M. V.
Walter, eds. ASM Press, Washington DC.
55.
Ma, S. W., Morris, V. L., and Cuppels, D. A. 1991. Characterization of a DNA region required
for
production
of the
phytotoxin
coronatine
by Pseudomonas
syringae pv. tomato. Mol. Plant-
Microbe Interact. 4:69-74.
56.
Manulis, S., Haviv-Chesner, A., Brandi, M. T, Lindow, S. E., and Barash, I. 1998. Differential
involvement of indole-3-acetic acid
of Erwinia herbicola pv.
57.
biosynthetic pathways
gypsophila.
in
pathogenicity
and
epiphytic
fitness
Mol. Plant-Microbe Interact. 11:634-642.
Marschner, P., and Crowley, D. E. 1996. Physiological activity of a bioluminescent Pseudomonas
fluorescens (strain 2-79)
{Capsicum
annuum
in the
rhizosphere
L). Soil Biol. Biochem.
of mycorrhizal and
28:869-876.
25
non-mycorrhizal
pepper
Chapter 1
58.
General introduction
Marschner, P., and Crowley, D. E. 1997. Iron
in the
pseudomonad
vulgare L.). Appl.
59.
rhizosphere
of white
stress and
pyoverdin production by
lupine (Lupinus
albus
Lukyanov,
fluorescent
L.) and barley (Hordeum
Environ. Microbiol. 63:277-281.
Matz, M. M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P.
and
a
S. A. 1999. Fluorescent
proteins
,
Zaraisky,
A. G.
Markelov, M. L.,
fromnonbioluminescent^4«^ozoa
species.
Nat.
Biotechnol. 17:969-973.
60.
Maurhofer, M., Keel, C, Haas, D., and Défago, G. 1994. Pyoluteorin production by
Pseudomonas fluorescens strain CHAO is involved in the
cress
61.
suppression
of Pythium
damping-off of
but not of cucumber. Eur. J. Plant Pathol. 100:221-232.
Maurhofer, M., Keel, C, Haas, D., and Défago, G. 1995. Influence of plant species
suppression by Pseudomonas fluorescens
strain CHAO with enhanced antibiotic
on
disease
production.
Plant
Pathol. 44:40-50.
62.
Maurhofer, M., Keel, C, Schnider, U., Voisard, C, Haas, D., and Défago, G. 1992. Influence of
enhanced antibiotic
production
in Pseudomonas fluorescens strain CHAO
suppressive capacity. Phytopathology
63.
on
its disease
82:190-195.
Maurhofer, M., Reimmann, C, Schmidli-Sacherer, P., Heeb, S., Haas, D., and Défago, G. 1998.
Salicylic
acid
induction of
biosynthetic
systemic
genes
expressed
resistance in tobacco
in Pseudomonas fluorescens strain P3
against tobacco
necrosis virus.
improve
the
Phytopathology
88:678-684.
64.
Meighen,
E. A. 1993. Bacterial bioluminescence:
organization, regulation,
and
application
of the
lux genes. FASEB J. 7:1016-1022.
65.
Meyer,
acid,
66.
a
J.
M., Azelvandre, P., and Georges, C. 1992. Iron metabolism in Pseudomonas: Salicylic
siderophore
Miller, W.G., Brandi, M. T., Quinones, B., and Lindow, S. E. 2001. Biological
availability:
67.
ofPseudomonas fluorescens CHAO. 1992. Biofactors 4:23-27.
Relative sensitivities of various reporter genes.
produced by Stenotrophomonas
damping-off disease. Appl.
68.
and
Nakayama, T., Homma, Y., Hashidoko, Y., Mizutani, J.,
xanthobaccins
Appl.
sensor
for
sucrose
Env. Microbiol. 67:1308-1317.
Tahara, S. 1999. Possible role of
sp. strain SB-K88 in
suppression
of sugar beet
Environ. Microbiol. 65:4334-4339.
Nowak-Thompson, B., Gould,
diacetylphloroglucinol by the
S. J., Kraus, J., and
Loper,
J. E. 1994. Production of 2,4-
biocontrol agent Pseudomonas fluorescens Pf-5. Can. J. Microbiol.
40:1064-1066.
69.
O'Sullivan, D. J., and O'Gara, F. 1992. Traits of fluorescent Pseudomonas
suppression
70.
of plant root
pathogens.
spp. involved in
Microbiol. Rev. 56: 662-676.
Pessi, G., Blumer, C. and Haas, D. 2001. lacZ fusions report gene expression, don't they?
Microbiology
147:1993-1995.
26
Chapter 1
71.
General introduction
Pierson, E. A., Wood, D. W., Cannon, J. A., Blachere, F. M., and Pierson, L. S., III. 1998.
Interpopulation signaling
rhizosphere.
72.
iV-acyl-homoserine
lactones among bacteria in the wheat
Mol. Plant-Microbe Interact. 11:1078-1084.
Pierson, L. S., Ill, Wood, D. W., Pierson, E. A., and Chancey, S. T. 1998. iV-acyl-homoserine
lactone-mediated gene
regulation
in
control
biological
fluorescent
by
pseudomonads:
Current
and future work. Eur. J. Plant Pathol. 104:1-9.
knowledge
73.
via
Porteous, F., Killham, K., and Meharg, A. 2000. Use of a lux-marked rhizobacterium
biosensor to
assess
changes
in
rhizosphere
C flow due to
pollutant
stress.
as a
Chemosphere
41:1549-
1554.
74.
Raaijmakers,
J.
M., Bonsall, R. F., and Weiler, D. M. 1999. Effect of population density of
Pseudomonas fluorescens
wheat.
75.
Phytopathology
on
production
of 2,4-diacetylphloroglucinol in the
rhizosphere
of
89:470-475.
Ravnskov, S., Nybroe, O., and Jakobsen, I. 1999. Influence of an arbuscular mycorrhizal fungus
on
Pseudomonas fluorescens DF57 in
rhizosphere
and
hyphosphere
soil. New
Phytol.
142:113-
122.
76.
Sacherer, P., Défago, G., and Haas, D. 1994. Extracellular protease and phospholipase C
controlled
by the global regulatory
gene
gacA
are
in the biocontrol strain Pseudomonas fluorescens
CHAO. FEMS Microbiol. Lett. 116:155-160.
77.
Sarniguet, A., Kraus, J., Henkels,
factor as affects antibiotic
M.
production
D., Muehlchen, A. M., and Loper, J. E. 1995. The sigma
and
biological
control
activity
ofPseudomonas fluorescens
Pf-5. Proc. Natl. Acad. Sei. 92:12255-12259.
78.
Schmidli-Sacherer, P., Keel, C, and Défago, G. 1997. The global regulator GacA of
Pseudomonas fluorescens CHAO is
not in Gramineae. Plant
79.
for
supression
of root diseases in
dicotyledons
but
46:80-90.
Schnider, U., Keel, C, Blumer, C, Troxler, J., Défago, G., and Haas, D. 1995. Amplification of
the
and
80.
pathol.
required
housekeeping sigma
improves
factor in Pseudomonas fluorescens CHAO enhances antibiotic
production
biocontrol abilities. J. Bacteriol. 177:5387-5392.
Schnider-Keel, U., Seematter, A., Maurhofer, M., Blumer, C, Duffy, B., Gigot-Bonnefoy, C,
Reimmann, C, Notz, R., Défago, G., Haas, D., and Keel, C. 2000. Autoinduction of 2,4-
diacetylphloroglucinol biosynthesis
repression by the
81.
bacterial metabolites
salicylate
and
pyoluteorin.
J. Bacteriol. 182:1215-1225.
Shah-Smith, D. A., and Burns, R. G. 1997. Shelf-life of a biocontrol Pseudomonas putida applied
to sugar beet seeds
82.
in the biocontrol agent Pseudomonas fluorescens CHAO and
using
commercial
coatings.
Biocontrol. Sei. Technol. 7:65-74.
Shanahan, P., O'Sullivan, D. J., Simpson, P., Glennon, J. D., and O'Gara, F. 1992. Isolation of
2,4-diacetylphloroglucinol
from
a
fluorescent
pseudomonad
and
investigation
parameters influencing its production. Appl. Environ. Microbiol. 58:353-358.
27
of physiological
Chapter 1
83.
General introduction
Shang, H., Chen, J., Handelsman, J.,
during
zoospores
and
Goodman, R. M. 1999. Behavior of Pythium torulosum
their interaction with tobacco roots and Bacillus
cereus.
Curr. Microbiol.
38:199-204.
84.
Silo-Suh, L. A., Lethbridge, B. J., Raffel, S. J., He, H., Clardy, J., and Handelsman, J. 1994.
activities of two
Biological
fungistatic
produced by Bacillus
antibiotics
cereus
UW85.
Appl.
Environ. Microbiol. 60:2023-2030.
85.
Snyder, L.,
and
Champness,
W. 1997. Molecular
genetics
of bacteria. ASM Press,
Washington,
DC.
86.
Stutz, E. W., Défago, G., and Kern, H. 1986. Naturally occurring fluorescent pseudomonads
involved in
87.
Tambong,
suppression
88.
T., and Höfte, M. 2001. Phenazines
J.
myriotylum
of black root rot of tobacco.
on
cocoyamby Pseudomonas
control: Mechanisms and
N. T. eds. Plant-Microbe
90.
76:181-185.
involved in biocontrol of Pythium
aeruginosa PNA1. Eur. J. Plant. Pathol. 107:511-521.
antifungal
metabolites.
Pages
use
of introduced bacteria for
187-235 in:
Stacey, G., Keen,
Interactions, vol. 1. Chapman & Hall, New York, NY.
Thrane, C, Nielsen, T. H., Nielsen, M. N., Sorensen, J., and Olsson, S. 2000. Viscosinamide-
producing
Pseudomonas fluorescens DR54 exerts
sugar beet
rhizosphere.
a
biocontrol effect
to
by
biological
a
on
Pythium
ultimum in
FEMS Microbiol. Ecol. 33:139-146.
Toyoda, H., Hashimoto, H., Utsumi, R., Kobayashi, H.,
fusaric acid
91.
are
Thomashow, L. S., and Weiler, D. M. 1996. Current concepts in the
biological
89.
Phytopathology
and
Ouchi, S. 1988. Detoxification of
fusaric acid-resistant mutant of Pseudomonas solanacearum and its
control of Fusarium wilt of tomato.
Toyota, K., Kimura, M., and Kinoshita,
colonization of tomato roots
by
T. 2000.
Phytopathology
78:1307-1311.
Microbiological
factors
affecting
application
the
Ralstoma solanacearum YUlRif431ux. Soil Sei. Plant Nutr.
46:643-653.
92.
Vali, G. 1971. Quantitative evaluation of experimental results
nucleation of
93.
van
heterogenous freezing
J. Atmos. Sei. 28:402-409.
bacteria. Annu. Rev.
Phytopathol.
36:453-483.
Vincent, M. N., Harrison, L. A., Brackin, J. M., Kovacevich, P. A., Mukerji, P., Weiler, D. M.,
and
Pierson, E. 1991. Genetic analysis of the antifungal activity of a soilborne Pseudomonas
aureofaciens
95.
the
Loon, L. C, Bakker, P. A. H. M., and Pieterse, C. M. J. 1998. Systemic resistance induced by
rhizosphere
94.
supercooled liquids.
on
strain.
Appl.
Env. Microbiol. 57:2928-2934.
Voisard, C, Bull, C. T., Keel, C, Laville, J., Maurhofer, M., Schnider, U., Défago, G., and Haas,
D. 1994. Biocontrol of root diseases
experimental approaches.
Dowling,
B.
by Pseudomonas fluorescens
In: Molecular
ecology
of rhizosphere
microorganisms.
Boesten, eds. VCH Publishers, Weinheim, Germany.
28
CHAO: Current concepts and
F.
O'Gara, D. N.
Chapter 1
96.
General introduction
Voisard, C, Keel, C, Haas, D., and Défago, G. 1989. Cyanide production by Pseudomonas
suppress black root rot of tobacco under
fluorescens helps
gnotobiotic
conditions. EMBO J.
8:351-358.
97.
Walsh, U. F., Morrissey, J. P., and O'Gara, F. 2001. Pseudomonas for biocontrol of
phytopathogens:
from functional
genomics
to commercial
exploitation.
Curr.
Opin.
Biotech.
12:289-295.
98.
Whipps,
J. M. 2001. Microbial interactions and biocontrol in the
rhizosphere.
J.
Exp.
Bot. 52:487-
511.
99.
Whistler, C. A., Stockwell, V. O., and Loper, J. E. 2000. Lon protease influences antibiotic
production
and UV tolerance ofPseudomonas fluorescens Pf-5.
Appl.
Environ. Microbiol.
66:2718-2725.
100.
Wüthrich, B., and Défago, G. 1991. Suppression of wheat take-all and black
by Pseudomonas fluorescens
Plant Growth
Défago,
101.
Promoting
strain CHAO: results of field and pot
Rhizobacteria
-
root rot of tobacco
experiments. Pages
Progress and Prospects. C. Keel,
B.
17-22 in:
Koller, and G.
eds. IOBC/WPRS Bulletin XIV/8.
Yeomans, C. V., Porteous, F., Paterson, E., Meharg, A. A., and Killham, K. 1999. Assessment of
lux-marked Pseudomonas fluorescens for
reporting
on
organic
carbon
compounds.
FEMS
Microbiol. Let. 176:79-83.
102.
Yuan, Z., Cang, S. Matsufuji, M., Nakata, K., Nagamatsu, Y., and Yoshimoto, A. 1998. High
production
grown
on
of pyoluteorin and
ethanol
as a
2,4-diacetylphloroglucinol by Pseudomonas fluorescens
sole carbon
source.
J. Ferment.
29
Bioeng.
86:559-563.
S272
30
Chapter 2
Biotic Factors
Affecting Expression
phlA
of the
2,4-Diacetylphloroglucinol Biosynthesis
in Pseudomonas fluorescens Biocontrol Strain CHAO in the
31
Gene
Rhizosphere
32
Chapter 2
Biotic factors
affecting
DAPG
biosynthesis
in the
rhizosphere
Abstract
Production of the
(DAPG)
is
a
biosynthetic
a
production
of the antimicrobial
dicots
(maize
and
(bean
and
gene
In
a
was
wheat), compared
the
transgenic
maize
maize
as a
expressing
pregerminated seedlings,
and
level
of plant
bacterial gene
expression
differences in
rhizosphere
comprehensive
in
on
expression
Significant
gnotobiotic
the Bacillus
model, expression of the phlA
throughout 22 days
determined
significantly greater
with gene
line. Plant age had
near-isogenic parent
as
in the
in the
dropped
'-
to a
growth.
a
on
thuringiensis
evaluation of how biotic factors that
agricultural systems (host genotype,
of key biocontrol genes for disease
on
was no
peaked
host age,
To
commonly
of two
of two
found among six
gene
by Pythium
plants.
and sand-based
difference
insecticidal gene cry 1Ab and
maize, and this
these host
direct extraction
bacterial gene
was
our
expression. Using
at 24 h
fourth ofthat value within 48
Root infection
accurately
rhizospheres
on
conditions. There
'lacZ reporter gene
of the phi
rhizospheres
were
significant impact
both cucumber and
colonization
differences
by
clay
a
We observed this host genotype effect
additional maize cultivars tested under
between
planta
expression
of the reporter gene
gnotobiotic system containing
also at the level of cultivars.
expression
used to monitor
was
rhizosphere. Expression
expression
cucumber).
2,4-diacetylphloroglucinol
of Pseudomonas fluorescens CHAO. Strain
of DAPG in vitro and in
compound.
soil, reporter
monocots
activity
translational phlA'-lacZ fusion
genes in vitro and in the
reflected actual
artificial
antimicrobial metabolite
factor in the biocontrol
key
carrying
CHAO
polyketide
after
planting
h, remaining
at
of
that
ultimum stimulated
independent
knowledge,
of
this is the first
confront bacterial inoculants
pathogen infection)
modulate the
expression
suppression.
Published 2001 in
Phytopathology
91:873-881
Introduction
Certain root-associated bacteria have the
caused
by
soilborne
greenhouse
fungal pathogens
conditions
performance
(8, 26).
ability
and to increase
In commercial
settings
to
reduce the
plant yield
and field
under
of diseases
laboratory
and
trials, however, inconsistent
from site to site and from year to year tends to be the
33
severity
disappointing reality (49).
Chapter 2
Biotic factors
Concerns about
performance reliability
bacterial-based biocontrol
Understanding
the
are a
found to affect the
performance
in biocontrol
(11, 35, 36).
Soil
suppressive
soils
specific
and
of bacterial
factors
long
of both
natural
containing
and mineral
(e.g., pH
fungal
important
in
root
(46).
Because
a
it is
(49),
pathogens
also affects root colonization and biocontrol
of the most
monitor bacterial gene
constraints. We have
biosynthesis
expression,
developed
in culture
a
compounds
antimicrobial
with
approximately
six
copies
genes
high sensitivity
and
are an
relatively
biosynthesis
DAPG in vitro
(43).
is
The vector
biocontrol
approach
to
a
lacZ translational
gene in the phlACBDE
on
cell
density
plasmid used, pME6010,
and
has
per chromosome and is maintained and stable in Pseudomonas
evaluated the influence of host
in the
result from
little technical
strongly dependent
(19).
The
objective
this reporter gene to examine the effect of biotic factors
we
available
quantitatively monitoring
promoter-proximal
in the absence of antibiotic selection
Specifically,
attractive
this reporter gene, which consists of
have shown that DAPG
positively auto-regulated by
compounds produced by
DAPG reporter construct for
(43). Using
are
suppression
performance might
activity
different hosts.
on
(21, 49). Reporter
fusion to the structural gene phiA, which is the
we
that variable
in
metabolite, 2,4-diacetylphloroglucinol (DAPG), has
important
strains of Pseudomonas fluorescens
mechanism of disease
primary
thought
antimicrobial
diseases
of disease
development
Host genotype affects
there
and the
concentrations)
against
are now
example,
evidence that host traits favorable for biocontrol
polyketide
expression
plant pathogens (12),
and bacterial inoculants
are
in the
recent
The
to use
be attributed to variations in
strains, with
as one
of
this obstacle. Recent
Genotype
of antimicrobial
fluorescens
can
overcoming
ways.
production
(1),
to
known to influence
antagonists (22).
variation in
cluster
key
variability
factors, particularly clay quality,
inherited
rhizosphere
large-scale application
of biocontrol agents but in different ways. For
activity
these bacteria is antibiosis
emerged
to
in the
nonspecific
quantitatively
to
same
clear link between abiotic soil factors
variability
biosynthesis
conditions, abiotic and biotic, that confront bacterial inoculants
of the
a
major impediment
of variability is
sources
rhizosphere. Many
is
DAPG
products.
evidence indicates that at least part of this
environmental
affecting
rhizosphere
species
evaluate the effects of other biotic factors that have
as
host age and host health when attacked
DAPG
and cultivar
and related this to biocontrol
to
on
activity.
previously
on
study
production
was
in situ.
DAPG gene
We used the reporter gene
received less attention such
by root-pathogenic fungi.
34
of the current
Chapter 2
Biotic factors
DAPG
affecting
biosynthesis
in the
rhizosphere
MATERIALS AND METHODS
Microorganisms, plants,
agar
and
(48)
CHAO
a
derivative
harboring plasmid pME6259 (43)
of King's medium B
plates
and culture conditions. Pseudomonas fluorescens strain
(KB) (23)
unless otherwise mentioned. Strain
were
routinely
and in Luria-Bertani broth
CHA0/pME6259
cultivated
(LB) (41)
at
on
27°C,
grown in the presence of
was
tetracycline (125 ug/ml).
ultimum strain 67-1
Pythium
Canada)
7
days.
was
cultured
For disease
1.5% malt agar
on
suppression
ultimum agar culture
was
assays,
placed
in
a
of incubation at 20°C in
1-mm diameter
(Phaseolus
wheat
(Triticum
(event 11)
cv.
Max
varieties. Seeds
water.
by
Seeds
and 10 ml of sterilized double-distilled water. After 7
pregerminated
of DAPG
containing
with 100 ul of
an
the
according
Miller
as
organic phase
pH
was
cvs.
bush bean
winter
Schlange),
Maïs Silex
170, Antares,
the
were
days
gene for
crylAb
period
toxin, and the other cultivars
and
on
thoroughly
(vol/vol)
grown in 500-ml
at 600 nm,
days.
after
sodium
of
silage
are
hypochlorite,
rinsed with sterile double-distilled
0.85% water agar at 24°C in darkness.
and phlA'-'lacZ
of 7
biosynthesis
expression
Erlenmeyer flasks
Bacterial
having
tetracycline.
growth
established
of phlA'-'lacZ
in vitro. Strain CHAO and
on
was
by
sealed with cotton
Media
shaking
were
at 160 rpm.
determined
dilution
by measuring
plating
plasmid pME6259
inoculated
the correlation
was
quantified
(32).
previously
with 2 M HCl to
were
Chinesische
LB culture and incubated at 27°C with
Production of DAPG
(HPLC)
for 3
ß-Galactosidase expression
to
mays
200 ml of KB medium without
over a
optical density (OD)
with CFU.
insecticidal
production
overnight
taken
(Zea
hybrids carrying
(vol/vol) H2O2,
CHA0/pME6259
were
and maize
Arina),
cv.
sieved. Particles of
was
and cultivars used
species
surface-disinfected for 15 min in 7%
wool stoppers
Samples
25 g of autoclaved millet seeds
containing
cucumber (Cucumis sativus
maize
10 min in 10%
Assays
its derivative
cv.
for
actively growing Pythium
an
darkness, the mycelium-covered millet
thuringiensis
were
were
dish
of
plug
at 20°C
88, N4660, and N4660 Bt). "Max 88" (event 176) and "N4660 Bt"
transgenic
are
abbreviated Bacillus
followed
Saxa),
aestivum
Corso, Magister,
0.7-cm
used to infest soil. Plant
were
sativus
a
Agriculture, Mississauga, ON,
plates (Difco Laboratories, Detroit, MI)
petri
(Biofarm, Kleindietwil, Switzerland)
days
from Allelix
(obtained
was
described
2 to
quantified using high-performance liquid chromatography
(10, 30). Aliquots
3, mixed with
separated
10 ml of
from the aqueous
of bacterial cultures
ethyl
acetate
(10 ml)
and shaken
phase by filtering through
35
were
vigorously.
acidified
The
silicon-coated filter
Chapter 2
paper
Biotic factors
and dried in
(Macherey-Nagel, Düren, Germany)
Heto Lab
Equipment, Allerad, Denmark).
analyzed by
a
The residue
with
liquid Chromatograph equipped
1090; Hewlett-Packard Co., Palo Alto, CA) and
Nucleosil 120-5-C18
Assay
for gene
expression
artificial soil
before
dissolved in 1 ml of methanol and
diode-array
column
(100
detector
on
the
rhizosphere.
maize
(300 ml) containing
age, and the presence of the
of the phlÄ-lacZ reporter gene
expression
different
were
was
monitored
grown in flasks
containing
(10%>, wt/wt)
plant species
75 g of artificial soil
LB cultures of
washed with 0.9% NaCl
were
CHA0/pME6259
solution, and diluted
concentration of 10 CFU/ml. The seeds
seedlings
and maize
were
soaked in the bacterial
inoculated with the
with 16-h
light
wild-type
were
to an
were
assayed
pregerminated
suspension
(160
at 18°C
uE/m2/s)
experiment evaluating plant
planting.
In both
age,
were
for 1 h.
sizes
(22).
The
and autoclaved in flasks
plant
randomized
expression
in the
design.
rhizosphere,
were
treatments
In the
by
at
day
Erlenmeyer
ß-galactosidase activity,
give
to
described
as
nutrient solution
a
final
previously.
were
planted
with
days
after
planting.
were
seedlings
growth
8-h darkness at 13°C. In the
Plant
chambers
experiment
In the
taken at 3 h and 1, 3, 5, 7, 10, 14 and 21
consisted of four
were as
grown in 1-liter flasks with 300 g artificial
sampled
Knop's
incubated in
were
experiment evaluating
conditions
age,
Thereafter, five seedlings
taken at 3, 5 and 10
samples
experiments,
completely
followed
for
OD6oo of 0.0125
strain CHAO. The flasks
comparing plant species, samples
block
particle
closed with cotton wool stoppers,
transferred to each flask and covered with soil. Control flasks
and
with
The influence of several biotic
cultivar, plant
moistened with double-distilled water
experiments comparing
(22). Overnight
were
(Hewlett Packard
mm) packed
4
x
autoclaved at 121°C for 30 min and amended with 10 ml of modified
after
rhizosphere
planting.
For
flasks
was
in the
centrifuge (HETOVAC;
of vermiculite and quartz sand of different
comprised
was
biosynthesis
a vacuum
conditions. Plants inoculated with bacteria
gnotobiotic
artificial soil
in the
(i.e., plant species,
pathogen Pythium ultimum)
an
a
a
DAPG
(Macherey-Nagel, Düren, Germany).
environmental factors
under
affecting
previously
replicate
arranged
maize cultivar effects
described except that
soil, watered with
6. Treatments consisted of four flasks
flasks
arranged
days
on
in
gene
seedlings
30 ml of Knop's
solution,
in
complete
a
randomized
design.
In the
Erlenmeyer
described
experiment examining
flasks
were
previously.
mixed into the soil at
filled with 300 g of artificial
Sieved millet
a
the effect of the
rate
pathogen
on
gene
expression,
soil, plugged, and autoclaved
powder completely
colonized
by Pythium
1-liter
as
ultimum
was
of 10 mg for cucumber and 100 mg for maize. Control flasks
36
a
Chapter 2
Biotic factors
without the
pathogen
received the
of Knop's nutrient solution
conditions described
activity
were
After 7
disease
days
severity,
severity
Plants
based
were
soil. Roots with
mechanically
root
in the
root
chamber under the
colonization and
same
ß-galactosidase
Pythium
reduction in
on
ultimum
seedling
soil
were
placed
the two host
gently
method recovered 97.1 ± 1.3% of the bacteria from wheat roots.
ß-Galactosidase activity
was
serial dilutions
initial wash
longer
a
measured
of plants inoculated with
wild-type
from calculations. Plants
were
(30 min)
using
or
on
were
loosely
vortexing (15
s) (data
suspension.
dry
determined
(22).
Our
Recovery efficiency
60
or
used to subtract
washed with tap water, blotted
was
agar media
appropriate
200 ul of the root
strain CHAO
we
in sterile flasks with saline solution
shaken for 5 min at 300 rpm. Bacterial root colonization
by
plants tested,
shaken to discard
washing
increased
In the
emergence and reduction of plant
removed from the flasks and
rhizosphere
on
resulting suspension by plating
not
milliliters
pregerminated,
were
from the
was
rhizosphere
powder. Thirty
seedlings
growth
in the
colonization, and ß-galactosidase activity.
visible lesions with
quantifiable
weight (24, 31).
and
previously,
severity rating,
estimated disease
adhering
planted.
biosynthesis
determined.
Disease
absence of
added. Cucumber and maize
was
inoculated with bacteria and
DAPG
of autoclaved millet
amount
same
affecting
not
shown).
Control treatments
background activity
with paper
tissues, and
o
weighed. ß-Galactosidase
calculated
according to
1000
Miller
(32)
[OD420 / (time [min]
x
Plasmid
determined
activities
stability
were
with the
x
sample
and inoculum
by replica plating
expressed
as
following
volume
units per 10
were
formula:
[ml])]
=
activity. Stability
onto KB agar
CFU. Unit values
supplemented
units of
ß-galactosidase
of plasmid
with
pME6259
tetracycline (125
was
ug
ml"1).
After 48 h at 27°C, the percentage of tetracycline-resistant colonies indicative of pME6259
was
evaluated.
experiments
ß-Galactosidase activity
was
determined.
Extraction and
in situ
quantification
ß-galactosidase activity
cucumber
seedlings
were
and
plants
experiments
After 10
days
treatment
colonization. All
with in situ
gnotobiotic
in the
of DAPG,
rhizosphere.
pregerminated
plants
weighed
were
Experiments
maize and
CHA0/pME6259
and
plants.
with maize consisted of 150
Each flask contained five
plants,
plants.
removed from the flasks. Plants from four flasks per
and assessed for
remaining plants
To compare
conditions. Control treatments consisted of maize
with cucumber consisted of 230
were
produced
production
grown without bacteria.
of growth,
(n=20)
of DAPG
inoculated with Pseudomonas fluorescens
grown in artificial soil under
and cucumber
of the bacterial inoculum used for in vitro and in situ
were
ß-galactosidase activity
and root
used for direct extraction of DAPG
37
(maize
n
=
130,
Chapter 2
cucumber
Biotic factors
n
210).
=
One
with 250-ml volumes of
for 30 min at 200 rpm. Soil and roots
acidified to
were
through hydrophobic
filter paper
washed and
The extract solvent
weighed.
Keel et al.
packed
column
The recovery
(21).
by adding synthetic
either
using
were
Systat
(SAS Institute, Cary, NC).
for final
further
protected (P
no
data
=
least
0.05)
2 with 2 M
evaporated
analyzed
soil
in
for DAPG
was
soil without bacteria
for
a
two to
quantity by
as
plants (data
three times. For each
or
was
a
described in
not
estimated
shown).
experiment,
analysis
of
PC SAS version 8.1
detected, data
were
pooled
for individual trials. In multifactorial
different
appropriate,
difference
re-
HPLC with
approximately 45%,
or
were
and the residue
growth ages), significant interactions
the influence of each level of the main effects
significant
mixed
Roots and shoots
trial-treatment interaction with
presented
at
were
extraction solution
vacuo
(Systat Inc., Evanston, IL)
species
When
procedure
rhizosphere
HCl, and vigorously shaken
by passing the
trial-treatment interaction
are
analyzed by examining
ages).
was
repeated
were
version 9.0
When
phase
for this
analyzed
effect of plant
maize at each of three
rhizosphere
mm) (Macherey-Nagel, Düren, Germany)
gnotobiotic
first
analysis. Otherwise,
experiments (e.g.,
were
4
x
experiments
data from all the trials
removed
were
efficiency
DAPG to
Statistics. All
variance
(250
pH
in the
biosynthesis
(Macherey-Nagel, Düren, Germany).
dissolved in 1 ml of methanol. Extracts
Nucleosil
DAPG
of roots with
hundred-gram portions
ethyl acetate,
affecting
means were
(LSD)
separated using
(e.g.,
Fisher's
test.
Results
Plasmid
on
grown in
plasmid pME6259 by
LB, which
was
was
expressed
used for inoculum
at
in inoculum. In the in vitro
strain CHAO
planta experiments,
the viable bacteria reisolated from the
amended agar. The reporter gene
was
ß-galactosidase activity
of growth in LB broth. In the in
generations
1.0%, based
and residual
retention of the reporter
experiments,
after 20
stability
rhizosphere
was
98.8 ± 0.9%
retention
that grew
low levels when strain
on
was
99.3 ±
antibiotic-
CHA0/pME6259
preparation. ß-Galactosidase activity
was
less
o
than 3 units per 10
CHA0/pME6259
the levels of
CFU detected in the
overnight
cultures of Pseudomonas fluorescens
used to inoculate either KB broth cultures
activity
that
were
detected reflect gene
experiments.
38
or
pregerminated plantlets. Thus,
activity during
the
course
of the
Chapter 2
Biotic factors
affecting
DAPG
biosynthesis
in the
rhizosphere
Fig. 1. Production of 2,4-diacetylphloroglucinol
(DAPG) and expression of a translational phlA'lacZ fusion in Pseudomonas fluorescens CHAO.
Strain
CHA0/pME6259
was
cultured in
medium broth at 27°C.
King's
taken
B
Samples
period of 7 days to A, determine bacterial
density (log CFU/ml), B, ß-galactosidase activity,
and C, DAPG production. Means ± standard
deviation from four experiments are shown. Some
of the error bars are too small to be distinguished.
were
over
a
40
80
Time
120
160
(hours)
Correlation of phlA'-'lacZ reporter gene
vitro.
Expression
levels of the
paralleled production
in KB broth
activity,
plasmid-borne
of DAPG
(Fig. 1). Reporter
and
peaked during
late
with DAPG
phlA'-'lacZ
determined
were
as
units of
first detected
exponential growth phase.
production
gene fusion
of Pseudomonas fluorescens
expression,
and accumulation of DAPG in media
growth phase
translational
during growth
gene
expression
in
directly
CHA0/pME6259
ß-galactosidase
during mid-exponential
The maximum concentration
o
of DAPG recovered from cultures
was
10.3 nmol per 10
CFU after 25 h
growth,
and the
o
maximum
ß-galactosidase activity
DAPG concentrations
steadily
was
5,600 units per
declined because of
39
10
CFU at 22 h
degradation by
growth. Thereafter,
the bacterium
(Fig. 1).
Chapter 2
Biotic factors
250
affecting
DAPG
biosynthesis
in the
rhizosphere
14
A
12
200
10
150
b
100
'-
50
a
a
0
8
b
ii
6
4
2
**
9^n
x
B
200
14
*
Ö
12
?
10
^<,
150
1
b
100
—
a
50
m
0
-
8
b
CO
b
E
II
6
o
8
4
£
£
2
Q.
</>
o
N
9^n
—
£
c
-
b
I
:
1
100
0
F
b
10
-
-
50
14
12
200
150
14.
a:
a
1
-
c
II
/
<b
8
c
i
6
2
^v
«"
<
ci>
x • ^
»2»
Fig. 2. Expression of a phlA'-'lacZ fusion in Pseudomonas fluorescens CHAO harboring plasmid
pME6259 in the rhizosphere of bean, cucumber, maize, and wheat. Plants were grown under
gnotobiotic conditions in artificial soil, and roots were assessed for ß-galactosidase activity and root
colonization after A and D, 3 days, B and E, 5 days, and C and F, 10 days. Each bar is the mean of
four independent experiments with four repetitions each. Bars with a common letter are not significantly
different according to Fisher's protected least significant difference test at P= 0.05.
40
Chapter 2
Biotic factors
Influence of
plant species
the level of reporter gene
When maize and wheat
example,
in the
after
days
at 3
were
compared,
levels of gene
CFU
higher
on
DAPG
There
maize and 90 units per 10
Q
than the level of gene
expression
CFU
measured
species
tested
(P
<
between monocots and dicots.
split
expression
the two
on
rhizosphere
dramatic difference in
was a
similar
were
(Fig.
was
approximately
dicots, which
C).
2A to
100 units
approximately
was
wheat. This
on
in the
biosynthesis
of the four crop
planting, ß-galactosidase activity
O
per 10
rhizospheres
Almost all of this difference could be
0.0001) (Fig. 2).
For
phlA expression.
on
expression
affecting
fourfold
was
o
approximately
though,
25 units per 10
CFU for either
There
species (Fig. 2).
was no
difference,
between bean and cucumber.
600
25
n
N
C
?n
-—*
U)
o
3
o
15
LL.
Ü
O
CI)
CO
CD
10
O
Q.
*—"
CO
5
O
N
0
.C
Ë
10
Time
15
10
15
Time
(days)
20
(days)
plant age on the expression of a phlA'-'lacZ fusion in Pseudomonas
CHA0/pME6259. Plants were grown in artificial soil and under gnotobiotic conditions in
Erlenmeyer flasks. Samples were taken at 3 h and 1, 3, 5, 7, 10, 14, 21 days after planting. Values
Fig.
3. Effect of maize
fluorescens
represent the
mean
of four flasks with ± standard deviation of the
Influence of
i.e.,
in the maize
expression (P
=
=
plant
age
rhizosphere,
0.009).
on
phlA expression.
there
was a
In
significant
For the other three
species,
one
mean.
experiment previously described,
influence of plant age
no
significant
on
age effect
0.561, 0.089, and 0.447 for wheat, bean and cucumber, respectively).
In
reporter gene
was
observed
(P
maize, ß-
o
galactosidase activity
doubled from
approximately
100 units per 10
CFU at 3 and 5
days
to
o
approximately
210 units per 10
CFU at 10
difference between maize and the other
Because the effect of plant age
CHA0/pME6259
although
gene
expression
gene
expression
on
after
days
plant species
was
this
most
was
over a
seedling
Not
surprisingly,
also greatest at 10
evident with
species
levels observed after
planting (Fig. 2).
maize,
we
the
days.
examined
longer growth period. Interestingly,
emergence
were
similar to those
o
observed in the
previous experiment (approximately
effect of age occurred
even
earlier in
plant growth.
100 units per 10
There
was a
rapid
CFU),
the greatest
increase in
ß-
o
galactosidase activity early
in
growth
that
peaked
at over 400
units per 10
CFU at 1
day
after
o
planting (Fig. 3).
Then
activity just
as
rapidly
declined to
41
approximately
70 units per 10
CFU
Chapter 2
by
Biotic factors
the next
sampling
of plant
throughout 22 days
expression
in the
days
planting,
108
in
activity
was
essentially
at
resulted
general
solely
we
differences in bacterial gene
difference
variation
mphlA
below 10
background levels,
phlA expression.
on
Maize
was
among six maize
expression
used
as a
'-
'lacZ
was
N4660
observed among the conventional
expression
transgenic N4660
on
Bt
model to
significant
from Switzerland
hybrids
Corso, and Magister) and the United States (Max 88, N4660, and
large
from phlA gene
sampled seedlings just
further examine the effect of host genotype at the cultivar level. We found
Whereas
level
(Fig. 3).
CFU
Influence of maize cultivar
4).
rhizosphere
negligible background activity
When
pregerminated seedlings.
to
in the
biosynthesis
and remained at that
early peak
planting, ß-galactosidase activity
units per
DAPG
and cannot be attributed to the
applied
was
after
This
growth.
rhizosphere,
in the inoculum that
3 h after
taken at 3
affecting
Bt) (P
=
cultivars, there
compared
with the
(Antares,
0.009) (Fig.
was no
near-isogenic
parent line N4660 (Fig. 4).
?
^ ,/
^
•
^
J
&
*
4
4?
^
J
<$f
<F
Fig. 4. Effect of maize cultivar on bacterial gene expression and rhizosphere colonization. Plants were
grown under gnotobiotic conditions in artificial soil and roots were assessed for ß-galactosidase
activity and root colonization after 6 days. Each bar represents the mean of eight replicate flasks with
five
seedlings each. Bars with a common letter
protected least significant difference test at P
are
=
Influence of
CHA0/pME6259
pathogen
was
monitored in the
infested with
Pythium
significantly
increased
sixfold
compared
stimulated
(Table 2).
infection
on
significantly
different
ß-galactosidase activity
in the
Whereas the increase in gene
rhizosphere
to Fisher's
(P
in the
<
expression
(P
<
42
Pathogen
in
expression
rhizosphere
of maize
on
by
of cucumber
cucumber
two to
was
0.031) (Table 1),
no
was
infection
0.047) (Table 1). Likewise,
rhizosphere
colonization
gene
of plants grown in artificial soil that
ultimum and in noninfested artificial soil.
ß-galactosidase activity
according
phlA expression. Reporter
rhizospheres
with noninfected controls
20-fold increase in
not
0.05.
by
three to
infection
fivefold
(P
<
accompanied by
such
0.003)
a
relationship
7- to
was
-P>.
a
of
seedlings (%)
per
CFU)
x
g"1)
seedlings (%)
z
y
x
w
per
(x108
CFU
x
g"1)y
64a
c
nd
nd
70 b
77
14c
+
-
131 7b
38 0 b
80 b
113c
19c
+
+
a
nd
ndz
100
6 81
24
100
690
347
a
a
a
a
a
1
nd
nd
100
a
408 b
127 b
Experiment
a
7 56
a
119 b
100
452 b
161b
in
in
the
a
nd
nd
100
531
275
a
716a
94a
100
367 b
89 b
-
+
Expen
c
c
nd
nd
65 b
107
28
+
-
nent 2
a
c
c
52 8 b
51 1 b
85
152
41
+
+
rhizosphere during damping-off
nd
nd
100
511
140
-
-
caused
a
a
a
a
a
a
a
a
a
a
8 59
45
100
511a
246
2
nd
nd
80 b
240 b
84 b
Experiment
a
8 95
a
106 b
100
347 ab
146 b
nd
nd
100
718
355
a
a
a
a
a
a
nd
nd
a
a
a
nd
nd
90 b
344 b
82 b
3
difference test
a
a
9 43
14
100
674
319
Experiment
w
90 b
59 b
15b
+
-
ultimum
45a
27a
100
469
125
-
+
3
ultimum
Experiment
by Pythium
rhizosDhere durina damDina-off caused bv Pvthium
a
maize
nd
nd
100
487a
123
-
-
the cucumber
CHA0/pME6259, a derivative of wild-type P fluorescens strain CHAO carrying a phlA '-'lacZ translational fusion on plasmid pME6259 (43)
same experiment within the same row followed by a different letter are significantly different at P < 0 05 according to Fisher's least significant
nd indicates that neither ß-galactosidase activity nor P fluorescens CHA0/pME6259 were detected
Plants were grown for 7 days under gnotobiotic conditions in artificial soil with or without the addition of P ultimum Fresh weight per plant
Percentage of emerged seedlings (100% =five plants)
ß-Galactosidase activity is indicated per 108 CFU
colonization
Means for the
Strain
Rhizosphere
ß-Galactosidase activity (units
of
735
weight (mg)x
Plant fresh
a
381a
Emergence
a
108a
100
420 b
99 b
-
+
Pseudomonas fluorescens strain CHA0/dME6259
nd
weight (mg)x
y
in
108 CFU)z
ohIA'-'lacZ fusion
CFU
a
ndz
100
Root fresh
P ultimum
a
(x108
CHA0/pME6259
ExDression of
colonization
P fluorescens
Table 2
Rhizosphere
ß-Galactosidase activity (units
Emergence
519a
weight (mg)x
a
Plant fresh
-
-
1
CHA0/pME6259
Experiment
Pseudomonas fluorescens strain
134
'
in
weight (mg)
y
fusion
108
phlA'-'lacZ
Root fresh
P ultimum
of
CHA0/pME6259
Expression
P fluorescens
Table 1
a
c
a
9 86
a
41 b
100
354 b
124 b
87 0 b
101 b
100
132
26 b
+
+
Chapter 2
observed
Pythium
Biotic factors
on
maize where bacterial colonization
ultimum
(P
reductions in root and
of cucumber
growth
Despite
plant
fresh
(Table 1)
consistent increases
both cucumber and
applied
to roots
seedling
affected than
severely
'lacZ reporter gene
'-
increased
slightly
(Table 1)
or
maize
in any trial for cucumber
caused
of maize
growth
expression
seedling
in the
as
(Table 2).
(Table 2).
or
Root fresh
(Table 1).
of Pythium ultimum
and those observed with the
background ß-galactosidase activity
Correlation of phlA
'-
rhizosphere.
We monitored gene
antimicrobial
compound
from the
on
growth
on
(data
in
one
weight
cucumber
of
seedling
of maize in the absence
reported
here for
as
controls
with DAPG
production
in the
with direct extraction of the
parallel
of
rhizospheres
not
shown).
not
expression
fresh
strain CHAO used
wild-type parent
measurement
expression
weight was
plant
adverse effect
was no
'lacZ reporter gene
in two of
pathogen, CHA0/pME6259
differences in the effects
were no
only
was
maize, and plant fresh weight was only
such adverse effect
no
There
(Table 2).
There
of
rhizospheres
emergence, but
reduction in root and
slight but significant
a
emergence. Bacterial treatment had
CHA0/pME6259
absence of
or
Clearly though,
emergence.
of three trials for cucumber. In the absence of the
cucumber in two of three trials
for
in the presence
same
rhizosphere
maize, the level of plant protection afforded by CHA0/pME6259
significantly
one
in the
of both maize and cucumber measured
and reduced
weights,
mphlA
three trials for either cucumber
increased in
growth
was more
inconsistent. Bacterial treatment
increased
the
was
biosynthesis
0.575) (Table 2).
>
infection reduced
Pathogen
DAPG
affecting
monocot
and
one
dicot.
ßo
Galactosidase
activity
of strain
CHA0/pME6259 ranged
between 185 and 229 units per 10
o
CFU in the maize
rhizosphere
rhizosphere (Table 3).
and between 11 and 22 units per 10
In the maize
detected at levels of 11.8 to 12.0
rhizosphere,
nmol/g
root.
produced by CHA0/pME6259
DAPG
Adjusted
CFU in the cucumber
for bacterial
colonization, this
was
was
o
equivalent
DAPG
to 0.60 to 0.69
was
nmol per 10
detected. DAPG
was
CFU
(Table 3).
also not detected in
In the cucumber
plants
rhizosphere,
no
without bacteria added. Our
o
detection limits for DAPG
fresh
weights
were
inoculation had
not
added
no
were
0.14
on
plant
fresh
rhizosphere
weights
colonization.
densities ofPseudomonas fluorescens
on
and 0.07 nmol per 10
CFU.
Average plant
in these
experiments,
where
pathogens
were
(Table 3).
Bacterial
than
root
1.2 g for maize and 0.72 g for cucumber. Bacterial
approximately
effect
nmol/g
dicots. For
example,
in the
Throughout
CHA0/pME6259
experiment
with
44
our
were
study, rhizosphere population
generally higher
parallel monitoring
on
of gene
monocots
expression
en
z
y
x
by
a
production
of
1
a
different letter
22 b
nd
229
ndw
Expt.
are
2
a
2
<0.07
nd
0.596
nd
Expt.
y
1
<0.14
nd
11.78
nd
Expt.
according
<0.14
nd
11.97
nd
Expt.
2
in the
g of root
different at P< 0.05
<0.07
nd
0.691
nd
1
108CFU
Expt.
significantly
11 b
nd
185
nd
Expt.
(units per 10£ CFU)X
nmol of DAPG per
2,4-diacetylphloroglucinol (DAPG)
ß-Galactosidase activity
assay and
1
a
a
a
2
a
1
g"1)
a
2
2.97 b
nd
14.52
nd
Expt.
CFU
difference test.
2.77 b
nd
13.56
nd
Expt.
(x 108
Root colonization
significant
743 b
729 b
1218
1215
Expt.
to Fisher's least
757 b
716 b
1279
1198a
Expt.
(mg)z
weight
of maize and cucumberu
Plant fresh
rhizosphere
ß-galactosidase activity nor DAPG nor P. fluorescens CHA0/pME6259 were detected.
Plants were grown for 10 days under gnotobiotic conditions in artificial soil.
Strain CHA0/pME6259, a derivative of wild-type P. fluorescens strain CHAO carrying a phlA'-'lacZ translational fusion on plasmid pME6259 (43)
ß-Galactosidase activity is indicated per 108 CFU. Expt. 1 and Expt. 2 are two independent experiments.
DAPG was extracted from the roots and the adhering artificial soil from 130 (maize) or 210 (cucumber) plants per treatment.
Fresh weight per plant.
nd indicates that neither
Means within columns followed
CHA0/pME6259
Cucumber
w
v
u
None
Cucumber
CHA0/pME6259
w
Maize
P. fluorescens
None
v
ß-galactosidase
Maize
Plant species
Table 3. Parallel
Chapter 2
Biotic factors
and DAPG
after 7
days,
and
colonization
There
days growth (Table 3).
and
species
3
production,
plant
age
in
as seen
colonization of maize
wheat
only
2A). By
was
5 and 10
0.0001) (Fig.
<
<
2B and
cucumber
on
between
same as
was
rhizosphere
plant
After
0.0001) (Fig. 2).
both dicots
colonization of either monocot
(P
maize than
on
gnotobiotic experiment (P
were
in the
biosynthesis
greater than that of bean but the
bean
or
CFU greater
significantly better than
days, however,
colonization of cucumber
log
1
DAPG
significant interaction, though,
was a
initial
our
was
colonized
was over
affecting
(P
that of
<
cucumber,
0.0001) (Fig.
significantly greater than
C).
Discussion
phlA
Our reporter gene system,
fluorescens phi gene expression
with DAPG extracted from the
that gene
expression
direct evidence for
maize
production
able to
production
predict
DAPG
of phenazine,
rhizospheres
concern
activity
we were
in the
production
of cotton,
at
procedures
pyoluteorin
cucumber,
time
study
ß-galactosidase
course
experiment
reflected
and
temporal
production
degrades ß-galactosidase,
strain CHAO to
levels
with
gene
and
a
This is also the first
biocontrol strain in the
pyoluteorin
have
rhizosphere,
our
previously
reporter gene
difficult and laborious. As
technically
oomycin
A
and
lacZ, have been used
product
to
production by Pseudomonas
should not be
dramatically
rose
overly stable,
expression
and then
expression.
Previous in vitro
suggested
but that the enzyme is
'-
DAPG
monitor
spp. in the
One
otherwise
of the target gene
gradually
maize, indicates that the phlA
by
an
beat, tomato, and wheat (6, 16, 20, 25, 51).
of DAPG have
fell from
day
(27).
to
day
'lacZ reporter gene used in
experiments comparing
gene
that the bacterium itself gradually
more
stable than DAPG, which is
degraded
monoacetylphloroglucinol (43).
Host genotype at the
species
level had
a
dramatic effect
the greatest differences observed between monocot and dicot
was
by
show, for the first time,
levels below that which could be measured
are
sugar
and
In the cucumber
including lux, inaZ,
genes,
able to
rhizosphere.
antimicrobial metabolite
an
levels will reflect cumulative rather than current
expression
by
of maize,
biosynthesis
with any reporter gene is that the
The fact that
this
rhizosphere. By comparing ß-galactosidase activity
(4, 21, 29, 39, 49).
alternative, various reporter
our
of
sensitive tool to monitor Pseudomonas
was a
rhizosphere. DAPG, phenazine-1-carboxylic acid,
extraction. Direct isolation
in
'lacZ,
rhizosphere
reflected DAPG
been isolated from wheat
was
in the
'-
consistently two
to
fourfold
higher
in the
rhizospheres
46
on
phlA
species.
gene
expression,
DAPG gene
with
expression
of maize and wheat than with bean
Chapter 2
Biotic factors
and cucumber in
expression
beet.
of
our
experiments. Georgakopoulos
phenazine biosynthetic
Interestingly, greater
effective
protection
different host
of monocots
susceptibilities
to
versus
that
a
threshold level
was
as
quantities
the
dicots,
obtained, and that
antimicrobial
compound over-producing transgenic
no
superior
disease
by CHAO,
thus does not offer
observed after inoculation with
CHAO
produces
extraordinary
an
which may exhibit
were
observed with
of each
(29, 30)
(Tables
among cultivars of the
model,
we
can
1 and
activity
found
preparation
study
same
to
traditional
and it
previously
slight but significant
wild-type.
which is also
the
same
plasmid.
effects
produced
we
several of
with
on
plasmid
cucumber
Inconsistent
CHA0/pME6259 might
programs
wild-type
been
offer
differences
emphasizes
breeding
optimal
(e.g., 'Magister'
N4660 and the
influence
reported
that while there
the cry 1Ab insecticidal gene from B.
an
production
might
varieties that support
noteworthy,
breeding
observed between
the
inhibition of cucumber
used to minimize phi gene
show that
species,
that has
selecting
bacteria. It is also
to
an
phytotoxic
was
secondary metabolites,
carrying
strain
2) by
interactions with biocontrol agents in crop
for
compared
pyoluteorin,
be ruled out, because the
CHAO not
Swiss maize cultivars. Our work further
approach
who found that
protection
be due to the
expression
at
the
plant experiment.
this is the first
in biocontrol
effects of the
cucumber, and it provided
slight growth
Host genotype at the cultivar level also had
knowledge,
to
or
array of antimicrobial
wild-type
method of inoculum
beginning
achieved with
reporter strain in the absence of Pythium (Table 1). Strain
reporter gene
our
of cucumber and maize
special
for
was
phytotoxicity, including hydrogen cyanide (8). Any problems
pME6259 carrying
growth
our
more
poor distribution of the
derivative of strain CHAO
phytotoxic
not
explanation
an
and sugar
cotton
out-weighed by phytotoxic
from DAPG
protection. Phytotoxicity
slightly higher
expected. Although
further benefit
Maurhofer et al.
supported by
over-producer was
rhizosphere
did not appear to translate into
maize and did not offer enhanced biocontrol of Pythium root rot
In contrast, the DAPG
in the
in the infection court may be involved. It is also
This
a
on
biosynthesis
also found
(16)
partly explain this,
extra DAPG.
to
is
DAPG
may have been
additional DAPG. Rather the benefits may have been
hypothesis
al.
production
dicots,
disease may
et
wheat than
on
genes
levels of antibiotic
enhanced antimicrobial metabolite
possible
affecting
were
vs.
of
one
an
on
gene
antimicrobial
47
To
compound
plausible explanation
(46). Using
'lacZ
on
mphlA
the
'-
expression
importance
of
our
varies
for variation
between cultivars
maize
as
six U.S. and
considering
programs, and offers
a
screening
antimicrobial metabolite
production by
differences among cultivars from
'Antares'),
no
such difference
near-isogenic transgenic
thuringiensis.
expression.
A recent note
was
line N4660 Bt
reporting
carrying
release of cry 1Ab
Chapter 2
Biotic factors
into the
by N4660
impact.
To
our
rhizosphere (42)
Bt maize in the
transgenic
raised considerable
this is the first
knowledge,
affecting
study
and
rhizosphere,
concern
in the
biosynthesis
rhizosphere
about unforeseen nontarget
address microbial interactions with
to
found
we
DAPG
no
evidence for any
impact, negative
or
positive.
The most obvious
species
explanation
for differences in DAPG
and crop cultivars is differences in the
Production of antimicrobial
abundantly
Rhizosphere pH,
metabolite
plant
which
production
exudates
root
in
can
root
can
and
be very
a
Elsas
(50)
presence of wheat,
(33) reported
after
days
Maize root exudates
reported
to
(2).
in Pseudomonas fluorescens R2f to
also has been
provided by
(lacZ transcriptional fusion)
exudates from
gene
mutants
expression
Overbeek and
van
of strain R2f for
was
enhanced in the
plants growing
production by B.
cereus
under natural conditions
problematic.
influence
on
DAPG gene
We observed very little difference for
whereas
planting,
were
in Pseudomonas fluorescens M.3.1. in vitro
expression
of
composition
grass roots but not in the presence of clover roots. Milner et al.
though, obtaining
an
The
produced (36, 45, 52).
exudates, and found that reporter
maize, and
Plant age had
dependent.
exudates, also affects bacterial antimicrobial
once
dicotyledonous plants
in vitro remains
testing
root
the total
(10, 14, 33, 34, 44).
that alfalfa sprout exudates stimulated zwittermicin A
UW85. In the end
for
production
who screened Tn5-B20
their response to root
by
by
acids, and other compounds
in bulk soil
lacking
species specific (7, 17, 37).
distinct response of gene
exudates of mono- and
van
be modified
possibly stability
enhance indole-acetic acid
Evidence for
exudates but
amino
nitrogen,
plant
of root exudates.
quality
or
between
biocontrol bacteria is modulated
compounds by
concentration and the type of carbon source,
found
and
quantity
production
expression
on
maize
but this
expression,
bean, cucumber,
or
approximately
was
was
host-species
wheat at 3, 5, and 10
doubled between 5 and
o
10
days,
was a
more
from 93 to 202
relatively
closely
small
ß-galactosidase
change compared
units per 10
CFU in
one
with that observed in
examined the effect of maize age. In this
a
experiment. However,
follow-up experiment
experiment,
this
that
expression
DAPG gene
o
rapidly
increased from nondetectable at
CFU at 1
day
after
planting.
Levels
planting
dropped by
3
to
peak
days
at 455
after
units per 10
ß-galactosidase
planting
to
approximately
50
ß-
o
galactosidase
that
units per 10
CFU, which
relatively large quantities
which may be essential for
onset
(28).
pathogen
of DAPG
was
can
stable for 20
be
controlling Pythium
days
produced early
root rot
of plant
on
during
(21)
can
be sustained
throughout plant growth,
48
the
This indicates
interaction,
and other disease that have
It also indicates that stable levels of antibiotic metabolite
inhibition
growth.
production
rapid
a
sufficient for
which may offer
longer
Chapter 2
term
Biotic factors
disease
DAPG
biosynthesis
in the
rhizosphere
There is evidence that the effect of plant age may be linked to
protection.
in root exudate
changes
affecting
and
quality
gene induction in Rhizobium
Richardson et al.
quantity (18, 47).
trifolii changed
with host
plant
(40)
found that nod
age.
Differential root colonization is another factor that may contribute to host genotype
variation in gene
can
vary in their
greatly
There is
expression.
ability
known that antimicrobial metabolite
sensing
to quorum
Autoinducing
oureofaciens
regulated by
mediated
by autoinducing
gene
peak
For
but there
in gene
as a
example,
wheat root
change
was no
expression early
on
in gene
least
direct effect
on
and cultivars
with
a
certain
This has been attributed
the bacteria.
produced by
CHAO, secondary metabolism is
a
cell-density-dependent manner (3),
autoinducer for the phi
explain
the differences
populations
were
lowest
on
bacterial gene
expression,
that time. In
'Antares', which
only
(43).
maize, there
was
an
after
days
rhizosphere populations
rather than
locus
observed for DAPG
we
decreased from 3 to 10
expression during
when
biosynthetic
were
was a
just
among the
levels among maize cultivars. This indicates that the host
expression
some
cell-density dependent,
up-regulated.
are
molecules
during growth,
increasing. Furthermore, populations
for gene
genes
In strain
positive
colonization alone does not
expression.
planting
be
can
the GacS/GacA two-component system in
root
species
positively regulate phenazine production by P.
rhizosphere (38).
and DAPG has been identified
However,
biosynthesis
biosynthetic
homoserine lactones
in the wheat
evidence that different
support bacterial rhizosphere populations (46). It is also
to
threshold of cells needed before
convincing
highest
had at
plant
indirect effect via
increased bacterial numbers.
Root infection with
cucumber and maize
by
ultimum stimulated phlA'-'lacZ gene
Pythium
two to
sixfold. Bull et al.
(5) reported
a
positive
expression
linear
between wheat root disease and bacterial root colonization. Mazzola and Cook
the
relationship
study changes
sizes. For
was
m
specific depending
phi gene expression
compounds
different
from
a
on
healthy
thoroughly
or
Our
study
CHA0/pME3090
not
were
always
a
was
on
spp.
(31)
found that
However, in
infected cucumber
devastated root system of cucumber
compounds
wild-type
of DAPG
Pythium
relationship
correlated with bacterial
increased
from
is the first to demonstrate that
critical biocontrol gene in
production
bacterial strain and
both
our
population
they
were
not
infected maize. It may be that the sudden release of plant
bacterium than the slower release of
that the
were
example, although populations
significantly
damage.
on
on
enhanced in the
pathogen
attack increases
our
antimicrobial metabolite
rhizosphere
49
beneficial to the
maize, which suffered less obvious
strain. These results support
by transgenic
was more
expression
previous
a
observations
over-producing
of wheat infected with
of
root
Pythium
strain
ultimum
Chapter 2
compared
ultimum
with
with
healthy plants (29).
Fedi et al.
mycelia repressed expression
DAPG
affecting
(13) reported
of 9 out of 5,000
biosynthesis
involved in
directly
that trehalose
another
and
rhizosphere
that contents leaked from
promoterless
genes
biological control, including
genes for DAPG
Pythium
randomly tagged
was a
Pythium-derived signal responsible
for
root-colonizing Pseudomonas fluorescens strain,
colonization. This offers
explanation
It also suggests, that in
Pythium.
probably
an
be attributed to
rather than to
a
direct
an
our
from the
Fusarium-Pseudomonas-tomato system
(15)
some
found evidence
trehalase genes in
and that these genes contribute to
for the link between bacterial root colonization
increased DAPG gene
study,
indirect effect of disease
signaling
stimulating
but
production,
of the genes may have been involved in root colonization. Gaballa et al.
root
in the
lacZ reporter gene in Pseudomonas fluorescens strain Fl 13. None of the affected genes
a
were
Biotic factors
pathogen,
(e.g.,
expression
can
increased release of root
which has
so
far
only
been
seen
exudates)
in the
(9).
Literature Cited
1.
Bangera,
for
M.
synthesis
G., and Thomashow, L. S. 1999. Identification and characterization of agene cluster
of the
fluorescens Q2-87.
2.
antibiotic
2,4-diacetylphloroglucinol
from Pseudomonas
J. Bacteriol. 181:3155-3163.
Benizri, E., Courtade, A., Picard, C, and Guckert, A. 1998. Role of maize
production
3.
polyketide
of auxins
by Pseudomonas fluorescens
root exudates in the
M.3.1. Soil Biol. Biochem. 30:1481-1484.
Blumer, C, Heeb, S., Pessi, G., and Haas, D. 1999. Global GacA-steered control of cyanide and
exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.
Proc. Natl. Acad. Sei. USA 96:14073-14078.
4.
Bonsall, R. F., Weller, D. M., and Thomashow, L. S. 1997. Quantification of 2,4-
diacetylphloroglucinol produced by
of wheat.
5.
79.
suppression ofGaeumannomyces graminis
Phytopathology
Chin-A-Woeng,
var. tritici
root colonization
by Pseudomonas fluorescens
strain 2-
81:954-959.
T. F.
C, Bloemberg, G. V., Van Der Bij, A. J., Van Der Drift, K. M. G. M.,
Schripsema, J., Kroon, B., Scheffer,
F.
rhizosphere
Environ. Microbiol. 63:951-955.
Bull, C. T., Weiler, D. M., and Thomashow, L. S. 1991. Relationship between
and
6.
Appl.
fluorescent Pseudomonas spp. in vitro and in the
R.
J., Keel, C, Bakker, P. A. H. M., Tichy, H. V., de Bruijn,
J., Thomas-Oates, J. E., and Lugtenberg, B. J. 1998. Biocontrol by phenazine-1-carboxamide-
producing
Pseudomonas
oxysporum f. sp.
chlororaphis
radicis-lycopersici.
PCL 1391 of tomato root rot caused
by Fusarium
Mol. Plant-Microbe Interact. 11:1069-1077.
50
Chapter 2
7.
Biotic factors
affecting
DAPG
biosynthesis
in the
rhizosphere
Cieslinski, G., Van-Rees, K. C. J., Szmigielska, A. M., and Huang, P. M. 1997. Low molecular
weight organic
acids released from roots of durum wheat and flax into sterile nutrient solutions. J.
Plant. Nutr. 20:753-764.
8.
and
Défago, G.,
borne
Keel, C. 1995. Pseudomonads
pathogens. Pages
as
biocontrol agents of diseases caused
137-148 in: Benefits and risks of introducing biocontrol
by
soil¬
agents. H.M.T.
Hokkanen, and J.M. Lynch, eds. Cambridge University Press, England.
9.
K., and Défago, G. 1997. Zinc improves biocontrol of Fusarium
Duffy,
B.
tomato
by Pseudomonas fluorescens
inhibitory to
10.
Duffy,
B.
bacterial antibiotic
and represses the
production
biosynthesis. Phytopathology
and root rot of
crown
of pathogen metabolites
87:1250-1257.
K., and Défago, G. 1999. Environmental factors modulating antibiotic and siderophore
biosynthesis by Pseudomonas fluorescens
biocontrol strains.
Appl.
Environ. Microbiol. 65:2429-
2438.
11.
Duffy,
B.
K., Ownley, B. H., and Weiler, D. M. 1997. Soil chemical and physical properties
associated with
suppression
of take-all of wheat
by
Trichoderma
koningu. Phytopathology
87:1118-1124.
12.
A. W. 1989. Soilborne
Engelhard,
microelements. American
13.
fluorescens
altered
signaling
between the
ecological
fitness.
Appl.
expression
macro-
and
Paul, MN.
of genes in P.
Appl.
ultimum and Pseudomonas
fluorescens
Fl
13, resulting in
Environ. Microbiol. 63:4261-4266.
Fiddaman, P. J., and Rossall, S. 1994. Effect of substrate
on
the
production
of antifungal volatiles
Bacteriol. 76:395-405.
Gaballa, A., Abeysinghe, P. D., Urich, G., Matthijs, S., De Grève, H., Cornells, P., and Koedam,
N. 1997. Trehalose induces
fluorescens
16.
St.
phytopathogenic fungus Pythium
Fl 13: P. ultimum represses the
from Bacillus subtihs. J.
15.
Phytopathological Society,
of diseases with
Fedi, S., Tola, E., Moënne-Loccoz, Y., Dowling, D. N., Smith, L. M., and O'Gara, F. 1997.
Evidence for
14.
plant pathogens: management
ATCC 17400.
Georgakopoulos,
expression
a
mutant
of a
D.
antagonism towards Pythium debaryanum
Appl.
Environ. Microbiol. 63:4340-4345.
G., Hendson, M., Panopoulos, N. J., and Schroth, M. N. 1994. Analysis of
phenazine biosynthesis
carrying
a
in Pseudomonas
locus of Pseudomonas
phenazine biosynthesis
aureofaciens
PGS12
on
locus-ice nucleation reporter gene fusion.
seeds with
Appl.
Environ. Microbiol. 60:4573-4579.
17.
Guckert, A., Chavanon, M., Mench, M., Morel, J. L., and Villemin, G. 1991. Root exudation in
Beta
18.
vulgaris:
a
comparison
with Zea mays. Dev.
Agric.
Man. For. Ecol. 24:449-455.
Haller, T., and Stolp, H. 1985. Quantitative estimation of root exudation of maize plants. Plant
Soil 86:207-216.
19.
Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C, Wade, J., Walsh, U., O'Gara, F., and Haas,
D. 2000. Small stable shuttle vectors based
negative, plant-associated bacteria.
on
the minimal
pVSl replicon
for
Mol. Plant-Microbe Interact. 13:232-237.
51
use
in gram-
Chapter 2
20.
Biotic factors
affecting
DAPG
biosynthesis
in the
rhizosphere
Howie, W. J., and Suslow, T. V. 1991. Role of antibiotic biosynthesis in the inhibition of Pythium
ultimum in the cotton
and
spermosphere
rhizosphere by Pseudomonas fluorescens.
Mol. Plant-
Microbe Interact. 4:393-399.
21.
Keel, C, Schnider, U., Maurhofer, M., Voisard, C, Laville, J., Burger, U., Wirthner, P., Haas, D.,
and
G. 1992.
Défago,
Suppression
of root diseases
by Pseudomonas fluorescens
CHAO:
Importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol. Plant-Microbe
Interact. 5:4-13.
22.
Keel, C, Voisard, C, Berling, C. H., Kahr, G., and Défago, G. 1989. Iron sufficiency,
prerequisite
for the
CHAO under
23.
King,
E.
gnotobiotic
conditions.
strain
79:584-589.
and fluorescin. J. Lab. Clin. Med. 44:301-307.
Kraus, J., and Loper, J. E. 1992. Lack of evidence for
by Pseudomonas fluorescens
Phytopathology
25.
Phytopathology
by Pseudomonas fluorescens
O., Ward, M. K., and Raney, D. E. 1954. Two simple media for the demonstration of
pyocyanin
24.
of tobacco black root rot
suppression
a
Pf-5 in
biological
a
control
role of antifungal metabolite
production
ofPythium damping-off of cucumber.
82:264-271.
Kraus, J., and Loper, J. E. 1995. Characterization of a genomic region required for production of
the antibiotic
pyoluteorin by the biological
control agent Pseudomonas fluorescens Pf-5.
Appl.
Environ. Microbiol. 61:849-854.
26.
Lemanceau, P., and Alabouvette, C. 1993. Suppression of Fusarium wilts by fluorescent
pseudomonads:
27.
Loper,
J.
Mechanisms and
Biocontrol Sei. Technol. 3:219-234.
applications.
E., and Lindow, S. E. 1997. Reporter
expression by
soil- and
microbiology.
C. J. Hurst, G. R.
plant-associated
gene
bacteria.
systems useful in evaluating in situ gene
Pages
482-492 in: Manual of environmental
Knudsen, M. J. Mc Inervey, L. D. Stetzenbach, and M. V.
Walter, eds. ASM Press, Washington DC.
28.
Martin, F. N., and Loper, J. E. 1999. Soilborne plant diseases caused by Pythium
epidemiology,
29.
and prospects for
biological
spp.:
control. Crit. Rev. Plant Sei. 18:111-181.
Maurhofer, M., Keel, C, Haas, D., and Défago, G. 1995. Influence of plant species
suppression by Pseudomonas fluorescens
Ecology,
strain CHAO with enhanced antibiotic
on
disease
production.
Plant
Pathol. 44:40-50.
30.
Maurhofer, M., Keel, C, Schnider, U., Voisard, C, Haas, D., and Défago, G. 1992. Influence of
enhanced antibiotic
production
in Pseudomonas fluorescens strain CHAO
suppressive capacity. Phytopathology
31.
its disease
82:190-195.
Mazzola, M., and Cook, R. J. 1991. Effects of fungal
of biocontrol strains of fluorescent
on
pseudomonads
root
pathogens
in the wheat
on
the
population dynamics
rhizosphere. Appl.
Environ.
Microbiol. 57:2171-2178.
32.
Miller, J. H. 1992. A Short Course in Bacterial Genetics: A laboratory manual and handbook for
Escherichia coli and related bacteria. Cold
Spring
52
Harbor
Laboratory Press, Plainview,
NY.
Chapter 2
33.
Biotic factors
affecting
DAPG
biosynthesis
in the
rhizosphere
Milner, J. L., Raffel, S. J., Lethbridge, B. J., and Handelsman, J. 1995. Culture conditions that
influence accumulation of zwittermicin A
by Bacillus
cereus
UW85.
Appl.
Microbiol. Biotechnol.
43:685-691.
34.
35.
Milner, J. L., Silo-Suh, L., Lee, J. C, He, H., Clardy, J., and Handelsman, J. 1996. Production of
kanosamine
by Bacillus
Ownley,
H., Weiler, D. M., and Alldredge, J. R. 1991. Relation of soil chemical and physical
B.
factors with
cereus
suppression
UW85.
of take-all
Appl.
Environ. Microbiol. 62:3061-3065.
by Pseudomonas fluorescens
2-79. IOBC/WPRS Bull. 14:299-
301.
36.
Ownley,
H., Weiler, D. M., and Thomashow, L. S. 1992. Influence of in situ and in vitro pH
B.
of
suppression
Gaeumannomy ce s
Phytopathology
37.
graminis
var. tritici
by Pseudomonas fluorescens
on
2-79.
82:178-184.
Pérez, F. J., and Ormeno-Nunez, J. 1991. Difference in hydroxamic acid
exudates of wheat
(Triticum
aestivum
L.) and
rye
(Secale
céréale
content in roots and root
L.): Possible role
in
allelopathy.
J. Chem. Ecol. 17:1037-1043.
38.
Pierson, L. S., Ill, Wood, D. W., Pierson, E. A., and Chancey, S. T. 1998. TV-acyl-homoserine
lactone-mediated gene
Raaijmakers,
J.
Phytopathology
fluorescent
pseudomonads:
Current
on
production
of 2,4-diacetylphloroglucinol in the
rhizosphere
of wheat.
on
the exudation from clover
of nodulation genes in Rhizobium
trifoln.
seedlings
of compounds that induced the
Plant Soil 109:37-47.
Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular cloning: A laboratory manual, 2n
ed. Cold
Spring
Harbor
Laboratory Press,
Cold
Spring Harbor,
NY.
Saxena, D., Flores, S., and Stotzky, G. 1999. Transgenic plants: Insecticidal toxin in
from Bt
43.
by
Richardson, A. E., Djordevic, M. A., Rolfe, B. G., and Simpson, R. J. 1988. Effects of pH,
expression
42.
control
89:470-475.
calcium and aluminum
41.
biological
M., Bonsall, R. F., and Weiler, D. M. 1999. Effect of population density of
Pseudomonas fluorescens
40.
in
and future work. Eur. J. Plant Pathol. 104:1-9.
knowledge
39.
regulation
corn.
root exudates
Nature 402:480.
Schnider-Keel, U., Seematter, A., Maurhofer, M., Blumer, C, Duffy, B., Gigot-Bonnefoy, C,
Reimmann, C, Notz, R., Défago, G., Haas, D., and Keel, C. 2000. Autoinduction of 2,4-
diacetylphloroglucinol biosynthesis
repression by the
44.
Slininger,
P.
of phenazine
in the biocontrol agent Pseudomonas fluorescens CHAO and
bacterial metabolites
salicylate
and
pyoluteorin.
J. Bacteriol. 182:1215-1225.
J., and Jackson, M. A. 1992. Nutritional factors regulating growth and accumulation
1-carboxylic
acid
by Pseudomonas fluorescens
2-79.
Appl.
Microbiol. Biotechnol.
37:388-392.
45.
Slininger,
nitrogen)
P.
J., and Shea-Wilbur, M. A. 1995. Liquid-culture pH, temperature, and carbon (not
source
fluorescens
2-79.
regulate phenazine productivity
Appl.
of the take-all biocontrol agent Pseudomonas
Microbiol. Biotechnol. 43:794-800.
53
Chapter 2
46.
Biotic factors
Soejima, H., Sugiyama, T.,
and
Phytopathol.
Akenohoshi. Plant Cell
in the
rhizosphere
37:473-491.
Physiol.
during ripening
of rice cultivars
contents of leaves and
Nipponbare
and
36:1105-1114.
Stutz, E. W., Défago, G., and Kern, H. 1986. Naturally occurring fluorescent pseudomonads
involved in
49.
biosynthesis
Ishihara, K. 1995. Changes in chlorophyll
in levels of cytokinins in root exudates
48.
DAPG
Smith, K. P., and Goodman, R. M. 1999. Host variation for interactions with beneficial plantassociated microbes. Annu. Rev.
47.
affecting
suppression
of black root rot of tobacco.
Phytopathology
Thomashow, L. S., and Weiler, D. M. 1996. Current concepts in the
biological
control: Mechanisms and
N. T. eds. Plant-Microbe
50. Van
antifungal
metabolites.
Pages
76:181-185.
use
of introduced bacteria for
187-235 in:
Stacey, G., Keen,
Interactions, vol. 1. Chapman & Hall, New York, NY.
Overbeek, L. S., and Van Elsas, J. D. 1995. Root exudate-induced promoter activity in
Pseudomonas fluorescens mutants in the wheat
rhizosphere. Appl.
Environ. Microbiol. 61: 890-
898.
51.
Wood, D. W., Gong, F., Daykin, M. M., Williams, P., and Pierson, L. S., III. 1997. 7V-acylhomoserine lactone-mediated
aureofaciens
52.
regulation
30-84 in the wheat
of phenazine gene
rhizosphere.
expression by Pseudomonas
J. Bacteriol. 179:7663-7670.
Yuan, Z., Cang, S., Matsufuji, M., Nakata, K., Nagamatsu, Y., and Yoshimoto, A. 1998. High
production
grown
on
of pyoluteorin and
ethanol
as a
2,4-diacetylphloroglucinol by Pseudomonas fluorescens
sole carbon
source.
J. Ferment.
54
Bioeng.
86:559-563.
S272
Chapter 3
Fusaric-Acid-Producing
glucinol Biosynthetic
Gene
Strains of Fusarium oxysporum Alter
Expression
2,4-Diacetylphloro¬
in Pseudomonas fluorescens CHAO In Vitro and
in the
55
Rhizosphere
of Wheat
56
Chapter 3
FA
producing
strains off. oxysporum affect DAPG
expression
Abstract
The
phytotoxic pathogenicity
diacetylphloroglucinol (DAPG),
factor fusaric acid
key
a
substantially.
by using
translational
a
with the
phlA
'-
'lacZ fusion.
mutant
a
strain
producing
The
their influence
on
and
one
a
sand and
repressed phlA expression
CHA638 than in the
wheat
or
on
of the
expression
rhizosphere
(242)
phlA
on
strongly
was
'-
'lacZ
correlated
expression
significantly,
was
of F. oxysporum
of wheat in
rhizosphere
phlA expression
whereas strain 242
altered
not
levels
wild-type CHAO, indicating,
by
was
repressor PhlF. One FAwere
a
tested for
gnotobiotic
mineral-based artificial soil. F. oxysporum strain 798
clay
in CHAO
798.
effect of FA
strain
in CHAO in the
phlF mutant CHA638, phlA expression
oxysporum strain 242
production
pathway-specific
non-producing
phlA expression
system containing
12 Fusarium oxysporum strains
F A-producing F. oxysporum strains could
repressing
that lacked the DAPG
(798)
of the biocontrol
activity
in strain CHAO and the FA concentration
degree ofphlA repression.
abolished in
Only
of 2,4-
production
operon of strain CHAO in culture media and in the wheat
production
suppress DAPG
production by
We measured the effect of FA
phlACBDE biosynthetic
represses the
factor in the antimicrobial
strain Pseudomonas fluorescens CHAO. FA
varied
(FA)
(FA")
(FA+)
did not. In the
the presence of either F.
were seven
to
eight
that PhlF limits phlA
times
higher
expression
in strain
in the
rhizosphere.
Published 2002 in
Applied and Environmental Microbiology
68:2229-2235
Introduction
Antibiosis is
overcome
an
the effects of soil-borne
diacetylphloroglucinol (DAPG)
produced by
fungi,
and helminths
indispensable
phlF gene,
precursor to
a
for
a
one
putative
which encodes
efflux
a
as
are
metabolite 2,4-
well
protein (phlE)
protein
and is effective
organized
as
Q2-87 and CHAO,
operon and
are
of DAPG. This operon is followed
and flanked
of DAPG
57
as an
against bacteria,
monoacetylphloroglucinol (MAPG),
degradation product
repressor
polyketide
In Pseudomonas fluorescens
phlACBD,
of DAPG
or a
The
to
of the most effective antimicrobial metabolites
pseudomonads (25, 40)
genes,
production
by plant-beneficial microorganisms
fungal pathogens (44).
(13, 18, 23, 30, 34, 39).
biosynthetic
for the
which is either
coding
is
strains of fluorescent
the four DAPG
gene
mechanism used
important
by
the
divergently
synthesis (5, 11, 38).
by
transcribed
a
Chapter 3
FA
producing
Environmental factors influence the
DAPG in fluorescent
pseudomonads.
bacteria has been attributed to
location and
host
cropping
time
strains ofF. oxysporum affect DAPG
of antimicrobial
production
Variation in the biocontrol
changes
performance
biotic
(14, 44). Complex
DAPG
such
as
of these
of biotic and abiotic factors associated with field
factors, such
plant species, plant
as
cultivar, and infection with the plant pathogen Pythium ultimum,
phlA expression (33).
compounds
expression
production
be influenced
can
by
the carbon
alter
significantly
can
age,
and minerals
sources
Q_i_
present in the bacterial environment. Fe
MAPG in P.
fluorescens
Fl 13
stimulated
by glucose (15, 34).
ethanol
the sole carbon
as
of DAPG is stimulated
important
role in the
by
(16),
but in P.
In strain
source
sucrose
increase
fluorescens
in P.
is also
of DAPG in that its
by
inhibited the
growth
other
Our
Yabuta et al.
of rice
(47)
from Fusarium
seedlings.
and bacteria
plants, fungi,
FA is
present study,
objective
media and
on
was
to
we
fluorescens
was
obtained with
CHAO the
Microbial metabolites
synthesis
(38).
is autoinduced and
The
heterosporum
produced by
can cause
fungal
production
play
an
repressed by
toxin fusaric acid
Nées
as a
compound
ability
wheat roots correlates with the
animal
the DAPG
that
interactions of FA with
toxicity (3, 12, 41, 46).
of these
suppression
was
many Fusarium spp. and is toxic to
screened 12 Fusarium oxysporum strains for FA
determine whether the
phlF mutant CHA638, lacking
investigate
CHAO, production is
(1, 8, 19, 20, 22, 31). Synergistic
naturally co-occurring mycotoxins
In the
of DAPG and
potent inhibitor of DAPG synthesis in P. fluorescens CHAO (14, 38). FA
a
first isolated
various
Pf-5 and
NH4Mo2+ (15).
and
other bacterial extracellular metabolites of strain CHAO
(FA)
production
S272, the highest DAPG yield
(48). Furthermore,
Zn2+, Cu2+,
regulation
and
fungi
of DAPG
pathway-specific
produce
to
FA in culture
production
PhlF,
repressor
production.
in CHAO. The
was
used to
the mode of action of the microbial interaction.
MATERIALS AND METHODS
Microorganisms, plants,
producing
strain isolated from the
suppressive
gene
to
Thielaviopsis
replacement (38).
Plasmid
six
copies
a
(43).
pME6259 is
tetracycline
(21).
a
Swiss soil
The phlF mutant CHA638
a
was
resistance gene
in P.
DAPG-
constructed
(38). pME6010
stably
a
naturally
derivative of pME6010 and contains
per chromosome and is maintained
absence of antibiotic selection
plasmid pME6259 by
of tobacco grown in
rhizosphere
basicola
7acZ translational fusion and
approximately
and culture conditions. P. fluorescens CHAO is
by
aphlA
is present at
fluorescens
in the
Earlier work has shown that retention of the reporter
strain CHAO is 98.8 ± 0.9 % after 20
58
generations
of growth in Luria-
'-
Chapter 3
Bertani
FA
(LB) (36)
(KB) (26)
broth
producing
All strains
(33).
strains ofF. oxysporum affect DAPG
routinely
were
and in LB with the addition of tetracycline
cultivated at 27°C
(125 ug/ml)
on
King's
required,
as
expression
B agar
unless
otherwise mentioned.
F. oxysporum strains
isolated from wheat roots
233, 234, 235, 236, 237, 238, 240, 241, 242, and
(Triticum
aestivum
cv.
Arina) by
Production, Changins, Switzerland. Strains
Centraal Bureau
voor
1.5% malt agar
798 and 801
Schimmelculture, Baarn, The Netherlands)
plates (Difco, Detroit, MI)
Seeds of winter wheat
(T
were
France. All F. oxysporum strains
Grignon,
were
the Swiss Federal Research
Station for Plant
cultivated with wheat in
410
(obtained
from
isolated from soil
were
routinely
cultured
on
at 24°C.
aestivum
cv.
followed
Arina)
were
10 min in 10%
7% sodium
hypochlorite (vol/vol),
thoroughly
rinsed with sterile double-distilled water. Seeds
by
surface disinfected for 15 min in
H2O2 (vol/vol), and then
for 3
pregerminated
were
days
on
0.85%) water agar at 24°C in darkness.
Production of FA
by
the F. oxysporum strains. Malt broth
(Oxoid, Hampshire,
ZnSÛ4
7H2O (Merck, Darmstadt, Germany) (250 ml in 500 ml baffled flasks) per liter
inoculated with
7-mm agar
incubated for 7
were
microconidia)
supernatant
2°C. In
2 M
one
was
days
addition,
phase
was
separated
(Macherey
and
through
a
50 ml of
a
min. FA
with
a
curve
a
was
detected
mobile-phase
of
synthetic
gradient
g)
and
pH
2 with
vigorously
were
a
in
and stored at
400 ul of
approximately
for 1 min. The
organic
silicone-coated filter paper
vacuo.
The residue
column
(4 by
125
mm) packed
and set at 50°C. The
from 25 to 100%) in 0.43%>
The retention time
collected, and the
samples
were
was
samples (10 ul)
acid
approximately
quantified against
was
Palo
with Nucleosil 120-
o-phosphoric
The fraction
presence of FA
59
was
high-performance liquid chromatography
(Acros Organics, Geel, Belgium).
was
with 89 mg
for 15 min. One part of the
brought to dryness
with
by monitoring A270nm-
standard from the HPLC
not
(Nalgene, Rochester, NY)
and shaken
flow rate of 0.7 ml/min. The
FA
or
liquid Chromatograph (Hewlett Packard Co.,
reverse-phase
linear methanol
x
acidified to
(Macherey-Nagel, Oensingen, Switzerland)
eluted with
amended
malt agar cultures. Cultures
7-day-old
phase by filtering through
analyzed
Hewlett Packard 1090
a
was
ethyl acetate,
Nagel, Düren, Germany)
Alto, CA) equipped with
5-C18
0.8-um-membrane
from the aqueous
(7)
rotary shaker (180 rpm). Fungal biomass (mycelia and
supernatant
dissolved in 1 ml of methanol and
(HPLC) using
taken from 5- to
by centrifugation (2,200
50 ml of the
HCl, mixed with
Kingdom),
at 24°C on a
collected
filtered
was
plug
and R2 broth
Dox
medium
•
United
(2%), Czapek
containing
a
over
2
12
min,
standard
the FA
qualitatively
were
and
Chapter 3
FA
confirmed
quantitatively
by
producing
strains ofF. oxysporum affect DAPG
spectrometry (MS)
mass
on a
expression
TSQ3000 triple quadrupole
mass
spectrometer with electrospray ionization (Thermo Finnigan, Bremen, Germany). The
experiment
was
carried out twice with similar results. Data from all trials
trial-by-treatment
interaction with
Evanston, IL). Data could
(see
Table
0.05)
least
Means of three
1).
plasmid pME6259,
fungal
optical density
an
inoculation. Cultures
cultures
appropriate,
fungal
were
at 600
received the
determined
same
on
established
by
were
filtrate
growth
dilution
was
2).
protected (P
0.05)
least
The
expression
on
an
autoclaving,
and
of three
assay in the
on
monitored
cotton
x
g)
the
on
One-liter
Czapek Dox
aliquots
(vol/vol)
stationary growth phases by
nm
medium
were
When
synthetic
ethanol
medium
used for
were
at 160 rpm.
'lacZ
'-
FA
was
(pH 6.5).
Controls
activities
were
the method of Miller
after the correlation with CFU
experiment
was
repeated
three
was
times, and
replicate
cultures
were
separated using
are
Fisher's
rhizosphere
expression
of wheat. The influence of the two F.
of phlA in P.
(24).
were
fluorescens
grown in artificial soil
The soil
Erlenmeyer flasks
CHAO and its phlF
were
was
containing
moistened with double-
filled with 300 g of artificial
wool stoppers, and autoclaved for 30 min at 121°C. After
for 15 min and the
were
or
experiments, ß-galactosidase
wheat roots. Plants
30 ml of sterile modified
Microconidia
translational phlA
difference test.
Knop's
oxysporum strains 242 and 798 grown in
(2,200
=
in vitro. P.
OD6oo of 1.5 and 2.5 of one representative experiment
significant
(10%>, wt/wt).
soil, plugged with
protected (P
glucose-ammonium
shaking
it in 20%
vermiculite and quartz sand of different fractions
distilled water
minimal
of 0.01, and 30-ul
KB agar. The
means
oxysporum strains 242 and 798
mutant CHA638 was
a
(Systat Inc.,
LB cultures of the bacterial strains
estimated from OD at 600
plating
Table
a
a
(described above)
by dissolving
use
of the solvent. In all
amount
presented (see
Gene
for
presented separately
Fisher's
amended with FA. A fresh stock solution of
were
similar. Values for
=
are
phlA'-'lacZ expression
CHA638, carrying
(ODöoo)
nm
throughout the exponential
Bacterial
separated using
incubated at 27°C with rotary
prepared immediately prior to
results
filtrates
tetracycline. Exponential-growth-phase
diluted to
(32).
were
grown in 24 ml of
were
amended with 6 ml of
(OSG) (38)
without
cultures
replicate
version 9.0
using Systat
and the two individual trials
CHAO and its mutant derivative
fluorescens
on
pooled,
of variance
analyzed
difference test.
significant
Influence of FA and
fusion
be
not
analysis
were
separated
nutrient solution
Czapek
Dox
as
in sterile
mycelia by filtering through
60
was
added. Cultures of F.
described above
resulting pellet resuspended
from
(24)
were
Knop's
sterile
glass
centrifuged
solution.
wool and
Chapter 3
FA
quantified by using
a
hemocytometer.
strains ofF. oxysporum affect DAPG
producing
The artificial soil
was
104
inoculated with
expression
conidia per
gram of soil.
Overnight
were
LB broth cultures of
washed with 0.9% NaCl solution and diluted with 0.9% NaCl to
concentration of 10
seedlings
to
each
discard
Is),
followed
design.
loosely
rhizosphere)
were
days later, plants
adherent soil. Roots with
in
growth
a
(0.9%)
on
King's
B agar
screened for
quantifiable
visible
from calculations. Plants
(27)
repeated
twice with four
variance
was
by-treatment
to
check for
replicate
flasks
were
interaction. Data could not be
(P
least
significant
ß-Galactosidase
was
or on
a
completely
gently
as
determined from the
malt agar amended
measured
200 ul of the
using
wild-type strain, CHAO,
were
washed with tap water,
were
surface sterilized and
(five plants/flask)
performed using Systat version
in Table 3. Means of four
0.05)
in
for five min.
plated
infection with F. oxysporum. The
endophytic
separately
=
arranged
lesions, blotted dry, and weighed. Representative samples of
and shoot tissue from all treatments
medium
was
Control treatments inoculated with the
background activity
transferred
were
this article defined
(in
at 300 rpm
rifampicin (100 ug/ml) (24). ß-Galactosidase activity
used to subtract
were
colonization with bacteria and Fusarium spp.
rhizosphere suspension.
seedlings
chamber with 16 h of light at 18°C
adherent soil
tightly
serial dilutions
final
a
removed from the flasks and shaken
were
shaken in sterile saline solution
resulting suspension by plating
root
for 60 min. Five
suspension
give
described above. Wheat
as
8 h of darkness at 13°C. The flasks
by
Seven
Rhizosphere
with
pregerminated
were
flask, covered with soil, and incubated
randomized
to
CFU/ml. The seeds
soaked in the bacterial
were
uE/m
(160
CHAO, CHA0/pME6259, and CHA638/pME6259
9.0
replicate
experiment
to
and two individual trials
flasks
were
separated using
was
analysis
per treatment. An
(Systat Inc., Evanston, IL)
pooled,
Komada's
onto
of
analyze
are
trial-
presented
Fisher's
protected
difference test.
assays and
plasmid stability. ß-Galactosidase
assays
were
o
conducted
values
1000
x
were
the method of Miller
calculated
by
of plasmid
(32).
Activities
the method of Miller
[OD420 / (time [minutes]
stability
with
by
pME6259
tetracycline (125 ug/ml).
tetracycline-resistant colonies,
x
was
sample
Plates
were
expressed
(32) following
volume
evaluated
were
[mililiters])]
by replica plating
as
units per 10
the formula
=
units of
ß-galactosidase.
onto KB agar
incubated at 27°C for 48
The
supplemented
h, and the percentage of
indicative of the presence of pME6259,
61
CFU. Unit
was
determined.
Chapter 3
FA
strains ofF. oxysporum affect DAPG
producing
expression
Results
Production of FA
by
12 strains of F. oxysporum in four different media. We
extracted FA from F. oxysporum cultures grown in four different media
medium, and
Dox
R2 broth with and without
ml of medium. F. oxysporum strains
FA in any of the four media
(data
least
(Table 1).
one
of the media tested
significant
amounts
small amounts in
of FA in both
only
236, 237, 238, 241, and
798
produced large
410
amounts
In malt
experiments.
of FA in this
saccharose-based medium R2, strain 241
also detected
abolished
(Table 1).
production
F. oxysporum
y
FA in at
produced
produced
broth, strains 233, 234,
Dox
medium,
FA
a
fusaric acid
production by
z
of FA,
reaching
strains 233, 798, and 801
not
was
shown).
different strains of Fusarium
Czapek Doxz
oxysporum)1
R2 with out zincz
Expt.1
233
<0.2
<0.2
6.8
a
<0.2
5.7
234
<0.2
<0.2
0.8
a
<0.2
<0.2
<0.2
236
<0.2
<0.2
5.9
a
<0.2
<0.2
<0.2
237
<0.2
<0.2
<0.2
0.9
a
<0.2
<0.2
238
1.4
<0.2
<0.2
0.5
a
<0.2
<0.2
114.7a
550.0 b
308.3 b
<0.2
<0.2
<0.2
0.9
a
1.4
a
2.3
a
7.7
a
a
Expt.
241
110.0b
410
<0.2
<0.2
798
<0.2
10.3 ab
801
<0.2
2.0 b
The F. oxysporum strains
for 7
days
were
47.1
2
a
concentration of 310 uM to R2 medium
(data
by
amounts
0.8 and 3.5 mM. In the
strains
respectively
z
Malt
produce
Strains 238, 798, and 801
Czapek
produced large
of FA in all strains tested
Phytotoxin
0.2 nmol per
detectable amounts of
strain 241
medium, ranging between
The addition of zinc at
Table 1. Production of the
In
was
small but detectable amounts of FA. Strains 801 and
produced
concentration up to 550 uM. In this
produce
liquid medium, only
experiments (Table 1).
of the two
one
242 did not
The other nine strains did
shown).
not
The FA detection limit
zinc).
235, 240, and
(malt broth, Czapek
a
Expt.1
100.0
1.6
Expt.
a
a
2,633.7 b
1,962.3
cultivated in 250 ml of
c
2
Expt.1
3,492.4 b
786.1
c
liquid malt, Czapek Dox,
or
a
2
Expt.
104.6
R2 without zinc
at 24°C and 180 rpm.
Production of fusaric acid
(FA) is indicated in nmol per milliliter of medium (uM). Detection limit was
(uM). Values have been adjusted regarding the 35% efficiency of extraction.
mean of three replicate cultures. Means for the same experiment within the same
0.2 nmol per milliliter
Each value is the
column followed
protected
least
a
by the same letter are not significantly
significant difference test.
62
different at P< 0.05
according
to Fisher's
Chapter 3
FA
Repression
of
a
phlA'-'lacZ fusion
concentration. The bacteria
under the
same
producing
conditions
in P.
strains ofF. oxysporum affect DAPG
fluorescens
grown in the presence of
were
with the culture filtrates of F. oxysporum
translational
phlA'-'lacZ gene
fusion
was
expressed
(Table 2).
from
in the absence of added FA
depends
100, 300, 500, and
(e.g. temperature, medium, inoculation,
experiments
stationary growth phase
CHAO in vitro
and
The
rpm)
expression
as
on
FA
750 uM FA
in the
plasmid-borne
mid-exponential
to
the
(Fig. 1). ß-Galactosidase
early
activities at
o
mid-exponential growth (ODöoo
of
0.638)
were
approximately
120 units per 10
CFU and
o
between 2,300 and 2,420 units per 10
phase). Reporter
to
the
strain
growth
gene
expression
was
CFU when the ODöoo
not
affected
was >
significantly by
medium. A concentration of 300 uM FA reduced the
CHA0/pME6259
and 750 uM blocked
to
^
_J
u_
>
Ü
(early stationary growth
the addition of 100 uM FA
ß-galactosidase activity
of
about half the amount of the control. FA concentrations of 500 uM
expression
of the phlA
addition of FA did not affect bacterial
_
2
'-
'lacZ fusion almost
growth (data
not
completely (Fig. 1).
The
shown).
3
+J
o
CO
<D
CO
o
CO
o
^
1_
Qi
CO
c
c
o
2
o
(0
(0
o
1
3
o
o
o
CO.
0
0
1
2
Growth
(OD 600)
Repression of expression of a phlA'-'lacZ translational fusion by the addition of synthetic
(FA) at different concentrations to a growing culture of P. fluorescens CHAO harboring
pME6259. Strain CHA0/pME6259 was grown in OSG-medium amended with Czapek Dox at a ratio
Fig.
1
fusaric acid
were added to the medium: none (A), 100 (aM (D), 300
(aM (). Throughout growth, ß-galactosidase activities and OD600 were
determined. Values are the means of three replicate cultures. Addition of FA to the medium had no
effect on cell growth. Error bars representing the standard errors of the means may be obscured by
symbols.
4:1 at 27°C. Different concentrations of FA
(aM (x),
500
(aM (A),
and 750
63
of
Chapter 3
FA
producing
strains ofF. oxysporum affect DAPG
Influence of culture filtrates of F. oxysporum
on
expression
of
expression
aphlA'-'lacZ
fusion in P. fluorescens strains CHAO and CHA638. Addition of F. oxysporum culture
filtrates stimulated bacterial
effects of bacterial
growth
ß-galactosidase
activities
incubation time
(Fig.
growth
caused
are
to
by
different extents
(data
shown).
not
In order to avoid
the addition of different F. oxysporum culture
always reported
1 and 2 and Table
for
a
certain cell
density (OD6oo)
filtrates,
rather than for
2).
Table 2. Influence of Fusarium oxysporum culture filtrates
on
phlA expression
in Pseudomonas
fluorescens CHAO and CHA638.
P. fluorescens
F. oxysporum
strainw
strainx
FA-concentration
in bacterial
medium
ODsoo
frM)y
=
per108CFU)z
1 -5
OD6oo
0
1510
a
2100
CHAO
240
0
1520
a
2000
CHAO
241
25
1480
a
1910
CHAO
242
0
1460
a
1950
CHAO
410
0
1640
a
1920
CHAO
798
700
220 b
590
CHAO
801
160
200 b
1520
CHA638
none
0
1850
a
1720
CHA638
240
0
1660
a
1390
CHA638
241
25
1590
a
1590
CHA638
242
0
1540
a
1560
CHA638
410
0
1740
a
1680
CHA638
798
700
1660
a
1490
CHA638
801
160
1680
a
1470
on
(wild-type)
plasmid pME6259.
The F. oxysporum strains
and CHA638
cultivated in
were
Filtrates from F. oxysporum cultures
20%
(vol/vol).
Fusaric acid
in the bacterial
z
(units
none
P. fluorescens CHAO
y
growth
activities
CHAO
fusion
x
ß-Galactosidase
growth
ß-Galactosidase
(FA)
were
(pWF-mutant) carrying
Czapek
a
translational
=
2.5
phlA'-'lacZ
Dox medium for 7
days at 24°C and 180 rpm.
growth medium (OSG) at a ratio of
added to the bacterial
content of culture filtrates
was
determined and final FA concentration
medium is indicated.
activities
were
determined in bacterial cultures grown to
an
OD at 600
nm
of 1.5 ±
0.1 and 2.5 ±0.1.
Data
represent
means
column followed
protected
least
of three
replicate
different letter
by
significant
a
are
FA in
Czapek
or
significantly
same
P. fluorescens strain within
different at P< 0.05
according
a
to Fisher's
difference test.
Six strains of F. oxysporum
(strains 798, 801)
cultures. Means for the
minimal
were
selected which
(strain 241)
Dox medium after 7
days
or
of
produced
undetectable
large
amounts
(strains 240, 242, 410)
growth (Table 1).
64
either
of FA
amounts
Filtrates from F. oxysporum
of
Chapter 3
FA
cultures grown in
bacterial
growth
Dox medium for 7
Czapek
medium
producing
(OSG)
at a
strains ofF. oxysporum affect DAPG
days (Table 1, Expt. 2)
ratio of 20%
(vol/vol),
were
expression
added to the
which gave final FA
concentrations of 0, 25, 0, 0, 700, and 160 uM for strains 240, 241, 242, 410, 798, and 801,
The effect of the
respectively.
CHA638
during
the
control treatment,
fungal
filtrates
fungal
Czapek
was
phlA expression
to
was
in strains CHAO and
monitored. In the
added to OSG medium at the
The addition of
growth compared
on
early stationary growth phase
Dox medium
(20%, vol/vol).
enhance bacterial
and
exponential
culture filtrates
growth
Czapek Dox
in
plain
OSG
to OSG
(data
ratio
same
medium did
not
slightly
shown).
Culture filtrates of F. oxysporum strains 240, 241, 242, and 410 did not affect
activities of strain
galactosidase
and
2.5) compared
to
the control
repressed phlA expression
activities of strain
CHA0/pME6259
to
(Table 2).
both cell densities
ßof 1.5
reported (OD6oo
Addition of culture filtrates of strains 798 and 801
different extents. At
CHA0/pME6259
at
the
as
an
ODöoo of 1.5, the ß-galactosidase
grown in the presence of filtrate from strain 798
801
or
o
dramatically repressed
was
sevenfold phlA
repression.
repression
At
an
was
only 30%,
CFU, which represents
a
OD6oo of 2.5, ß-galactosidase activity measured in
grown with 801 filtrate
CHA0/pME6259
but
and did not exceed 220 units per 10
whereas
was
still
repression
lower than that of the
significantly
in the presence of 798 filtrate
was
control,
70%
^..•J
-i-
_
activ ty CFU)
b
108
en
actosidae units
per
9
CO.
b
o
--
^
n
n
I
I
I
1
2
3
u.u
(D
Growth
Fig.
2
(C)D
60())
Influence of the addition of filtrates of F. oxysporum strain 801 grown in
for 7
days at 24°C
growing culture of
at 180 rpm
differing
in fusaric acid
(FA)
content
P. fluorescens CHAO. Filtrates of strain 801
on
were
Czapek-Dox medium
phlA'-'lacZ expression of a
added to OSG medium at
a
ratio
|aM (D) and 157 (aM () respectively. The control
treatment (O) received the same amount of Czapek Dox. ß-Galactosidase activities and OD600 of
strain CHA0/pME6259 were determined throughout growth. Values are the means of three replicate
of 1:4
giving
a
final FA-concentration of 392
cultures. Error bars
represent the standard
errors
of the
to be shown.
65
means.
Some of the
error
bars
are
too small
Chapter 3
FA
The
(Table 2).
were
was
follow-up experiment,
a
expressed
Czapek
Dox
400 and 160 uM in the bacterial
on
the phlA
in the phlF mutant CHA638
expression
'lacZ fusion
'-
(Table 2).
tested the effect of culture filtrates of F. oxysporum
we
strain 801 with different FA contents
from cultures grown in
strains ofF. oxysporum affect DAPG
effects of culture filtrates 798 and 801
repressing
abolished when the fusion
In
producing
onphlA expression
(Table 1)
growth
were
medium.
in P. fluorescens CHAO. Filtrates
used to obtain final FA concentrations of
in P.
Average ß-galactosidase activity
o
with culture filtrate of Expt. 1
from
Expt.
2
(160 uM)
was
CFU in medium amended
remained below 680 units per 10
fluorescens CHA0/pME6259
(400 uM) (Fig. 2).phlA expression
repressed
in medium with filtrate
smaller extent; the maximal
to a
ß-galactosidase
o
in this treatment
activity
control
was >
on
1,550 units per
of
a
phlA'- 'lacZ fus ion
rhizosphere
Table 3.
an
OD6oo of 2.7, whereas that of the
in strains CHAO and CHA638 in the
of wheat in the presence of two different F. oxysporum strains. After 2 weeks
wheat roots, retention of plasmid
65.8 ± 4.3%)
CFU at
10
2,000 units per 108 CFU.
Expression
rhizosphere
was
pME6259
based
inphlF mutant CHA638,
that grew
on
was
wild-type
strain CHAO and
the viable bacteria reisolated from the
on
tetracyline-amended
97.3 ± 2.1%> in
agar.
ß-Galactosidase
activities
Expression of a phlA'-'lacZ fusion in Pseudomonas fluorescens strains
rhizosphere with or without Fusarium oxysporum strain 242 or 798.
were
CHAO and CHA638 in
the wheat
Bacterial
Microorganisms
ß-Galactosidase activity
P fluorescens
w
"
F oxysporum
y
z
(units
per
Expt
1
108CFU)Z
Expt
(x 107CFU
(g)2
2
1
Expt
rhizosphere
colonization
weight
Expt
2
Expt
1
x
g'1)z
Expt
none
none
nd
nd
27
a
23
a
nd
nd
none
242
nd
nd
31
a
24
a
nd
nd
none
798
nd
nd
31
a
27
a
nd
CHA0/pME6259
none
a
26
a
26
a
27
a
1 4
a
CHA0/pME6259
242
304 b
56 b
28
a
26
a
09
a
1 1
a
CHA0/pME6259
798
38
22
c
26
a
26
a
1 9
a
1 1
a
CHA638/pME6259
none
460 b
287 d
29
a
26
a
25
a
1 5
a
CHA638/pME6259
CHA638/pME6259
242
514 b
365 d
30
a
27
a
1 7
a
1 0
a
798
420 b
252 d
30
a
26
a
24
a
08
a
65
on
34
a
c
Pseudomonas fluorescens strains CHAO
translational fusion
x
y
Plant fresh
(wild-type)
and CHA638
(phlF mutant) carrying
plasmid pME6259.
Fusarium oxysporum strains 242
or
798
were
added to the
gnotobiotic system
104
at
per gram of soil.
Plants were grown for 14 days under gnotobiotic conditions in artificial soil. Fresh
plants (always 5) in one flask are presented.
Values
represent the
mean
of four
replicate
flasks with 5
plants
66
are
significantly
nd
a
phlA'-'lacZ
microconidia
weight
each. Means for the
experiment within the same column followed by a different letter
according to Fisher's protected least significant difference test.
2
from all
same
different at P< 0.05
Chapter 3
FA
calculated per CFU still
between 2.6 to 3.1 g in
the wheat
on
and
plants,
interaction
was
around 1.9
105
x
experiments (data
colonization
was
cells
fluorescens
In both
significantly
treatment
or
shown).
not
leafs
indicating
not
estimated from 0.9
were
measured in
10
7
10
x
and 1.5
7
to 2.7
10
x
7
10
x
7
mutant CHA638
rhizosphere
F. oxysporum strain 242
was
enhanced two- to
ß-Galactosidase
significantly
in both
experiments (Table 3). Rhizosphere
CHA638
(Table 3), suggesting
mediated
by
activities
no
Expt.
798, i.e.,
Expt.
1
2. No P.
(Table 3).
measured in strain CHAO
lowered
was
ofthat of the control
to 60%
242, ß-galactosidase activity
fourfold, compared
were seven
when Fusarium
wild-type
798 had
or
or
withi7. oxysporum
CFU per g of rhizosphere in
In the treatment with F. oxysporum
than in the
were
endophytic
an
CFU per g of rhizosphere in
in the treatment withi7. oxysporum strain
without Fusarium added.
ranged
Komada's medium
on
detected in the treatments without bacteria added
CHA0/pME6259
did not differ
2. No visible lesions
the absence of
Colonization of the
experiments, ß-galactosidase activity
(Table 3).
Expt.
expression
No statistical differences could be found for bacterial
shown).
x
weight
shown in Table 3 and
CFU per g of rhizosphere and did not differ
between 0.8
ranged
Plant fresh
F. oxysporum could be reisolated
no
colonization between the treatments the two
rhizosphere
and
(data
experiments
1 and between 2.3 to 2.7 g in
Expt.
from the inside of wheat roots, stems,
pathogenic
strains ofF. oxysporum affect DAPG
carrying reporter plasmid pME6259.
between the treatments in both
significantly
detected
producing
was
not
significant influence
on
that the effects of F. oxysporum
to
the treatment
eightfold higher
to
in the phlF
added. The amendment with
ß-galactosidase activity
the
on
wild-type
CHAO
in
are
PhlF.
Discussion
F. oxysporum is found worldwide. This
due to its
ability
to
survive winter in the
normal crop rotation is not
saprophyte
produce
the
FA
impact
model
as
well
as a
a
wilt
practical
pathogen
(2, 4, 10, 35, 42).
In this
FA
markedly
mycelial
control
study
production by
measure
we
and
persist
almost
(6).
plants,
The
indefinitely
state
species
on a
in soil
with the result that
occurs as a
soil
and many of the strains isolated
addressed in vitro and in the wheat
gene critical for the biocontrol
rhizosphere
activity
of the
CHAO.
F. oxysporum is strain
between F. oxysporum strains and media
production (10, 28),
can
chlamydospore,
or
of many crop
of different F. oxysporum strains
organism P. fluorescens
fungus
indeed,
dependent. Production of FA varied
(Table 1).
Sucrose is
F. oxysporum strains 798 and 801 did
67
reported
to
favor FA
produce large
amounts
Chapter 3
of FA in
Czapek
supposed
used in
FA
favor FA
to
our
broth, which is
Dox
producing
In the
four strains
addition of zinc to this medium abolished FA
Fusarium strains
DAPG
Synthetic
500 uM
FA represses
as
in CHAO is
biosynthesis
observed
by
concentration of 300 uM gave
concentration of 100 uM,
Strains of the
such
no
species
evidence show that FA is the
repression, (i) Repression
of
amount
of
one
of two different
growing
culture filtrate
significant repressive
F. oxysporum
and
2).
of gene
fungal
synthetic
by
FA
(R2)
has been shown for other
and
our
phlA expression through
a
almost
an
Here
fungal
we
cause
complete
expression,
a
fungal
same
only
160
filtrate
three lines of
likely
experiments,
to
be
increasing
those reached
Fusarium
for phlA
responsible
contents
of FA in the
similar concentrations
by
addition of the
Addition of
completely.
FA
Amendment
FA concentration in the bacterial
almost
same
strain, 801, with different
a
growth
different
uM, resulted in partial repression only (Fig.
same
concentration of
intact phlF gene is needed for
Schnider-Keel et al.
(Fig. 1).
plants,
well with those obtained with the
an
a
metabolites
repressed phlA expression differentially.
was
FA
and at
secondary
(Table 2, Fig. 1). (ii) Moreover,
expression
block at
diverse responses in
increased with
filtrates from the
metabolite FA.
show that
wide range of
correspond to
Evidence that
provided by
the
by
effect could be observed in vitro
(Table 1, Expt. 1) brought the
(Fig. 1). (iii)
FA has been
In
expression
culture of CHAO
correspond
(38).
metabolite most
secondary
in which FA-concentration
These effects
as
in vitro
produce
mycotoxins
medium to 400 uM and blocked phlA
filtrate,
production,
intermediate inhibition of phlA
FA added to the medium
contents to a
detectable amounts of FA. The
of DAPG in CHAO, with
filtrates. The levels of repression
synthetic
produced
repressed
animals, humans, and microorganisms (29).
fungal
modified form of this medium
Schnider-Keel et al.
an
Phytotoxins, pigments,
as
slightly
(9, 14, 17).
biosynthesis
(Fig. 1),
expression
sucrose-based medium. Richard's medium is also
a
production (9, 37).
experiments (7), only
strains ofF. oxysporum affect DAPG
(38). Similarly,
the addition of fungal filtrates
was
in
our
repression ofphlA
study, repression
of
abolished in the phlF mutant
CHA638(Table2).
Interactions between F. oxysporum strains and wheat.
strains
were
visible
lesions,
plant
isolated from wheat roots
material
no
reduction in
strongly
cultivar used in
our
indicate that there
strains
from fields cultivated with
plant weight,
plant experiments.
specialized pathogenic
or
are
Although
wheat, the absence of
and lack of detection of the
are no
pathogenic
F. oxysporum is
a
fungus
inside the
interactions with the wheat
cosmopolitan
causal agents of vascular wilts and
68
the F. oxysporum
soil
saprophyte,
and
damping-off diseases.
Chapter 3
They
(6).
FA
exhibit
In
a
high degree
case, either the strains
our
infection did not take
by
of host
reisolation of the
specificity
from the
strains ofF. oxysporum affect DAPG
and many formae
nonpathogenic,
are
place. However,
fungi
producing
or
speciales
host genotype
the strains colonized the wheat
have been described
recognition
CHAO
differentially compared
knowledge,
from in vitro
altered phlA
the
rhizosphere. Previously,
rockwool system
(14).
of
impact
pathogenic
effect is caused
suppressing
supported by
the
binds to the
proposed to
promoter(s)
of phlA
been demonstrated in vitro
leading
to
increased
be excluded that
DAPG
synthesis
in CHAO
a
ultimum
phlA
might
exudates from
interactions
the
fungus.
It is
pathway-specific
(5, 11, 38).
was
To
gene
be
an
of FA in
hypothesize
We
that
The latter is
synthesis
in that it
FA has
repression by
interact with the repressor,
might
act
higher affinity
it cannot
upstream of phlF and that
of
a
FA-PhlF
this is the first report to show
a
complex
to
PhlF-mediated
rhizosphere.
expression
in CHAO.
expression
Previously,
in CHAO
on
roots.
By contrast,
observed, the change
in the current
in gene
that strain 242
an
enhancing
an
study,
is
effect of
Pythium
both cucumber and maize
expression
produces
even an
have shown that
we
indirect consequence of disease due to
possible
radicis-
PhlF-promoter complex. However,
a
of
abolished in the phlF mutant
A PhlF-mediated DAPG
knowledge,
expected
specialis
rhizosphere.
repressor of DAPG
It is conceivable that FA
our
forma
rhizosphere.
of FA in the
effect
gene critical
through production
effect of F. oxysporum 798 contrasted with
damaged
were
repressive
characteristics of the
stimulate phlA gene
can
effect that
in the
repressing
on
a
production
FA may not be the result of
region.
suppression
strain 242
(38).
binding
repression by
The
the
on a
our
nonpathogenic FA-producing
other, yet unidentified, regulatory mechanisms
the phlA promoter
DAPG
by
be
fluorescens
interactions. As
tomato-pathogenic
Our results show that
the observation that the
CHA638. PhlF is
P.
repressed phlA expression
strain of F. oxysporum represses phlA in CHAO in the wheat
this
in
Fusarium strain
a
strain 798 also
FA-producing
has been shown to repress DAPG
hydroponic
expression
rhizosphere.
the control treatment without Fusarium added. To
of wheat without any
rhizosphere
experiments,
CHAO in the wheat
a
(FA)
this is the first report to address the
for biocontrol in the
lycopersici
to
shown
as
rhizosphere.
and 798
(FA")
failed and
rhizosphere,
Interactions between F. oxysporum strains and CHAO in the wheat
F. oxysporum strains 242
expression
(33),
an
increased release of root
in which
interpreted
no
as a
pathogenic
direct effect of
inducer that binds to PhlF and
thereby
prevents the interaction of the repressor protein with the phi promoter. The fact that the
presence of strain 242 did not alter phlA
expression
hypothesis.
69
in the phlF mutant CHA638 supports this
Chapter 3
FA
strains ofF. oxysporum affect DAPG
vitro, phlA expression of the phlF mutant
In
type (Table 2). However,
CHA638
producing
compared
expression
wheat roots,
on
phlA
CHA638
was seven-
not
was
rhizosphere
of wheat
in the wild-
eightfold overexpressed
to
with CHAO. This observation suggests that PhlF
of the DAPG operon in the
higher than
expression
more
generally
profoundly
in strain
restricts
than in
liquid
cultures.
Implications
for biocontrol. Even
had
no
key
factor in biocontrol of beneficial bacteria
pathogenic impact
on
in
nearly universally present
be found in almost any soil
secondary
the
though
plants,
the
fungi
the F. oxysporum strains used in this
did influence the
co-occurring
soil, which suggests that
sample
taken. The
at
least
suppressive
metabolite critical in biocontrol may affect the
biocontrol bacteria
against
consideration when
using
any soil-borne
pathogen.
biocontrol strains in
in the
production
rhizosphere.
some
of DAPG,
performance
on
the
a
F. oxysporum is
FA-producing
effect of FA
study
strains will
production
of
a
of FA-sensitive
This fact should be taken into
agricultural systems
where FA
producers
are
present. Consistent biocontrol of soil-borne diseases might be achieved by applying beneficial
microorganisms,
or
mutants
formulations offer another
strains
capable
repressors
of
thereof
approach to improve
detoxifying
(e.g. PhlF)
(e.g. phlF-mutants),
could
that
biocontrol
are
performance: coapplication
FA and amendments with zinc
improve
the level and
insensitive to FA. Efficient
reliability
or a
with
of substance that neutralizes
of biocontrol
(14, 45).
Literature Cited
1.
Abbas, H. K., Duke, S. O., Shier, W. T., Badria, F. A., Ocamb, C. M., Woodward, R. P., Xie, W.,
and
Mirocha, C. J. 1997. Comparison of ceramide synthase inhibitors with other Phytotoxins
produced by Fusarium species.
2.
J. Nat. Toxins 6:163-181.
Abbas, H. K., Mirocha, C. J., Kommedahl, T., Vesonder, R. F., and Golinski, P. 1989. Production
of trichothecene and non-trichothecene
Minnesota
3.
(USA). Mycopathologia
108:55-58.
by injection
into fertile chicken egg.
Mycopathologia
B-l and
129:29-35.
Bacon, C. W., Porter, J. K., Norred, W. P., and Leslie, J. F. 1996. Production of fusaric acid by
Fusarium
5.
isolated from maize in
Bacon, C. W., Porter, J. K., and Norred, W. P. 1995. Toxic interaction of fumonisin
fusaric acid measured
4.
mycotoxins by Fusarium species
Bangera,
for
species. Appl.
M.
synthesis
Environ. Microbiol. 62:4039-4043.
G., and Thomashow, L. S. 1999. Identification and characterization of a gene cluster
of the
fluorescens Q2-87.
polyketide
antibiotic
2,4-diacetylphloroglucinol
J. Bacteriol. 181:3155-3163.
70
from Pseudomonas
Chapter 3
FA
producing
strains ofF. oxysporum affect DAPG
genus Fusarium. Commonwealth
expression
6.
Booth, C. 1971. The
7.
Braun, R. 1960. Über Wirkungsweise und Umwandlungen der Fusarinsäure. Phytopath. Z.
Agricultural Bureau,
London and
Reading.
39:197-241.
8.
Capasso, R., Evidente, A., Cutignano, A., Vurro, M., Zonno,
Fusaric and
Phytochemistry
9.
methyl
C, and Bottalico A. 1996.
esters from Fusarium nygamai.
41:103 5 -103 9.
Chakrabarti, D. K., and Chaudhary, K. C. B. 1980. Correlation between virulence and fusaric acid
production
10.
acids and their
9,10-dehydrofusaric
M.
in Fusarium oxysporum f. sp. carthami.
Phytopathol.
Z. 99:43-46.
Davis, D. 1969. Fusaric acid in selective pathogenicity of'Fusarium
oxysporum.
Phytopathology
59:1391-1395.
11.
Delany, I., Sheehan,
production
Fl 13:
12.
of the
M.
M., Fenton, A., Bardin, S., Aarons, S., and O'Gara, F. 2000. Regulation of
antifungal
metabolite
of phlF
genetic analysis
as a
2,4-diacetylphloroglucinol
transcriptional
in Pseudomonas fluorescens
repressor. Microbiol.
Reading.
146:537-546.
Dowd, P. F. 1988. Toxicological and biological interactions of the fungal metabolites fusaric acid
and
kojic
acid with xenobiotics in Hehothis
zea
and
Spodoptera frugiperda (J.
E.
Smith).
J. Chem.
Ecol. 15:249-254.
13.
Dowling,
D.
N., and O'Gara, F. 1994. Metabolites of Pseudomonas involved in plant disease.
Trends Biotechnol. 12:133-141.
14.
K., and Défago, G. 1997. Zinc improves biocontrol of Fusarium
Duffy,
B.
tomato
by Pseudomonas fluorescens
inhibitory to
15.
Duffy,
B.
bacterial antibiotic
and represses the
production
biosynthesis. Phytopathology
crown
and root rot of
of pathogen metabolites
87:1250-1257.
K., and Défago, G. 1999. Environmental factors modulating antibiotic and siderophore
biosynthesis by Pseudomonas fluorescens
biocontrol strains.
Appl.
Environ. Microbiol. 65:2429-
2438.
16.
Dunne, C, Delany, I., Fenton, A., and O'Gara, F. 1996. Mechanisms involved in biocontrol by
microbial inoculants.
17.
Egli,
T. A. 1969. Studies
lycopersici
18.
Agronomie
the influence of heavy metals
and progress of tomato wilt.
Phytopathol.
on
Fusarium oxysporum f. sp.
Z. 66:223-253.
Fenton, A. M., Stephens, P. M., Crowley, J., O'Callaghan, M., and O'Gara, F. 1992. Exploitation
of gene(s) involved in
capability to
19.
on
16:721-729.
a
2,4-diacetylphloroglucinol biosynthesis
Pseudomonas strain.
Gapillout, I., Milat,
cultivars resistant
M.
or
Appl.
to confer
biocontrol
Environ. Microbiol. 58:3873-3878.
L., and Blein, J. P. 1996. Effects of fusaric acid
susceptible
a new
to Fusarium oxysporum f. sp.
on
lycopersici.
cells from tomato
Eur. J. Plant Pathol.
102:127-132.
20.
Gäumann, E., Naef-Roth, S., and Kobel, H. 1952. Über Fusarinsäure, ein zweites Welketoxin des
Fusarium
lycopersici
Sacc.
Phytopathol.
Z. 20:1-38.
71
Chapter 3
21.
FA
producing
negative, plant-associated bacteria.
Jullien, M. 1988. Effects of the
in
23.
mesophyll
on
use
officinalis.
Plant
Physiol.
in gram-
Biochem. 26:713-722.
Keel, C, and Défago, G. 1997. Interactions between beneficial soil bacteria and
ecological impact,
p. 27-46. In: A. C.
Gange
and V. k. Brown
root
pathogens:
(ed.), Multitrophic
Science, London, England.
Keel, C, Voisard, C, Berling, C. H., Kahr, G., and Défago, G. 1989. Iron sufficiency,
prerequisite
for the
CHAO under
25. Keel
suppression
gnotobiotic
as
antagonists
diacetylphloroglucinol
King,
by Pseudomonas fluorescens
of tobacco black root rot
conditions.
Phytopathology
a
strain
79:584-589.
C, Wirthner, P., Oberhansli, T., Voisard, C, Burger, U., Haas, D., and Défago, G. 1990.
Pseudomonads
E.
in the
of plant
pathogens
suppression
in the
rhizosphere:
role of the antibiotic 2,4-
of black root rot of tobacco.
Symbiosis
9:327-341.
O., Ward, M. K, and Raney, D. E. 1954. Two simple media for the demonstration of
pyocyanin
27.
for
pVSl replicon
Fusarium spp. toxins and selection of crude toxin resistant strains
interactions in terrestrial systems. Blackwell
26.
the minimal
Mol. Plant-Microbe Interact. 13:232-237.
cell cultures of Asparagus
Mechanisms and
24.
expression
Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C, Wade, J., Walsh, U., O'Gara, F., and Haas,
D. 2000. Small stable shuttle vectors based
22.
strains ofF. oxysporum affect DAPG
and fluorescin. J. Lab. Clin. Med. 44:301-307.
Komada, H. 1975. Development of a selective medium for quantitative isolation of Fusarium
oxysporum from natural soils. Rev. Plant Prot. Res. 8:114-125.
28.
Luz, J. M., Paterson, R. R. M., and Brayford, D. 1990. Fusaric acid and other metabolite
production
29.
Mycotoxicology.
The
Pennsylvania
production
State
Mégnégneau, B., and Branchard,
M. 1988.
Fusarium
University Press, University
Toxicity
Physiol.
species. Identity
Park and London.
on
its disease
of fusaric acid observed
on
callus cultures of
Biochem. 26:585-588.
Miller, J. H. 1992. A Short Course in Bacterial Genetics: A laboratory manual and handbook for
Escherichia coli and related bacteria. Cold
Spring
Harbor
Laboratory Press, Plainview,
NY.
Notz, R., Maurhofer, M., Schnider-Keel, U., Duffy, B., Haas, D., and Défago, G. 2001. Biotic
factors
affecting expression
of the
2,4-diacetylphloroglucinol biosynthesis
Pseudomonas fluorescens biocontrol strain CHAO in the
34.
Microbiol. 11:141-144.
82:190-195.
various Cucumis melo genotypes. Plant
33.
Appl.
in Pseudomonas fluorescens strain CHAO
suppressive capacity. Phytopathology
32.
Lett.
Maurhofer, M., Keel, C, Schnider, U., Voisard, C, Haas, D., and Défago, G. 1992. Influence of
enhanced antibiotic
31.
vasinfectum.
Marasas, W. F. O., Nelson, P. E., and Tousson, T. A. 1984. Toxigenic
and
30.
in Fusarium oxysporum f. sp.
Nowak-Thompson, B., Gould,
diacetylphloroglucinol by the
S. J., Kraus, J., and
gene
phlA
in
rhizosphere. Phytopathology
Loper,
91:873-881.
J. E. 1994. Production of 2,4-
biocontrol agent Pseudomonas fluorescens Pf-5. Can. J. Microbiol.
40:1064-1066.
72
Chapter 3
35.
FA
and
pineal gland
expression
Fusarium
corn, and feeds toxic to livestock and the neurochemical effects in the brain
of rats. J. Nat. Toxins 3:91-100.
Sambrook, J., and Russell, D. W. 2001. Molecular cloning: A laboratory manual, 3rd ed. Cold
Spring
37.
strains ofF. oxysporum affect DAPG
Porter, J. K, Bacon, C. W., Wray, E. M., and Hagler Jr, W. M. 1995. Fusaric acid in
moniliforme cultures,
36.
producing
Harbor
Laboratory Press,
Cold
Spring Harbor,
N.Y.
Savard, M. E., Miller, J. D., Ciotola, M., and Watson, A. K. 1997. Secondary metabolites
produced by
a
strain of Fusarium oxysporum used for
Striga
control in West Africa. Biocont.
Science Technol. 7:61-64.
38.
Schnider-Keel, U., Seematter, A., Maurhofer, M., Blumer, C, Duffy, B., Gigot-Bonnefoy, C,
Reimmann, C, Notz, R., Défago, G., Haas, D., and Keel, C. 2000. Autoinduction of 2,4-
diacetylphloroglucinol biosynthesis
repression by the
39.
in the biocontrol agent Pseudomonas fluorescens CHAO and
bacterial metabolites
salicylate
and
pyoluteorin.
J. Bacteriol. 182:1215-1225.
Shanahan, P., O'Sullivan, D. J., Simpson, P., Glennon, J. D., and O'Gara F. 1992. Isolation of 2,4-
diacetylphloroglucinol
from
a
fluorescent
pseudomonad
and
investigation
of physiological
parameters influencing its production. Appl. Environ. Microbiol. 58:353-358.
40.
Sharifi-Tehrani, A., Zala, M., Natsch, A., Moënne-Loccoz, Y,, and Défago, G. 1998. Biocontrol of
soil-borne
fungal plant
pseudomonads
diseases
by 2,4-diacetylphloroglucinol-producing
with different restriction
profiles
fluorescent
of amplified 16S rDNA. Eur. J. Plant Pathol.
104:631-643.
41.
Smith, T. K, McMillan, E. G., and Castillo, J. B. 1997. Effect of feeding blends of Fusarium
mycotoxin-contaminated grains containing deoxynivalenol
consumption
42.
and fusaric acid
on
growth
and feed
of immature swine. J. Anim. Sei. 75:2184-2191.
Smith, T. K, and Sousadias, M. G. 1993. Fusaric acid
content of swine feedstuffs. J.
Agr.
Food
Chem. 41:2296-2298.
43.
Stutz, E. W., Défago, G., and Kern, H. 1986. Naturally occurring fluorescent pseudomonads
involved in
44.
suppression
of black root rot of tobacco.
Thomashow, L. S., and Weiler, D. M. 1996. Current concepts in the
biological
control: Mechanisms and
N. T. eds. Plant-Microbe
45.
antifungal
metabolites.
biological
by
a
Pages
76:181-185.
use
of introduced bacteria for
187-235 in:
Stacey, G., Keen,
Interactions, vol. 1. Chapman & Hall, New York, NY.
Toyoda, H., Hashimoto, H., Utsumi, R., Kobayashi, H.,
fusaric acid
46.
Phytopathology
and
Ouchi, S. 1988. Detoxification of
fusaric acid-resistant mutant of Pseudomonas solanacearum and its
control of Fusarium wilt of tomato.
Phytopathology
application
to
78:1307-1311.
Voss, K. A., Porter, J. K, Bacon, C. W., Meredith, F. I., and Norred, W. P. 1999. Fusaric acid and
modification of the subchronic
toxicity
to rats of fumonisins in F.
Food. Chem. Toxicol. 37:853-861.
73
moniliforme
culture material.
Chapter 3
47.
producing
strains ofF. oxysporum affect DAPG
expression
Yabuta, T., Kambe, K, and Hayashi, T. 1934. Biochemistry of the bakanae-fungus. I. Fusarinic
acid,
a new
Abstr.
48.
FA
product
of the
bakanae-fungus.
J.
Agr.
Chem. Soc.
Japan.
10:1059-1068.
(Chem.
29:1132).
Yuan, Z., Cang, S., Matsufuji, M., Nakata, K, Nagamatsu, Y., and Yoshimoto, A. 1998. High
production
grown
on
of pyoluteorin and
ethanol
as a
2,4-diacetylphloroglucinol by Pseudomonas fluorescens
sole carbon
source.
J. Ferment.
74
Bioeng.
86:559-5.
S272
Chapter 4
Differential
2,4-Diacetylphloroglucinol
Production
by
Two
Ecologically
and
Genetically
Different Biocontrol Pseudomonads in response to Fusaric Acid
75
76
Chapter 4
Differential response in DAPG
production
of two
pseudomonads
Abstract
Production of 2,4-diacetylphloroglucinol
biocontrol traits of the two
genetically
strains CHAO and Q2-87. DAPG gene
of either lacZ
and
(CHAO
promoter-proximal
Q2-87)
high
Fusarium oxysporum f. sp.
repression
expression
inaZ
or
ecologically
(CHAO)
to
show
a
radicis-lycopersici
mediated
had
no
was
DAPG
expression
expression
repressed phlA expression
22
measured
using transcriptional
e.g. ARDRA group
fusions
the structural gene phlA, which is the
to
(FORL22)
region
of Q2-87
with inaZ
in CHAO in
a
in
of either CHAO
as a
appeared
(FA)
to
or
reporter gene. By
or
Q2-87
less sensitive to the
was
of CHAO. In
significant influence onphlA expression
background,
different Pseudomonas fluorescens
amendments of FA and FORL22 could be shown with
through
demonstrate in vitro that DAPG
FORL22
important
effect of either fusaric acid
repressive
lacZ. Two fusions of lacZ to the phlA promoter
effect of FA than
was
of the most
one
gene in the phlACBDE cluster of both strains. This promoter
transcribe at levels too
contrast,
and
is
(DAPG)
were
used to
repressing
addition, amendments of FA-producing
tomato-rockwool system whereas FORL22
Q2-87. Correlation with the genetical
affiliation, is discussed.
Introduction
It is
(DAPG)
is
now
one
in biocontrol
biosynthetic
33)
and its
factor
g
(35, 39).
well established that the
of the most
against
soilborne
antimicrobial
metabolite
compounds
2,4-diacetylphloroglucinol
of fluorescent
pseudomonads
plant pathogens (4, 5, 12, 21, 18, 17, 32, 42, 44).
The DAPG
locus is conserved among root associated fluorescent Pseudomonas strains
expression
and the
The
production
important
polyketide
is
subject
to
stationary phase
complex regulations,
factor
specific transcriptional
g
play
a
role in
A
global
control
on
factors such
regulating
repressor PhlF exerts
of P. fluorescens Q2-87, Fl 13, and CHAO
region (1, 7, 38).
g
a
negative
by binding
biocontrol factors
the
to
including
as
the
housekeeping
production
control
(20,
on
g
of DAPG
DAPG
thephlA-phlF intergenic
DAPG is exerted
by
the
two-component system gacS/gacA (reviewed in 15) involving the small RNA binding protein
RsmA and
a
regulatory RNA, RsmZ,
Environmental
signals
at a
posttranscriptional
that act upon DAPG
level
regulatory
(2, 14).
cascades in biocontrol
Pseudomonads have been identified for different strains in several studies.
factors that
commonly
modulate DAPG
confront bacterial inoculants in
expression
in the
rhizosphere.
agricultural systems
Notz et al.
77
(31)
Complex
biotic
have been
have shown that the
seen
to
Chapter 4
Differential response in DAPG
of DAPG
expression
and that it is stimulated
metabolites
also
play
that
analog
an
autoinduced and
have
an
impact
pathogenicity
species
and
cultivar,
ultimum. Microbial
plant pathogen Pythium
regulation by /V-acyl-homoserine
is not
synthesis
fluorescens
regulated by
production
signal
CHAO is affected
repressed by salicylic
on
pseudomonads
regulation
of antimicrobial
biocontrol bacteria. One of the best-documented systems is the cell-
DAPG
of P.
host age, host
by
molecules in the
signal
as
solvent-extractable extracellular
synthesis
DAPG
role
important
an
gene
fluorescens CHAO,
infection with the
during
compounds produced by
density-dependent
genes is influenced
biosynthetic
of two
production
acid and
lactones
its
own
might
be involved
(FA)
is
pyoluteorin (Pit) (38). Fungal
produced by
implies
(14).
extracellular metabolites: it is
of metabolites relevant for biocontrol. The
factor fusaric acid
In P.
AHL's but recent evidence
molecule
by
(AHL's) (11).
metabolites
can
phytotoxic
different Fusarium strains and acts
as a
potent inhibitor of DAPG synthesis in P. fluorescens CHAO (9, 30, 38). By contrast, enhanced
production
DAPG
roots
without
was
the presence of the FA" F. oxysporum strain 242
triggered by
interactions
pathogenic
(30).
The type of carbon source,
and minerals have been shown to modulate DAPG
some
environmental factors
example,
can
be strain
while others have
dependent
amendments of zinc enhanced DAPG
in
production
an
amino
nitrogen,
production (8, 32, 39, 45).
ecologically
wheat
acids,
The effect of
general
a
on
effect. For
and
genetically
diverse collection of 42 P. fluorescens strains but the level of stimulation varied among
strains
stimulated DAPG
(8). Furthermore, glucose
exception
analysis
of the Irish isolate Fl 13
of restriction patterns of
(8, 39).
and Pit and groups 2 and 3 strains
were
not
linked to any
(ARDRA
group
'-lacZ
additional
2)
work,
were
P.
used
are
affiliated with
superior biocontrol
has
produced
producers only (20). Response
(8). However,
cluster
(ARDRA)
1 contained all strains that
group of strains
monitor phlA
transcriptional
fluorescens
as
models to
fusion
transcriptional
quantitatively
phlA
DAPG
for 16S rRNA
by
to
DAPG
zinc and
there is evidence that
abilities than those of
(40).
In the current
phlA
were
particular
strains of ARDRA group 2 and 3
group 1
(20). Group
in all strains with the
characterization
genotypical
amplified DNA coding
clustered these strains into 3 groups
glucose
The
production
the
was
(ARDRA
regulation
group
of DAPG
1)
and Q2-87
production.
A
constructed and used earlier to
of CHAO in culture
(38).
We have
now
developed
fusions of the reporter genes inaZ and lacZ to the structural gene
constructs to
different biocontrol
study
pME6710
expression
of strains CHAO and Q2-87,
reporter gene
on
strains CHAO
respectively.
The aim of the present
study phlA expression patterns
pseudomonads
CHAO and
Q2-87.
78
We
in two
study
was
ecologically
provide
to use
and
these
genetically
evidence that CHAO and
Chapter 4
Differential response in DAPG
Q2-87 respond differently
sp.
radicis-lycopersici
amendments of FA in vitro and to
to
strain 22
of inaZ and lacZ
disadvantages
(FORL22)
as
in the tomato
of two
production
pseudomonads
FA-producing F.
oxysporum f.
The
and
rhizosphere.
advantages
reporter genes for monitoring DAPG biosynthetic genes
are
discussed.
Materials and Methods
and inoculum
Microorganisms
in this
study
37°C
was
,
on
respectively,
grown in
a
King's
B
(KB)-agar (22)
at 27°C
and Luria Bertani
if not mentioned otherwise. For gene
minimal
glucose-ammonium
medium
were
(LB)-agar (36)
expression studies,
(OSG)
used
plasmids
described in Table 1. P. fluorescens and Escherichia coli strains
are
cultivated
routinely
The bacterial strains and
production.
at
P. fluorescens
described in Schnider-Keel et al.
(38).
F. oxysporum f. sp.
INRA, Dijon, France)
was
produced by inoculating
growing
shaker
radicis-lycopersici
strain 22
routinely
2% malt agar. Inoculum of FORL22
grown
250 ml malt broth
on
FORL22 malt agar culture. Cultures
biomass
(180 rpm). Fungal
centrifugation
DNA
at 4000 rpm
or
x
g)
were
and
for 15
routinely
was
Inserts
microconidia)
gel electrophoresis,
DNA
EcoRI restriction site
amplification
stability
DNA Purification
were
used for
added to
in strain CHAO
was
primers
GTGGATCCTTCTGACTTGTTCGCTCTCG-3')
and
AGGTTC-3')
and
or
E. coli
primer Q2-87phlF (30-mer
reverse
blender.
plasmids.
alkaline
System (Catalysis,
by CaCb
were
treatment
(36).
generated by
A BamHI
respectively.
reverse
PCR
or a
PCR
5'-TC
primer phlAproml (30-mer
For PCR
amplification
5'-
in
5'-TCGTGAATTCGCAGCAGGAAATTG
primer Q2-87phlA (30-mer
79
by
a
primer phlAprom2 (30-mer
GTCTGGATCCGTCATAGGGATTGGTGCAGG-3') (Fig. 1).
strain Q2-87 forward
in
of constructed
of CHAO and Q2-87
with forward
rotary
restriction, dephosphorylation,
of CHAO and Q2-87,
performed
actively
an
at 24°C on a
Polymerase (Catalysis, Wallisellen, Switzerland).
was
was
washed, collected by
pseudomonads
and transformation ofE. coli
containingphlA-phlF mtergenic regions
using Promega Pfu
and
isolated from fluorescent
Promega Wizard® Plus Minipreps
agarose
was
days
of
Alabouvette,
min, and then briefly homogenized
Wallisellen, Switzerland). Standard techniques
ligation,
plugs
incubated for 14
manipulations, plasmid mobilization,
Plasmid DNA
lysis (36)
(2,200
(mycelia
with three 0.7-cm
(2%)
of C.
(FORL22) (a gift
5'- GTCTGAATTCGGGAGTCATAT
Chapter 4
Differential response in DAPG
Table 1. Bacterial strains and
Strain
used in this
plasmids
of two
production
pseudomonads
study
Genotype/phenotype
Reference
CHAO
Wild-type
41
CHA638
pWF::Q-Km, Kmr
38
Q2-87
Wild-type/Phl+,
44
or
plasmid
Strains:
P. fluorescens
Pit
DH5a
AthsdRU
F
FendA1hsdR17
E. coli
end
i
(rK
mK
) supE44
thi-1 recA1
36
gyrA96relA1
<|)80d/acZAM15X"
leu thi pro
HB101
SrrT lacY rK mK recA
3
Plasmids:
RK2-Mob+RK2-Tra+, Kmr
pRK2013
OriColEI
pPROBE'-gi/p[tagless]
Derivative of
pJL6230
pPROBE'-g/p[tagless]
Kmr
pJL6244
pJL6230
Kmr
plasmid pBBR1 MCS-2, Kmr
with
gfp
removed
29
by
a
Hind\\\
This
study
site,
This
study
phlA'-
This
study
deletion,
with the 3.8 kb InaZ gene at the Hind\\\-BamH\
pJL6244 with a 1.1 kb fragment
transcriptional fusion, KrrT
pSLII8
13
of CHAO
containing
a
inaZ
pME6016
vector for construction of
Cloning
derived from
transcriptional
lacZ
38
fusions,
pME6010, Tcr
pME6016 with a 1.1-kb BglW fragment of CHAO, containing a
phlA'-lacZ transcriptional fusion at the BglW site in phlA, Tcr
pME6710
pME607E
pME6016 with a 1.1 kb fragment
transcriptional fusion, Tcr
Kmr: kanamycin, Tcr: tetracycline
of Q2-87
containing
a
38
This
phlA'-
study
lacZ
a
resistances:
GGGTTGGTG-3')
from GenBank
used
were
(accession
no.
(Fig. 1).
All
synthesized by Microsynth, Balgach,
agars
gels using QIAquick
respective
confirmed
into P.
to
standard
P.
fluorescens
broth
was
repeated
strains
after
ligation
from sequences available
were
CHAO
or
Q2-87
was
respective
of newly constructed
evaluated
vector.
days. Samples
were
Plasmids
or
by growing CHA0/pSLII8
digested
techniques
were
and
was
introduced
pME607E
in
and Q2-
Subsequently,
24 h-old bacterial culture and this
50 ml of LB-
procedure
taken at the time of inoculation. Bacterial
80
and
from
electroporation according
plasmids pSLII8
in LB-broth without antibiotic selection at 27°C.
a
purified
(Qiagen AG, Basel, Switzerland)
into the
Primers
Q2-87) (1,38).
PCR-products
by triparental mating, using pRK2013,
inoculated with 100 ul of
for 7
Switzerland.
Gel Extraction kit
protocols (36). Stability
87/pME607E
developed
restriction enzymes. Absence of mutations due to PCR
by sequencing
fluorescens
were
AF207529 for CHAO and U41818 for
were
with the
primers
was
growth
was
Chapter 4
Differential response in DAPG
determined
plating
by measuring
OD at 600
with the
supplemented
onto KB agar
and
nm
percentage of antibiotic-resistant colonies
integrity
2
are
the
of the
plasmids
of 3
means
expression
replicate
in vitro.
conferred
activity (INA)
checked
of DAPG and
Assays
gene
was
to
Miller
87/pME607E
were
(28).
determined. At the end of the
experiment
of phlA
'-inaZ
on
was
of
period
pSLII8 according
conferred
hyphlA
LB cultures diluted to
approximately
'-lacZ
2 to
3, mixed with
phase
was
separated
HPLC
using
a
Ca) equipped
(100
x
4
ethyl
from the aqueous
mobile-phase
independent
representative experiment
Influence of FA
87/pME607E
Cultures
were
were
on
or
growth
production
were
or
pME607E
and Q2-
as
120
aliquots
Samples
were
determined
of
taken
by
of DAPG and MAPG.
acidified with 2 M HCl
vigorously
a vacuum
for 1 min. The
centrifuge (HETOVAC,
(Hewlett-Packard Co.,
Palo
Heto
analyzed
Packard Co., Palo
Alto, CA) and
a
with
Alto,
column
(Macherey-Nagel, Düren, Germany)
samples (10 ul)
o-phosphoric
were
organic
silicon-coated filter paper
dissolved in 1 ml of methanol and
were
eluted with
acid in 12 min. DAPG
approximately
flow rate of 0.7 ml/min.
cultures. The results from
is
of 0.1.
liquid Chromatograph (Hewlett
the A270nm- The retention times
of at least three
a
was
with Nucleosil 120-5-C18
a
nm
(10 ml)
and shaken
and dried in
controlled at 50°C. The
min for MAPG with
ice nucleation
inoculated with 1.5 ml
phase by filtering through
detector
from 25 to 100%) in 0.43%
monitoring
acetate
The residue
diode-array
mm) packed
thermostatically
gradient
a
and
of bacterial cultures
Hewlett Packard 1090
with
pME6710
OD at 600
Fig.
and phlA
Loper and Lindow (26)
to
on
in
presented
quantified using high-performance liquid
was
10 ml of
Equipment, Denmark).
experiment,
Erlenmeyer flasks containing
was
phlA expression,
at 600 nm,
(Macherey-Nagel, Düren, Germany),
Lab
by measuring
200 h for evaluation of bacterial
chromatography (HPLC). Aliquots
pH
an
by replica
twice.
fluorescens CHA0/pSLII8, CHA0/pME6710,
Production of DAPG and MAPG
to
Data
performed
was
grown at 24°C and 160 rpm in 300 ml
measuring optical density
mapping.
evaluated
ml of OSG without selective antibiotics. The medium
over a
evaluated
was
monoacetylphoroglucinol (MAPG) production
hyphlA
exponential growth-phase
pseudomonads
antibiotic. After 24 h at 27°C the
restriction enzyme
by
Expression
P.
of plasmids
respective
was
cultures. The
measuring ß-galactosidase activity
according
stability
of two
production
a
linear methanol
was
detected
by
7.4 min for DAPG and 2.0
Reported
values
duplicate experiments
are
the
were
means
similar and
presented.
phlA'-lacZ expression. P. fluorescens CHA0/pME6710
and Q2-
grown in 30 ml of OSG without selective antibiotics at 24°C and 160 rpm.
amended with 150 ul stock solution of
81
synthetic FA,
which
was
prepared
Chapter 4
Differential response in DAPG
immediately prior to
use
it in 20%
by dissolving
ethanol
(vol/vol)
of two
production
(pH 6.5),
concentration of 500 uM in the medium. Control treatments received the
solvent.
Exponential growth-phase
optical density
OD at 600
Galactosidase activities
phase.
the
Bacterial
growth
of three
means
experiments.
The
determined
was
87/pME607E
strains
(pSLII8
both
a
and
second
pregerminated
containing
20 rockwool cubes
fluorescens
tomato
seeds
on
was
4
x
days
at 24°C in
cm
deep,
Grodania
2.5
x
106
CFU per
ml, and,
if
was
at 22°C
four trays each
(160
design.
After 2 and 5
days 8,
and 12, trays
water was
tomato
added
seedlings
scale of 0 to 4
(0
as
lesions around the
followed
plants
20
and
were
by
were
days, approximately
keep
carefully
per
ml,
or
hypocotyl
both. The
and
1
arranged
in
a
on
a
chambers with
growth
randomized
250 ml of OTCMG1
were
few brown lesions around the
the roots, 3
82
=
extensive
complete
block
added to each tray. At
between, sterile deinonized
the rockwool saturated. Fourteen
=
pregerminated
8 h darkness at 18°C. Treatments consisted of
days
removed from the rockwool. Disease
symptomless,
with FORL22 at
appropriate,
amended with 250 ml of OTCMG2. In
needed to
were
=
plus mycelial fragments
uE/m2/s)
containing
A/S, Hedehusene,
inoculated with strains of P.
in the indentations of the rockwool and incubated in
light
darkness. Plastic trays
saturated with 800 ml of sterile OTCMG1
seeds
placed
surface
were
rinsed several times with sterile deionized
rockwool, the nutrient solution
approximately
on
the Office of Horticultural Production of Geneva, Switzerland
designed by
the
AG, Zuchwil, Switzerland)
surface
cm
and Q2-
esculentum Mill.
(Tycopersicum
0.85%> water agar for four
(3.5
onphlA
'-inaZ and phlA '-lacZ reporter
microconidia
16 h
using
carried out in the
were
approximately 106
were
are
independent
calculated
were
ß-
values
Reported
derived from 5
values
an
evaluated for their response to FORL22
were
hypochlorite,
autoclaved. The rockwool
saturating
at
nm.
the influence of FORL22
experiment, phlA
Samen und Pflanzen
water, and
Prior to
are
experiments
experiments,
pME6710)
disinfected for 30 min in 1% sodium
(40).
4
diluted to
of the
stationary growth
CHA0/pME6710, CHA638/pME6710,
experiments,
'Supermarmande', Wyss
nutrient solution
Fig.
R-squared (R )
Two kinds of
first set of
evaluated. In
was
were
the OD at 600
by measuring
amount
same
were
and
final FA
a
used for inoculation,
throughout the exponential
trendline and
a
fluorescens
tomato roots. For
Denmark)
were
give
to
97 SR-2.
of CHAO
constructs
aliquots
cultures. Data shown in
replicate
rockwool-tomato system. In
of P.
determined
were
rockwool-bioassays.
Tomato
expression
of 0.1 and 1500-ul
nm
logarithmic
Microsoft® Excel
LB cultures of the bacterial strains
pseudomonads
crown
after
severity
planting,
was
hypocotyl,
and root
2
rated
=
on a
bigger
necrosis,
4
=
Chapter 4
plant
dead
Differential response in DAPG
nearly so).
or
In all
sterile saline solution and
ml of the
media
mechanically
resulting suspension
(19).
with the
experiments,
Plasmid
respective
pME607E
stability
determined
in sterile flasks with 10 ml of
serial dilutions
by plating
by replica plating
200 ul of the root
agar
supplemented
by plasmids pME6710
Unit values
suspension.
appropriate
on
onto KB agar
conferred
ß-Galactosidase activity
using
pseudomonads
shaken for 15 min at 300 rpm. Numbers of CFU per
evaluated
was
antibiotic.
measured
was
was
placed
roots were
of two
production
and
calculated
were
o
according
to
Miller
inoculated with
(28)
wild-type
background activity
freezing
and
expressed
strains
as
the
lacking
The
weighed.
plasmids
CFU. Control treatments of plants
always
were
from calculations. Number of ice nuclei
assay at -5°C and ice nucleation
previously (26).
units per 10
Shoots
was
washed with tap water, blotted
were
experiments
activity (INA)
were
repeated
was
determined
calculated
dry
and results of both
used to subtract
by
the
droplet
described
as
with paper tissues and
experiments
shown in Tables
are
2 and 3.
Results
Construction and
removed from
inaZ
was
one
the
insert
with BamHI restriction sites
mphlA
sequenced to
direction
a
pME607E.
as
by
a
EcoRI site. The PCR
site of pME6016.
product
Screening
the phlA '-inaZ construct
Parental vectors of pSLII8
the addition of
was
was
pJL6230.
A 0.9 kb-
generated by
PCR. Primers
PCR-product was purified,
were
mapping
pJL6244.
screened for the presence of
and PCR. Positive clones
possibly produced during
with the
(Fig. 1)
was
exception
purified, digested
that
CHAO
retention
was >
dropped
maintained
90.7
were
PCR. For the
stably:
antibiotics, retention of pPROBE'-g^? by
Retention of pJL6244, which contains the
promotorless
83
as
were
was
an
into the EcoRI-
described above. Retention of
after 68
after 68
CHAO
extended with
ligated
3.1% after 35
±
to 8.7 ± 6.4%>
more
primers
with EcoRI, and
and selection of clones occurred
were
pSLII8, gfp
kb-fragment containing the Q2-87-phlA promoter region
pSLII8 by
(Fig. 2A). Gradually,
in
resulting
the ends. The
restriction enzyme
0.9
described above
on
To obtain
üz'raüll-deletion which resulted in
pJL6244. Colonies
exclude those with mutations
construction of pME607E,
broth
and
CRAO-phlA promoter region (Fig. 1)
with BamHI, and cloned into
generated
pSLII8
inserted at the HindlU-BamHl-site of pJL6230
designed
digested
of
pPROBE'-g^>[tagless] (29) by
fragment containing
were
stability
generations
generations (Fig. 2A).
generations
was
inaZ gene,
in LB
97.7
was
±
in LB without
2.1%
46.3
±
(Fig. 2A).
12.0 after 68
Chapter 4
Differential response in DAPG
production
of two
pseudomonads
phlAproml
phlAprom2
CHAO
phlF
phlA
Q2-87phlA
Q2-87phlF
Q2-87 -4
phlF
phlA
1 kb
Fig.1. Physical
generations
87
location of the
of growth
much
were
pME607E
more
pME607E after
mapping (data
£.100
±
amplify phlA-phlF intergenic region.
pME6016 and its derivative pME607E introduced into Q2-
generations,
5.8% and 86
68 and 65
not
2 A).
used to
stable. After 65
still 92
was
(Fig.
primers
±
retention of plasmids
pME6016 and
6.9%, respectively (Fig. 2B). Integrity of pSLII8 and
generations, respectively,
was
demonstrated
by
restriction enzyme
shown).
-
c
o
I
75-
<d
*-»
<d
£
50-
E
!8
25
-
-m—
CHAO/pPROBE-gfp
-e—
CHA0/pJL6244
-A^-
CHA0/pSLII8
o—
Q2-87/pME6016
Q2-87/pME607E
_
Q.
10
30
50
70
10
Generations
30
50
70
Generations
Stability of plasmid pSLII8 (A) and pME607E (B) and their parental vectors in P. fluorescens
Q2-87, respectively, was evaluated by growing fluorescent pseudomonads in LB-broth
without antibiotic selection at 27°C. Subsequently, 50 ml of LB-broth was inoculated with 100 pi of a
24 h-old bacterial culture and this procedure was repeated for 7 days. Samples were taken at the time
of each inoculation. Bacterial growth was determined by measuring OD at 600 nm and stability of
plasmids was evaluated by replica plating onto KB agar supplemented with the respective antibiotic.
Values are the means of at least three replicate cultures. Error bars represent the standard deviation
of the means; some of the error bars are obscured by graph symbols.
Fig.
2.
CHAO and
84
Chapter 4
CHAO
Differential response in DAPG
production
of two
pseudomonads
(phlA'-inaZ)
Q
o
.E
D)
8
B
m
100
CHAO
100
150
(hours)
Time
Time
1 50
200
1 50
200
1 50
200
(hours)
(phlA'-lacZ)
Q
o
1
D)
m
100
Time
Q2-87
150
100
(hours)
Time
(hours)
(phlA'-lacZ)
Q
o
i
m
0
c<
100
Time
Time
(hours)
(hours)
Expression of transcriptional phlA reporter constructs (A) and production of DAPG () and
(D) in Pseudomonas fluorescens CHAO and Q2-87. Strain CHAO carrying a phlA'-inaZ fusion
on plasmid pSLII8, A and B, strain CHAO carrying a phlA'-lacZ fusion on pME6710, C and D, and
strain Q2-87 carrying a phlA'-lacZ fusion on plasmid pME607E, E and F, were cultivated in OSG
medium at 24°C and 160 rpm. Samples were taken over a period of 200 hours to determine bacterial
growth (•), DAPG and MAPG production, and reporter gene expression. Values are the means of
three replicate cultures. Error bars represent the standard deviation of the means; some of the error
bars are obscured by graph symbols.
Fig.
3.
MAPG
DAPG
and phlA gene
Q2-87/pME607E. Expression
and
INA
and
production
or
ß-galactosidase activity,
Q2-87/pME607E growing
expression
of the phlA
was
in
CHA0/pSLII8, CHA0/pME6710,
biosynthetic
monitored in strains
in OSG medium
85
gene,
as
determined either
by
CHA0/pSLII8, CHA0/pME6710,
(Fig. 3A,C,E). Biosynthesis
of DAPG and
Chapter 4
MAPG
Differential response in DAPG
was
determined in
similar. Cultures grew
for about 50 h after inoculation until
OD600 of 3.9 (CHA0/pSLII8 and CHA0/pME6710, Fig. 3A,C)
cultures went into
87/pME607E, Fig. 3E). Thereafter,
aphlA
'-lacZ
transcriptional
fusion
of two
pME6710
on
pseudomonads
Growth kinetics of all three strains
parallel (Fig. 3B,D,F).
exponentially
production
or an
they
were
reached
an
OD600 of 2.8 (Q2of
stationary growth phase. Expression
in strain CHAO increased from 40 to 3321
o
units per 10
CFU from
Galactosidase
activity
mid-exponential
declined
early stationary growth phase (Fig. 3C). ß-
to
only slowly during stationary growth phase
units per
to 2290
o
10
CFU at 191 h of
growth (Fig. 3C). Similarly, expression
of
aphlA
'-lacZ
transcriptional
o
fusion carried
by pME607E
in strain
Q2-87
rose
from 56 to 2627 units per 10
CFU from
o
mid-exponential
191 h of
early stationary growth phase
to
growth (Fig. 3E).
transcriptional
fusion
exponential growth
expression
-0.36
(Fig.
on
A different
pSLII8
expression pattern
in strain CHAO. INA
log(nuclei/cell).
exponential
to
INA declined thereafter to -2.86
3 A). Production of DAPG and MAPG
was
was
and measurements started with -1.08
reached at the transition of
was
and declined to 1758 units per 10
observed for the phlA '-inaZ
already quite high
in
log(nuclei/cell) (Fig.
early
3 A). Maximal
and reached
stationary growth phase
at 198 h
log(nuclei/cell)
in all three strains in
peaked
CFU at
of growth
early stationary
o
growth phase
and maximal concentrations DAPG
CFU for strains
CHA0/pSLII8, CHA0/pME6710,
were
and
5.23, 6.44, and 9.10 nmol per
10
Q2-87/pME607E, respectively.
o
Maximal concentrations of MAPG
and
CHA0/pSLII8, CHA0/pME6710,
CHA0/pSLII8
of bacterial
and
were
CHA0/pME6710,
0.92, 1.03, and 3.87 nmol per
10
CFU, for strains
Q2-87/pME607E, respectively (Fig. 3B,D,F).
neither DAPG
growth (Fig. 3B,D). By contrast,
nor
in strain
In strains
MAPG could be measured after 70 h
Q2-87/pME607E,
minimal DAPG
o
concentrations
(0.02
nmol per 10
small MAPG concentrations
(0.09
CFU)
nmol per
FA-mediated/>M4-repression
expression
in two
ecologically
and
was
108 CFU)
genetically
trendlines
CHA0/pME6710
were
were
calculated for
1.5, expression of phlA '-lacZ in the
treatment
for
repression by
Q2-87/pME607E
were
as
well
as
growth (Fig. 3F).
Q2-87. To evaluate if phlA
or
CHA0/pME6710
and Q2-
without the addition of 500 uM FA
ß-galactosidase
activities.
Corresponding
0.76 for the treatment without the addition of FA and
growth
of phlA
up to 191 h of
'-lacZ fusions in strains
0.74 with the addition of FA. When
extent
up to 106 h of growth
different P. fluorescens strain is affected
monitored in OSG medium with
(Fig. 4). Logarithmic
R -values for
quantified
in strains CHAO and
differentially by FA, expression ofphlA
87/pME607E
could be
of
CHA0/pME6710
with FA
was
reached
repressed by
an
OD at 600
62%
nm
(Fig. 4A).
of
The
FA went down to 58% at ODöoo of 2.5. R -values to trendlines
0.87 for the treatment without the addition of FA and 0.79 with the
86
Chapter 4
Differential response in DAPG
production
of two
pseudomonads
ACHA0/pME6710
ACHA0/pME6710+FA
o
A
n.
o
o
o
£
4
I
b
o
ro
O
3
o
I
40 %
28 %
Q2-87/pME607E
R7
=
0.87
Q2-87/pME607E+FA
</>
ro
o
2
2
o
ro
O
R2
=
0.79
^ +^0>+
0.5
1.5
2.5
Bacterial
3.5
growth (OD 600)
Fig. 4. Effect of fusaric acid (FA) on the expression of transcriptional phlA'-lacZ fusions
pME6710 and pME607E in the two ecologically and genetically different P. fluorescens
A
(A,
and
A)
and Q2-87
(B,0 ), respectively.
pME607E, respectively,
symbols)
or
with
were
Strains CHAO and
the addition of
synthetic
by
strains CHAO
Q2-87, harboring plasmids pME6710
grown in OSG-medium at 24°C and 160 rpm without
(closed symbols)
carried
FA to
give
a
(open
final concentration of 500 uM
in the medium. Throughout exponential and early stationary growth, ß-galactosidase activities and
OD600 were determined. Values are the means of three replicate cultures. Logarithmic trendlines with
corresponding Revalues are displayed for treatments without (-, R2) and with (—, R2) FA amendment.
The extent of p/?//A-repression through FA is indicated in % at OD600 of 1.5 and 2.5.
addition of FA. At OD6oo of 1.5,
in
repression ofphlA
'-lacZ
was
40% and at OD6oo of 2.5 28%
Q2-87/pME607E (Fig. 4B).
Suppression
of Fusarium
crown
CHA0/pME6710, CHA638/pME6710,
phlA '-lacZ transcriptional
strains
and
fusions. In both
Q2-87/pME607E
experiments,
provided significant protection against F.
of disease index
compared
to
protect the
(Table 2).
was
tomato
(Table 2).
plants significantly
was
fluorescens
expression
and
oxysporum f. sp.
of their
radicis-lycopersici
gave
The PhlF mutant
Expt.
1 and
87
to 91.7%
CHA638/pME6710
did not
wild-type regarding
2, respectively (Table 2).
significant better protection
in terms
Expt. 2, the disease index for
better from Fusarium than its
89.6% and 84.6% in
Q2-87/pME607E
P.
Expt. 1, CHA0/pME6710 lowered the disease index
lowered to 81.3%
index, which
contrast,
In
by
all three fluorescent Pseudomonas
the FORL22 control treatment without bacteria. In
this treatment
disease
and root rot of tomato
than did
the
In
CHA0/pME6710,
with
a
Chapter 4
Differential response in DAPG
production
of two
pseudomonads
Table 2.
Suppression of Fusarium crown and root rot of tomato by Pseudomonas fluorescens CHA0/pME6710,
CHA638/pME6710, and Q2-87/pME607E and expression of their phlA'-lacZ transcriptional fusions
Disease index
of FORL22
(%
v
P fluorescens
91 7
+
CHA638/pME6710
(=
2
Shoot fresh
control)
(%
2
Expt
100
94)
1
Expt
100
(=2 86)
z
weighty
of FORL22
100
±
7 9
81 3
130
±
115 ±
28
89 6
±
2 6
84 6
2 7
±
123 ±
11
121
nd
10
69 9
+
±
5 2
64 8
2 9
±
146 ±
33
128 ±
74 ±
18
65 ±
14
±
372
1518 ±
668
1743 ±
329
789
1053 ±
111
±
42
33 ±
14
81
±
34
24 ±
27
26
(wild-type), CHA638 (phlF mutant), and Q2-87 (wild-type) are carrying a transcriptional phlA'-lacZ
plasmids pME6710 (CHAO, CHA638) or pME607E (Q2-87)
Fusarium oxysporum f sp radicis-lycopersici strain 22 (FORL22) was added to the rockwool system at 4 x 107 microconidia
plus mycelial fragments per plant
After 2 weeks of growth, disease was rated on a scale of 0 to 4, whereas 0
plant healthy and 4 plant dead Disease index
was always 0 in the absence of the pathogen (data not shown)
In the absence of the pathogen, shoot fresh weight per plant was 329 ± 21 mg (Expt 1) and 382 ± 26 mg (Expt 2) The
addition of fluorescent Pseudomonas strains did not influence shoot weight significantly (data not shown)
Values represent the mean of 4 replicates ± standard deviation of the mean nd
not detected
on
=
z
23
P fluorescens strains CHAO
fusion
y
nd
132 ±
-
Q2-87/pME607E
2
19
15
±
Expt
119 ±
1551
+
Q2-87/pME607E
1
Expt
(=218)
117 ±
8CFU)Z
per 10
(units
2
Expt
(=213)
ß-Galactosidase activity
control)
-
CHA638/pME6710
x
100
z
-
CHA0/pME6710
w
1
Expt
+
none
CHA0/pME6710
v
w
FORL22
x
=
=
disease index of 69.9%> in
pseudomonads
15%>).
had
no
Shoot fresh
significant impact
weight
of tomato
2
Expt.
was
plants
on
in all but
shoot fresh
enhanced
weight
treated with
did not differ
weight
and 128%
to 146
significantly between
1
Expt.
Addition of
(115
±
in this treatment
CHA638/pME6710
was
28%>),
(117
±
significantly
respectively. Q2-87/pME607E
Protection in terms of shoot fresh
(Table 2).
treatments
one case:
in
significantly
the FORL22 control and reached 123 and 121%,
enhanced shoot fresh
Treatment with fluorescent
(Table 2).
weight significantly
Expt. 2, shoot fresh weight
higher than
weight
1 and 64.8%> in
enhanced shoot fresh
CHA0/pME6710
whereas in
Expt.
with different P. fluorescens strains
(Table 2).
Significant
FORL22
lowered
were
only
activities
effect of FORL22
was
Expt.
1 and 49.2%> in
Expt.
abolished in the phlF mutant CHA638.
2
(Table 2).
not FORL22 was
added
(Table 2).
were
not
significantly
pME607E
was
was
99.5
growth
assessed
±
lowered
on
tomato
by Q2-87
was
±
4.3%
93.1
±
The
wild-type,
ß-galactosidase activity
of Q2-
the presence of FORL22.
roots, average retention of plasmids pME6710
by replica plating
0.9% and 74.9
retention of pME607E
Values of
through
without
ß-Galactosidase
in the phlF mutant CHA638 than in the
After two weeks of
pME6710
to 62.3%> in
or
where the presence of FORL22
higher
87/pME607E
and
CHA0/pME6710
between treatments with
11 to 13-fold
were
or
ß-galactosidase activity
found with strain
ß-galactosidase activity
repressing
whether
differences in
on
tetracycline
CHAO and
by
4.3%.
88
amended
KB-agar. Stability of
CHA638, respectively, and
Chapter 4
Differential response in DAPG
FA
by
or
FORL22. Table 3 shows
by
fusions of phlA to either inaZ
transcriptional
introduced into CHAO in OSG medium
on
tomato roots
(without
activities conferred
nuclei/CFU)
an
measured.
by
at an OD
OD of 600
nm
or
or
a
comparison
lacZ
(without
or
on
pseudomonads
of 600
nm
on
expression
pSLII8
lower with the amendment of FA
FORL22. In
and
log(ice
logfice nuclei/CFU)
significantly
ß-galactosidase
or
FA)
between -0.72 and -0.64
were
differences in INA could be found
without the amendment of FORL22. In contrast,
pME6710
vitro, ice nucleation
of 1.5 and between -0.25 and -0.17
significant
or
of
with the amendment of 500 uM
with the amendment of FORL22). In
the phlA '-inaZ fusion
no
of the
plasmids, pSLII8
of 3.5. The addition of FA did not alter INA
Similarly,
significantly
of two
of the two reporter genes inaZ and lacZ to reflect />M4-repression
Suitability
mediated
production
at
both ODöoo
with
tomato roots
on
activities
at
were
or
always
vitro, the addition of FA
to
the
o
medium reduced
growth
ß-galactosidase activity
to
from 1254 to 437 units per 10
CFU at
an
o
OD6oo of 1.5 and from 2705
to 1381
the presence of FORL22 lowered
per
108
units per 10
CFU at
ß-galactosidase activity significantly
phlA expression
activity (log(nuclei/CFU))
plasmid pME6710 in vitro
In vitro
Amendment of
in Pseudomonas fluorescens CHAO conferred
from
y
Tomato
+
CHA0/pME6710
was
or
0 03
-0 25
±
0 17
-0 72
+
0 23
-017
±
0 12
1254
+
130
2705
±
116
437
+
201
1381
±
695
grown
the medium
was
in
per
OSG medium at 24° C without
Samples
were
taken at
an
0 04
±0 08
+
-0 13
±0 10
322
±
33
+
222
±
28
by quantifying ice nucleation activity (log(ice nuclei/CFU))
108 CFU) from plasmid pME6710
(-) or with (+) the addition of fusaric acid to a final concentration of
600 nm of 1 5 and 3 5 Values represent the mean of 3 replicate
measured either
ß-galactosidase activity (units
cultures ± standard deviation of the
'-
+
+
plasmid pSLII8
in
rhizosphere
OD 3 5
-0 64
gene expression of P fluorescens CHAO
|xM
from
FORL22
CHA0/pME6710
500
either ice nucleation
x
CHA0/pSLII8
Strain CHAO
by
plasmid pSLII8
by ß-galactosidase activity (units per 108 CFU)
rhizosphere of tomatoes.
OD1 5
'
from 322 to 222 units
and in the
CHA0/pSLII8
from
roots,
or
Fusaric acid
P fluorescens
phlA
tomato
CFU.
Table 3.
'
OD6oo of 3.5. On
an
OD at
mean
inoculated with P fluorescens strains CHA0/pSLII8 and CHA0/pME6710 at 108 CFU per plant Fusarium
radicis-lycopersici strain 22 (FORL22) was added to the rockwool system at 4 x 107 microconidia plus
mycelial fragments per plant Plants were harvested after 2 weeks of growth Values represent the mean of 4 replicates with
20 plants each ± standard deviation of the mean
Tomato
plants
were
oxysporum f sp
89
Chapter 4
Differential response in DAPG
production
of two
pseudomonads
Discussion
P. fluorescens CHAO and
biocontrol bacteria
(20, 34, 40).
biosynthetic
as
phlG
genes
and phlH
phlH of Q2-87).
transcriptional
as
well
Q2-87
was
the
to
activity
bacteria
if DAPG
of
expression
studies
pSLII8,
was
vectors
used in microbial
The
(43).
impractical,
parental
vector
as
on
are
over more
plasmid pME6016
in CHAO
are
than 50
(>
95%
retention)
70
of these
AF207529 for phiA,
and
phlF, phlG,
of these two strains
to
as
tomato
generations.
was more
After the
Similar
or more
Reporter
stable
the presence of each
roots,
a new
region
of strain CHAO
region
of
stably
numbers of
CHAO and
This
proved
of both inaZ
seems
expression
vector
number of
in
constructed
Gram-negative
fori3.
true
due to the metabolic
CHAO and
slightly
or
derivatives, pJL6244
protein. pME6010
fluorescens
plasmids
was
maintained in various
and its
were
constructed
lower in strain Q2-87
pME607E
generations,
generations
at 86%>
was
its
parental
reached in the in vitro studies
than 13 at which retention of both vectors is
was over
rhizospheres
study,
ecology
inaZ and lacZ
Q2-87 in vitro and
of various
90
were
reported
in
than sufficient
growth
on
obtained in strain CHAO
plants (14, 31).
should be sensitive and their
were
on
more
93% after two weeks of
stabilities of pME6010 derivatives
weeks in the
In this
pPROBE'-gj^,
instability might be
in P.
same
particularly important
stable with 92% retention but did not reach the levels obtained
genes used in microbial
(26).
fluorescens
high
is
generations (29).
constructed phlA '-lacZ
Retention of pME607E in Q2-87
grown for two
P.
newly
were never more
tomato roots.
overly
stable
(99%>). However,
this paper
forphlA,phlF,
e.g., in studies in natural habitats
of the 153 kDa InaZ
pVSl replicon (16). Stability
approximately
(> 95%>).
multiple copies
highly
retention of the
after
well
ecology
of pSLII8,
much lower. We suggest that this
burden of producing
as
and
fusion of the lacZ reporter gene to the DAPG promoter
(e.g. pseudomonads)
with the
no.
regulation
in media and
of promoter-probe vectors, which
set
derivatives
analysis
U41818 for phiA,
fluorescens CHA0/pPROBE'-gj^ but, surprisingly, stability
and
the level of DAPG
on
GenBank accession
no.
different
genetically
constructed.
expression
a
restriction
FA-producer FORL22,
when antibiotic selection of plasmids is
within
and
fusion of the inaZ reporter gene to the DAPG promoter
Stability
gene
(34,
investigates
DAPG promoter
transcriptional
as a
ecologically
forphlD by
sequences
The current work
measure
two
of CHAO, GenBank accession
responds differently to FA,
other. To
are
Genetic differences also appear
has been shown
by comparing
phlF, phlG, andphlH
Q2-87
products
used to report phlA promoter
tomato roots.
Both
transcriptional
not
activity
lacZ
of
Chapter 4
Differential response in DAPG
fusions
represented
growth
very
DAPG
production
obtained
well,
the increase of DAPG
as
their
expression
of their hosts
a
in
declined much
Q2-87. Conversely, DAPG production,
in strain CHAO
was
about
one
offer
explanation
an
for CHAO
the lower
for the
normalizes
sample
discrepancy
not
the
only
different strains.
increases in
growth
Expression
beginning
During
cell
of
that
under
of phlA,
in
provided
an
Q2-87 might explain
measured with the
as
a
until the
values leveled around -0.5
genomic/?/^ '-inaZ fusion expressed
in P.
of DAPG
tool to monitor
beginning
of
were
high
from the
stationary phase (Fig.
which
means
Loper (23) report similar
fluorescens
very
Pf-5 grown in
a
levels too
high
advantages
detection,
our
study (Fig.
INA values for
Pit
promoting
of the inaZ reporter gene, such
can
turn
into
production
went
which represents
dropped
high sensitivity
to a
value of-3
be
at
and its wide range of
quantitative
when DAPG
log(nuclei/CFU)
within 170
h,
300-fold reduction of ice nuclei per CFU. This suggests that InaZ is not
stable in strain CHAO, which is
earlier that INA,
can
medium like OSG. The
(26). During stationary growth phase,
down to zero, INA
a
its
DAPG-promoting
when the promoter is too strong for
disadvantages
the inaZ reporter
by
as
a
a
medium.
suggests that the DAPG promoter is transcribed
for this reporter gene, at least in
assessment
overly
3 A,B). This
3 A).
that every third
However, the phlA '-inaZ fusion reports phlA transcription before DAPG production
detected in
These
conditions.
log(LNA/CFU)
active ice nucleus. Kraus and
produced.
quantification
helpful
levels of the inaZ reporter gene fusion in CHAO
exponential growth phase
phase,
varying
compound,
lower for Q2-87 than
was
plasmid present
Nevertheless, they represent
Maximal
activities. Yet, this does
factor to determine the amount of DAPG
transcriptional activity
these studies
(Fig. 3D,F). Dividing through
results show that the reporter gene systems cannot be used for direct
production by
were
than those measured
higher
ß-galactosidase
expression
to
direct extraction of the
by
to
(31, 38).
growth (Fig. 3C,E).
third
one
since bacterial
activities. However,
ß-galactosidase
reporter constructs, is
about
A lower copy number of the
(Fig. 3C,E).
slowly compared
third lower than that of Q2-87
the amount of cells present in the
not
were
parallel
'lacZ fusion in CHAO
more
measured
as
'-
early stationary
in
expression
pseudomonads
confirms earlier data that
after 200 h of
expression
activities measured in CHAO
ß-galactosidase
maximal
finding
translational phlA
Interestingly, ß-galactosidase activity
and leveled around 70% of maximal
to
zero
This
(Fig. 3C,E).
kinetics of
by monitoring
and
production during exponential
from
rose
of two
production
even
transcriptional activity
of cells that
at
are
a
prerequisite
not
least within 24 h
for reporter genes. It has been shown
actively growing,
(25, 26).
91
reacts to
changes
in
Chapter 4
Differential response in DAPG
INA measured in the
Stunningly,
from the in vitro
experiments (Table 3). They
level which is
one
levels of gene
expression
shown with
our
galactosidase
nucleus per cell
[=
would be
activities in the
much smaller INA in the
0
were
around
were
or
log(INA/CFU)] (26).
for
expected
higher than
did
resides in
give
a
INA
rhizosphere
This is
surprising,
rhizosphere experiments.
than in vitro. Another
pit '-inaZ fusion
growth stage
example
or
the assay since cells
That could
can
retain InaZ for
explain why
Ice nucleation
some
that
proteins present
exposed
waterlogged
no
a
reported
The
reason
CHAO without
early exponential
to an
addition,
cells could have been assessed with
losing
above
were
or
ß-
DAPG could be measured. In
time after
in dead
their
zero
viability/culturability
in the
rhizosphere
were
present in the
nonculturable cells could have conferred
observed VBNC cells of CHAO when the strain
(27)
combination of low redox
to a
comparable
ice nuclei than viable cells
more
nor
(23).
case
It would be conceivable that strain CHAO
(VBNC)
INA values
some
the additional ice nuclei. Mascher et al.
was
shown).
viable-but-nonculturable
experiments (Table 3), meaning
sample.
not
high although
was
for such
in P. fluorescens Pf-5 which also
the tomato roots that is
in vitro where INA
ice nuclei of dead
(24).
on
lower
as
This is the
of cucumber and cotton than in vitro
rhizospheres
background (data
growth stage
those obtained
above maximum detectable
for this difference is not obvious. Neither the tomato-rockwool system
pSLII8
pseudomonads
'-lacZ reporter, which gave 4-to 30-times lower
transcriptional phlA
is the chromosomal
relationship
plant experiments
of two
production
potential
and oxygen limitation which mimics
soil. The rockwool saturated with nutrient solution
might represent
a
a
similar
environment favorable to generate VBNC.
The
tested in CHAO to the
3).
FA is
a
suppressing
pathogenicity
suppress DAPG
factor
production
both FA and FORL22 had
on
tomato
nor
in
of the two reporter gene fusions of inaZ and lacZ to phlA has been
responsiveness
roots,
a
vivo, when expressing the phlA
in
general
possibility
(e.g.
to
too
high
detect
to
was
show
well
the
as
CHAO
on
FA-producer FORL22 (Table
species
(9, 38). Using
and has been shown to
lacZ
as a
ß-galactosidase activity
reporter gene,
in OSG medium and
differences could be measured neither in vitro
no
'-inaZ fusion in CHAO. lacZ represents phlA promoter
it remains stable
more
from -5 to
effect
In contrast,
as
many Fusarium
fluorescens
repressing
strong promoters like phlA that
assay
produced by
in P.
respectively.
strength well, although
was
effect of FA
over
long period.
used in this
suppressive
study
inaZ
and
seems
expression
effects mediated
by
to
be too sensitive for
of the phlA '-inaZ fusion
either FA
or
differences could be to raise the temperature for the
-2°C),
as
then,
more
InaZ
92
proteins
are
required
to
form
FORL22. One
ice-nucleating
an
aggregate.
Chapter 4
Differential response in DAPG
Furthermore, the phlA
DAPG
'-inaZ fusion
P. fluorescens
take-all-suppressive
from
a
soil
Q2-87
strains differ in
abolished DAPG
ecological
basicola in
and
genetical
traits.
completely,
that strains that
group
(20).
to
was
Duffy
and
seems
not
Défago (8)
surprising
that the
have shown that
Q2-87. Conversely, phosphate
whereas it
lowered DAPG
only
a
production
worldwide collection of Pseudomonas strains showed
were
produced
against Pythium damping-off (40).
An
tomato
explanation
plants
of the two strains to
Fusarium,
FA has been shown to suppress DAPG
postulated
strain Q2-87, but to
CHAO and not in
expression
high
in
a
signals coming
better
FORL22 than CHAO
against
from the
production
in CHAO
(9, 38)
synthesis
to 73%
of the
DAPG
of
case
and
Duffy
and
The present
dependent.
only significantly suppressed
Q2-87 (Table 2). Although the
Q2-87
in the
pathogens. Indeed,
that response to FA is ARDRA group
FORL22
and root
crown
This fact repeats itself in the current
in the ARDRA2
much smaller extent than in the ARDRAl strain CHAO
producing
roots, FA
and
(ARDRA2
against Fusarium
shows for the first time that FA also inhibits in vitro-DAPG
tomato
analyzed
could be found in differential responses in DAPG
regulation
study
separate
a
part of ARDRA2 (20). Interestingly, strains of ARDRA 2 and 3 proved
(ARDRAl) (Table 2).
have
were
DAPG but not Pit
strains of ARDRAl in their biocontrol of tomato
Défago (10)
similar and form
genetically highly
formed with strains that
Q2-87 (ARDRA2) protected
as
are
when the restriction patterns of their 16S ribosomal DNA
and of cucumber
work
of
both DAPG and Pit
produce
Two clusters
superior
rot
(6). Analysis
(ARDRAl)
and Q2-87
3)
(Fig. 4).
production
On
in
presence of FORL22 did lower DAPG gene
control, differences could
not
be ensured
due
statistically
standard deviations. In CHAO, the amendment of FORL22 lowered DAPG gene
expression
to 62
and 49%,
respectively,
and this
standard deviations. These data follow the
modifications: neither does FA
is DAPG
synthesis
sensitivity
on
isolated
was
Q2-87 (8). One major difference between CHAO and Q2-87 is that CHAO produces Pit in
addition to DAPG
to
whereas strain CHAO
a
Morens, Switzerland (41). Considering
in CHAO but not in
in CHAO
of
production
of wheat grown in
rhizosphere
it therefore
agricultural origin,
production
production
isolated from the
Quincy, Washington (44)
and
pseudomonads
very low.
originally
Thielaviopsis
geographical
zinc stimulates DAPG
in
to
of two
still be useful in environments where
might
naturally
was
soil from
suppressive
their different
two
strains is
by investigated
production
DAPG
to FA
of
an
ARDRA2 strain
could be
expression
in
completely
explained
on a
the/'MF-mutant
was
postulate
statistically
of Duffy and
block DAPG
entirely
genetic
despite high
Défago (10)
production
in
an
with two
ARDRAl-strain
nor
insensitive to FA. This differential
level. Amendment of FORL22 had
of CHAO, CHA638
93
different
(Table 2).
no
Evidence that
effect
an
Chapter 4
Differential response in DAPG
intact phlF gene is needed for
studies in media and
is therefore
thought
repressor PhlF
on
be mediated
to
(30, 38). Suppression
through
(38). Comparing the phlF
the
of DAPG
identical.
different
sensitivity
of the two P.
on
fluorescens
in earlier
provided
biosynthetic
sequences of strains CHAO and
these differences based
Regarding
pseudomonads
genes
by
FA
transcriptionally acting pathway-specific
of nucleotide sequence and 85% of the amino acids
identity
of two
of DAPG in CHAO has been
FA-repression
wheat roots
production
sequence
forming
data, it
Q2-87 reveals 76%
the PhlF
only
can
be
protein
are
speculated
that
strains CHAO and Q2-87 is due to the
differences in amino acid sequence of the PhlF repressor since it is not known whether these
changes
lead to another
protein
unidentified, regulatory mechanisms
FA may not be the result of higher
region. Although
DAPG gene
provide significantly
suppression
production
In
However, it
structure.
of Fusarium root-rot
of the FA-PhlF
affinity
of CHA638
CHAO
tomato
on
conclusion,
long period.
much
was
is mediated
lacZ has been shown to report increases
Under
experimental
transcribes at levels too
which
for the phlA promoter
complex
(phlF)
repression by
higher
it did not
by
another mechanism than
of DAPG.
CHAO well in environments favorable for DAPG
producing
other, yet
than CHAO which may suggests that
protection
by
be excluded that
may act in addition to PhlF and that DAPG
expression
better disease
cannot
FORL22
might
on
high
conditions
expression, although
applied
for inaZ to report the
DAPG
production.
contribute in part to
superior
DAPG
mphlA promoter activity
in this
study,
repressive
regulation
biocontrol
over a
the phlA promoter
effect
in
it stays stable
of
by
either FA
or
FA
Q2-87 is less sensitive
to
FA,
performance.
Literature Cited
1.
Bangera,
for
M.
synthesis
G., and Thomashow, L. S. 1999. Identification and characterization of a
of the
fluorescens Q2-87.
2.
polyketide
antibiotic
2,4-diacetylphloroglucinol
gene cluster
from Pseudomonas
J. Bacteriol. 181:3155-3163.
Blumer, C, Heeb, S., Pessi, G., and Haas, D. 1999. Global GacA-steered control of cyanide and
exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.
Proc. Natl. Acad. Sei. USA 96:14073-14078.
3.
Boyer,
H.
W., and Roulland-Dussoix D. 1969. A complementation and analysis of the restriction
and modification of DNA in Escherichia coll. J. Mol. Biol. 41:459-472.
4.
Cronin, D., Moënne-Loccoz, Y., Fenton, A., Dunne, C, Dowling, D. N., and O'Gara, F. 1997.
Ecological
interaction of a biocontrol Pseudomonas fluorescens strain
94
producing 2,4-
Chapter 4
Differential response in DAPG
diacetylphloroglucinol
with the soft rot potato
pathogen
of two
production
Erwinia carotovora
pseudomonads
subsp. atroseptica.
FEMS Microbiol. Ecol. 23:95.
5.
Cronin, D., Moënne-Loccoz, Y., Fenton, A., Dunne, C, Dowling, D. N., and O'Gara, F. 1997.
Role of 2,4-diacetylphloroglucinol in the interactions of the biocontrol
with the potato cyst nematode Globodera rostochiensis.
6.
C. H.,
Défago, G., Berling,
Pseudomonas fluorescens:
B.
7.
Delany, I., Sheehan,
production
Fl 13:
8.
of the
M.
Duffy,
B.
and mechanisms.
D.
Hornby,
R. J.
Pages
by
93-108 in:
P. and
strains of
Biological
Cook, Y. Henis, W. H. Ko, A. D. Rovira,
Scott, eds. CAB international.
M., Fenton, A., Bardin, S., Aarons, S., and O'Gara, F. 2000. Regulation of
metabolite
antifungal
genetic analysis
Environ. Microbiol. 63:1357-1361.
root rot of tobacco and other root diseases
potential applications
plant pathogens.
and P. R.
Schippers,
strain Fl 13
Burger, U., Haas, D., Kahr, G., Keel, C, Voisard, C, Wirthner,
Wüthrich, B. 1990. Suppression of black
control of soil-borne
Appl.
pseudomonad
of phlF
as a
2,4-diacetylphloroglucinol
transcriptional
in Pseudomonas fluorescens
repressor. Microbiol. UK 146:537-546.
K, and Défago, G. 1999. Environmental factors modulating antibiotic and siderophore
biosynthesis by Pseudomonas fluorescens
biocontrol strains.
Appl.
Environ. Microbiol. 65:2429-
2438.
9.
K, and Défago, G. 1997. Zinc improves biocontrol of Fusarium
Duffy,
B.
tomato
by Pseudomonas fluorescens
inhibitory to
10.
Duffy,
B.
bacterial antibiotic
and represses the
biosynthesis. Phytopathology
Dunlap,
Pages
69-106 in: Bacteria
of pathogen metabolites
87: 1250-1257.
87:S26.
lactone autoinducers in bacteria:
multicellular
as
organisms.
J. A.
Shapiro
Unity
and M.
and
diversity.
Dworkin, eds. Oxford
London.
Fenton, A. M., Stephens, P. M., Crowley, J. O'Callaghan, M., and O'Gara, F. 1992. Exploitation of
gene(s)
to
13.
TV-acyl-homoserine
P. V. 1997.
University Press,
12.
and root rot of
K, and Défago, G. 1997. A Fusarium pathogenicity factor blocks antibiotic biosynthesis
by antagonistic pseudomonads. Phytopathology
11.
production
crown
a
involved in
2,4-diacetylphloroglucinol biosynthesis
Pseudomonas strain.
Figurski,
plasmid
D.
Appl.
to confer
a new
biocontrol
capability
Environ. Microbiol. 58:3873-3878.
H., and Helinski, D. R. 1979. Replication of an origin-containing derivative of
RK2
dependent on
a
plasmid
function
provided
press. A
regulatory
in
trans. Proc.
Natl. Acad. Sei. USA
76:1648-1652.
14.
Heeb, S., Blumer, C, and Haas, D.
dependent global
15.
RNA
as a
mediator in GacA/RsmA-
control in Pseudomonas fluorescens CHAO. J. Bacteriol.
Heeb, S., and Haas, D. 2001. Regulatory roles of the GacS/GacA two-component system in plantassociated and other
16.
in
gram-negative
bacteria. Mol. Plant-Microbe Interact. 14:1351-1363.
Heeb, S., Itoh, Y., Takayuki, N., Schnider, U., Keel, C, Wade, J., Walsh, U., O'Gara, F., and
Haas, D. 2000. Small stable shuttle
negative plant-associated
vectors based
on
the minimal
pVSl replicon
bacteria. Mol. Plant-Microbe Interact. 13: 232-237.
95
for
use
in gram-
Chapter 4
17.
Differential response in DAPG
of two
Keel, C, and Défago, G. 1997. Interactions between beneficial soil bacteria and
Mechanisms and
Systems:
ecological impact. Pages
the 36th
University
18.
production
Symposium
of London.. A. C.
27-46 in:
of the British
Gange,
Multitrophic
pseudomonads
root
pathogens:
Interactions in Terrestrial
Ecological Society, Royal Holloway College,
Brown, eds. Blackwell Science, Oxford.
V. K.
Keel, C, Schnider, U., Maurhofer, M., Voisard, C, Laville, J., Burger, U., Wirthner, P., Haas, D.,
and
G. 1992.
Défago,
Suppression
of root diseases
by Pseudomonas fluorescens
CHAO:
Importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol. Plant-Microbe
Interact. 5:4-13.
19.
Keel, C, Voisard, C, Berling, C. H., Kahr, G., and Défago, G. 1989. Iron sufficiency,
prerequisite
for the
CHAO under
20.
suppression
gnotobiotic
locations.
geographic
locus among fluorescent
Appl.
Environ. Microbiol. 62:552-563
Keel, C, Wirthner, P. H., Oberhansli, T. H., Voisard, C, Burger, U., Haas, D., and Défago, G.
1990. Pseudomonads
as
antagonists
2,4-diacetylphloroglucinol
King,
E.
in the
of plant
pathogens
suppression
in the
rhizosphere:
of black root rot of tobacco.
Role of the antibiotic
Symbiosis
9: 327-341.
O., Ward, M. K, and Raney, D. E. 1954. Two simple media for the demonstration of
pyocyanin
23.
79:584-589.
2,4-diacetylphloroglucinol biosynthesis
Pseudomonas strains from diverse
22.
Phytopathology
strain
Keel, C, Weiler, D. M., Natsch, A., Défago, G., Cook, R. J., and Thomashow, L. S. 1996.
Conservation of the
21.
by Pseudomonas fluorescens
of tobacco black root rot
conditions.
a
and fluorescin. J. Lab. Clin. Med. 44:301-307.
Kraus, J., and Loper, J. E. 1995. Characterization of a genomic region required for production of
the antibiotic
control agent Pseudomonas fluorescens Pf-5.
pyoluteorin by the biological
Appl.
Envviron. Microbiol. 61:849-854.
24.
Lindow, S. E. 1983. The role of bacterial ice nucleation in frost injury
Phytopathol.
25.
Loper,
J.
plants.
Annu. Rev.
21:363-384.
E., and Henkels, M. D. 1997. Availability of iron
rhizosphere
to
and bulk soil evaluated with
to Pseudomonas fluorescens in
ice nucleation reporter gene.
an
Appl.
Environ.
Microbiol. 63: 99-105.
26.
Loper,
J.
E., and Lindow, S. E. 1997. Reporter
expression by
soil- and
microbiology.
C. J. Hurst, G. R.
eds. ASM Press,
27.
plant-associated
Washington
gene
bacteria.
systems useful in evaluating in situ gene
Pages
482-492 in: Manual of environmental
Knudsen, M. J. Mc Inervey, L. D. Stetzenbach, and M. V. Walter,
DC.
Mascher, F., Hase, C, Moënne-Loccoz, Y., and Défago, G. 2000. The viable-but-nonculturable
state induced
by
abiotic stress in the biocontrol agent Pseudomonas fluorescens CHAO does not
promote strain persistence in soil. Appl. Environ. Microbiol. 66:1662-1667.
28.
Miller, J. H. 1992. A Short Course in Bacterial Genetics: A laboratory manual and handbook for
Escherichia coli and related bacteria. Cold
Spring
96
Harbor
Laboratory Press, Plainview,
NY.
Chapter 4
29.
Differential response in DAPG
Miller, W. G., Leveau, J. H. J., and Lindow, S. E. 2000. Improved gfp and
promoter-probe
30.
production
inaZ
pseudomonads
broad-host-range
vectors. Mol. Plant-Microbe Interact. 13:1243-1250.
Notz, R., Maurhofer, M, Dubach, H., Haas, D., and Défago, G.
strains of Fusarium oxysporum alter
Pseudomonas
of two
Fusaric-acid-producing
2,4-diacetylphloroglucinol biosynthetic
CHAO in vitro and in the
fluorescens
2002
rhizosphere
gene
of wheat.
expression
Appl.
in
Environ.
Microbiol. 86:2229-2235.
31.
Notz, R, Maurhofer, M., Schnider-Keel, U., Duffy, B., Haas, D., and Défago, G. 2001. Biotic
factors
of the
affecting expression
2,4-diacetylphloroglucinol biosynthesis
Pseudomonas fluorescens biocontrol strain CHAO in the
32.
Nowak-Thompson, B., Gould,
diacetylphloroglucinol by the
S. J., Kraus, J., and
gene
rhizosphere. Phytopathology
Loper,
phlA
in
91:873-881.
J. E. 1994. Production of 2,4-
biocontrol agent Pseudomonas fluorescens Pf-5. Can. J. Microbiol.
40:1064-1066.
33.
Raaijmakers,
producing
34.
M., Weiler, D. M., and Thomashow, L. S. 1997. Frequency of antibiotic-
Pseudomonas spp. in natural environments.
Appl.
Environ. Microbiol. 63:881-887.
Ramette, A., Moënne-Loccoz, Y., and Défago, G. 2001. Polymorphism of the polyketide synthase
gene
phlD
in biocontrol fluorescent
comparison
35.
J.
of PhlD with
pseudomonads producing 2,4-diacetylphloroglucinol
plant polyketide synthases.
Sarniguet, A., Kraus, J., Henkels,
factor as affects antibiotic
M.
and
Mol. Plant-Microbe Interact. 14:639-652.
D., Muehlchen, A. M., and Loper, J. E. 1995. The sigma
production
and
biological
control
activity
of Pseudomonas fluorescens
Pf-5. Proc. Natl. Acad. Sei. 92:12255-12259.
36.
Sambrook, J., and Russell, D. W. 2001. Molecular cloning: A laboratory manual, 3r ed. Cold
Spring
37.
Laboratory Press,
Cold
Spring Harbor,
NY.
Schnider, U., Keel, C, Blumer, C, Troxler, J., Défago, G., and Haas, D. 1995. Amplification of
the
and
38.
Harbor
housekeeping sigma
improves
factor in Pseudomonas fluorescens CHAO enhances antibiotic
production
biocontrol abilities. J. Bacteriol. 177: 5387-5392.
Schnider-Keel, U., Seematter, A., Maurhofer, M., Blumer, C, Duffy, B., Gigot-Bonnefoy, C,
Reimmann, C, Notz, R., Défago, G., Haas, D., and Keel, C. 2000. Autoinduction of 2,4-
diacetylphloroglucinol biosynthesis
repression by the
39.
in the biocontrol agent Pseudomonas fluorescens CHAO and
bacterial metabolites
salicylate
and
pyoluteorin.
J. Bacteriol. 182:1215-1225.
Shanahan, P., O'Sullivan, D. J., Simpson, P., Glennon, J. D., and O'Gara, F. 1992. Isolation of 2,4-
diacetylphloroglucinol
from
a
fluorescent
pseudomonad
and
investigation
of physiological
parameters influencing its production. Appl. Environ. Microbiol. 58:353-358.
40.
Sharifi-Tehrani, A., Zala, M., Natsch, A., Moënne-Loccoz, Y., and Défago, G. 1998. Biocontrol of
soil-borne
fungal plant
pseudomonads
diseases
by 2,4-diacetylphloroglucinol-producing
with different restriction
profiles
104:631-643.
97
fluorescent
of amplified 16S rDNA. Eur. J. Plant Pathol.
Chapter 4
41.
Differential response in DAPG
suppression
of black root rot of tobacco.
control: Mechanisms and
N. T. eds. Plant-Microbe
van
der
Bij,
A. J., de
antifungal
metabolites.
Pages
76:181-185.
use
of introduced bacteria for
187-235 in:
Stacey, G., Keen,
Weger,
L.
A., Tucker, W. T., and Lugtenberg, B. J. J. 1996. Plasmid stability
rhizosphere. Appl.
Environ. Microbiol. 62:1076-1080.
Vincent, M. N., Harrison, L. A., Brackin, J. M., Kovacevich, P. A., Mukerji, P., Weiler, D. M.,
and
Pierson, E. 1991. Genetic analysis of the antifungal activity of a soilborne Pseudomonas
aureofaciens
45.
pseudomonads
Interactions, vol. 1. Chapman & Hall, New York, NY.
in Pseudomonas fluorescens in the
44.
Phytopathology
Thomashow, L. S., and Weiler, D. M. 1996. Current concepts in the
biological
43.
of two
Stutz, E. W., Défago, G., and Kern, H. 1986. Naturally occurring fluorescent pseudomonads
involved in
42.
production
strain.
Appl.
Env. Microbiol. 57:2928-2934.
Yuan, Z., Cang, S. Matsufuji, M., Nakata, K, Nagamatsu, Y., and Yoshimoto, A. 1998. High
production
grown
on
of pyoluteorin and
ethanol
as a
2,4-diacetylphloroglucinol by Pseudomonas fluorescens
sole carbon
source.
J. Ferment.
98
Bioeng.
86:559-563.
S272
Chapter 5
General Conclusions
100
Chapter 5
General conclusions
Production of the
substantially to
polyketide 2,4-diacetylphloroglucinol (DAPG)
the antimicrobial
activity
of many biocontrol
about environmental factors that influence the
and
knowledge
understanding
beneficial effect of the
of these factors is crucial to
applied pseudomonads.
factors with which bacterial inoculants in
and which influence DAPG
fluorescens
biosynthesis
the roots of many
pseudomonads
not
are
and
plant species
expression
with gene
compared
host genotype effect
exudates is
(e.g.
species
DAPG gene
sustained
plant
or
plant
specific
in the
expression.
biosynthesis
of the
environmental
some
may be
Chapter
an
can
and
confronted
commonly
Pythium
modulate the
production ability
of two monocots
of two dicots
production
(cucumber
from the
DAPG gene
a
roots
were
and/or
of root
quality
biocontrol strains for certain
inhibition
expression
and
by
DAPG
highest
by P.
by P.
an
can
be
provided.
were
that have
had
no
either isolated from wheat roots
indirect effect caused
by
the
ultimum.
on
DAPG gene
rhizosphere
pathogenic impact
or
a
ultimum stimulated DAPG
of DAPG is correlated with the fusaric acid
chapter
on
be
can
values
controlling pathogens
probably
devastated
production
of different F. oxysporum strains in vitro and in the
The F. oxysporum strains used in this
This
bean).
composition
quantity
direct effect of Fusarium oxysporum strains
repression
wheat)
cucumber, bean and wheat had little influence
ultimum. Root infection
plant
and
and
reports
in CHAO.
expression
(maize
2
of DAPG in strain CHAO. These
by choosing specific
longer-term pathogen
on
associated with
are
pathogens. Chapter
and the differences in their
both cucumber and maize. This is
3 shows
plant only. They
influence DAPG gene
This may be essential for
in strain CHAO. The
although they
can
rhizospheres
specific
impact
planting.
compounds
one
This indicates that stable levels of DAPG
age did have
on
for
rhizospheres
cultivars. The age of
disease onset like
expression
consistency
also observed at the cultivar level in maize. The
was
during plant growth
release of
This thesis identifies
able to suppress several
are
in the
expression
minerals)
measured 24 h after
rapid
the
improve
The
lacking.
in the model biocontrol strain Pseudomonas
should be taken into consideration
plant species
Maize
higher
was
and cultivar
carbon source,
findings
of DAPG has been
agricultural systems
that host genotype, host age and host health
gene
Information
pseudomonads.
CHAO.
Biocontrol
phlA
biosynthesis
contributes
on
the
(FA)
of wheat.
plants,
from soil cultivated with wheat.
However, the fungi did influence the DAPG production of CHAO while co-inhabiting the
wheat
rhizosphere.
repressive
effect
on
The distribution of FA-producing Fusarium strains is worldwide and their
DAPG may affect biocontrol
performance
systems. In agricultural systems where FA-producers
101
are
in many
plant-pathogen-
present, better disease control might
Chapter 5
General conclusions
be achieved
using
biocontrol strains insensitive to FA-mediated DAPG inhibition
by using
strains
of
capable
pathway-specific
degrading
repressor
rhizosphere. Nevertheless,
Chapter
4
of this strain
was
pseudomonad
which
rhizosphere,
than
was
did not
production
producing
in the
exhibit the desired
methods.
of the
biosynthesis
strain Q2-87
less sensitive to FA and the FA
against FORL22
(CHA638),
appropriate screening
by
the DAPG
lacking
overexpressed phlA
even
might naturally
up this idea and the DAPG
picks
of CHAO
insensitive to FA and
was
radicis-lycopersici (FORL22).
tomato
mutant
a
other biocontrol strains
different fluorescent
genetically
CHA638,
and could be detected with
phenotypes
sp.
PhlF,
FA.
or
was
ecologically
studied. DAPG
tomato
pathogen F.
Amendments with Q2-87 resulted in
a
better
and
biosynthesis
oxysporum f.
protection
of
than amendments with CHAO. However, the phlF mutant of CHAO
insensitive to FORL22 and
provide
of DAPG
better disease
might
overexpressed phlA
protection.
in the tomato
This suggests that mechanisms other
be involved in biocontrol of tomato
and root rot
crown
by
this strain.
Throughout this study,
DAPG
biosynthetic
constructed
inaZ is
was
quantifiable
fusion
long
which
was
(chapter 4)
to
simultaneously
case
stable in CHAO than
in
were
for lacZ. The gene
aphlA
of the
This work shows that DAPG
can
be strain
fluorescens
produced
and
in
agricultural
dependent.
application
be demonstrated
aphlA
of
(e.g.
protein,
accurately
to
appeared
addition of FA
'-lacZ fusion
to
can
reported
be affected
impact
necessarily implicate
might
by
less
reflect
be to strong for
one
FORL22)
nucleus
could not
the FA-mediated
biotic factors that
of these
factors,
biosynthetic
as
genes in
a
in the
are
case
of
P.
that the antimicrobial substance is
be of relevance for
isolation, formulation
appropriate
promising. Negative impacts
102
or
was
ß-galactosidase.
environments. The
seems
a
by extraction,
around maximum detectable level of
of biocontrol agents. A careful choice of the
specific plant-pathogen system
important
the DAPG promoter
biosynthesis
in all environments. These results
was
in two different strains.
of inaZ, the ice nucleation
The presence of the DAPG
soil isolate does not
to
and this would be
higher stability
of
expression
could be measured with the phlA '-inaZ
treatments
'-inaZ fusion whereas
repression well, despite
FA,
were
Therefore, differences between
commonly present
monitor
up to 6000-fold fewer cells than lacZ for
product
transcriptional activity. However,
be shown with
to
phlA expression
high enough
ß-galactosidase (lacZ)
inaZ, specially in planta, where values
per cell.
reporter gene
measure
requiring
before amounts of DAPG
the
as a
(1). Here, promoter activity
response
not
used
genes. An additional fusion of phlA to the reporter gene inaZ
very sensitive reporter gene
a
changes
lacZ
biocontrol agent for
of other
microorganisms
a
Chapter 5
General conclusions
could be avoided
mutants),
that
are
by applying
beneficial
a
biocontrol
performance: Co-application
substances
(e.g. FA),
substances
or
changing
and
environment. Efficient formulation is
with strains
amendments of minerals
an
capable
(e.g. zinc)
thereof
more
of
microorganisms
in
large-scale
field
to
more
approach to improve
degrading DAPG-inhibiting
production
(e.g. PhlF) might
reliable biocontrol. However, other mechanisms
required
(e.g. phlF-
might be
additional
that block
of substances that suppresses DAPG inhibitors
biocontrol and additional progress will be
biocontrol
mutants
or
less sensitive. Mixtures of strains with different traits
reliable in
improved
pseudomonads,
can
fully implement
of inhibiting
lead to
an
be involved in
the beneficial
impact
of
application.
Literature cited
1.
Miller, W.G., Brandi, M. T., Quinones, B., and Lindow, S. E. 2001. Biological
availability:
Relative sensitivities of various reporter genes.
103
Appl.
sensor
for
sucrose
Env. Microbiol. 67:1308-1317.
104
Acknowledgements
Mein Dank
geht
Gogo: danke,
an...
dass Du mir diese Diss
ermöglicht hast,
für die Geduld und
guten Tipps für die Wissenschaft und das restlichen Leben.
Tatkräftige Hilfe
bei grossen und kleinen
und
durchgelesen
korrigiert hast,
Manuskripte.
grosses Dankeschön
Arbeitsklima
•
für die moralische
angenehm
so
merveilleux ami.
•
macht!
•
being
Alban: pour
m'apprendre
wilde Zwitserse meid! Het
die kritische Durchsicht meiner
Manuskripte.
•
my co-referent.
•
Gruppe Phytopathologie: danke,
Bernie: danke für Deine Hilfe und Zeit.
avonturen te störten met een
Seiten, die
Du
Art.
•
Kritik, die Diskussionen und fürs „Durchackern"
alle Mitarbeiter der
an
von
Unterstützung und Deine positive
Bruce McDonald: thank you for
geht
Monika: danke für Deine
Problemen, für die hunderten
Dieter Haas: danke für Deine konstruktive
diverser
•
Unterstützung und
Dank
•
subjonctif (!)
dass Ihr das
et pour être un
Celeste: dank je wel
heel leuk!
was
an
le
Ein ganz
•
om
je
in
Matthias: danke für
Andi, Andrea, Andreas, Artemis,
Brion, Carmen, Carsten, Cesare, Champa, Christian, Dani, Danilo, Davide, Eve, Fabio, Flori,
Francesca, Franziska, Giovanni, Hampi, Isa, Lazaro, Lilo, Luca, Marcello, Marco, Manuela,
Michaela, Michel, Michèle, Miriam, Miro, Phil, Reinhard, Rex, Röbi, S0ren, Stéphanie, Ueli,
Uli, Vicente und
auch bei meiner
Yvan für Eure gute Gesellschaft!
Diplomandin,
Dani und Christian.
•
Broadhagen
beaucoup
des
au
and
copains
questions
Ursula.
•
Cheryl
me
the secrets of
du Laboratoire de
Meine Eltern:
you for
Whistler: thank you
moléculaires:
Herzlich Bedanken möchte ich mich
Semestrandin und bei meinen
Joyce Loper: thank
Nancy Chaney for teaching
•
letting
cloning
so
me come
and for your
much for the
Biologie
Lehrlingen: Helen, Simone,
good
to your
lab.
friendship.
time in
Microbienne à Lausanne
•
•
qui
danke, dass
Ihr meinen
Werdegang
bomen. Dank je
Wohnen!
voor
Merci
m'ont aidé
immer unterstützt habt.
Eure Freundschaft und
bedanken möchte ich mich auch bei meiner
„heimelige"
•
avec
Birgit, Caroline, Christoph, Cécile, Eric, Karin, Katja, Stephan
Katrin, Momo, Felix, Hampi, Thömes: Danke für
für's
Marion
Oregon.
und Familie: Euch möchte ich danken für die offenen Türen, Ohren und Herzen.
Speziellen
I thank
•
105
Sibylle
Baba,
Unterstützung!
•
Im
WG-Familie, Baba und Hampi: danke
Guido: omdat wat jij met
alles.
•
•
et
mij doet,
de lente doet met de kersen
Publications
Notz, R., Maurhofer, M., Dubach, H., Haas, D., and Défago, G. 2002. Fusaric-acid-producing
strains of Fusarium oxysporum alter
expression
Appl.
2,4-diacetylphloroglucinol biosynthetic
in Pseudomonas fluorescens CHAO in vitro and in the
gene
of wheat.
rhizosphere
Env. Microbiol. 86:2229-2235.
Notz, R., Maurhofer, M., Schnider-Keel, U., Duffy, B., Haas, D., and Défago, G. 2001. Biotic
factors
affecting expression
of the
2,4-diacetylphloroglucinol biosynthesis
Pseudomonas fluorescens biocontrol strain CHAO in the
gene phlA in
rhizosphere. Phytopathology
91:873-881.
Schnider-Keel, U.,
Reimmann,
A.
R.
Seematter,
M.
Maurhofer,
Blumer,
B.
Duffy,
C.
Gigot-Bonnefoy,
C.
Notz, G. Défago, D. Haas, and C. Keel. 2000. Autoinduction of 2,4-
diacetylphloroglucinol biosynthesis
CHAO and
C.
repression by
in the biocontrol agent Pseudomonas fluorescens
the bacterial metabolites
salicylate
and
pyoluteorin.
J.
Bacteriol. 182:1215-1225.
Notz, R., and Walter, T. 1998. Zur Entwicklung der Tagfalter-Gemeinschaften nach
Räumungen
am
Beispiel
der Niederholzes
(Kanton Zürich).
Schweiz. Z. Forstwes.
149:808-821.
Ryser, P., and Notz,
density.
R. 1996.
Competitive ability
of three
ecologically contrasting
Bulletin-of-the-Geobotanical-Institute-ETH. 1996; 62:3-12.
106
grass tissue
Curriculum
vitae
Regina Edith Notz
Born July
1997-2002
6th 1971
in
Mannedorf, ZH
PhD candidate and research assistant at the Institute of Plant
Science/Phytopathology
(EHTZ), supervised by
2000
Two month
training
group, Swiss Federal Institute of
Prof. Dr. Geneviève
program to construct
a
Department of Botany and Plant Pathology,
1996-1997
research
lab, Oregon
Practical
training
consultant,
State
followed
University (Dr.
in Natural
reporter gene fusion
at
the
USDA-ARS Horticultural
J. E.
Crops
Loper)
as an
(ANL)", Aarau,
environmental
Switzerland
Sciences,
1996
Diploma
1996
Diploma thesis
1991-1996
Studies of Natural
1991
Federal Matura, type B
1988-1989
Foreign exchange
1986-1991
Kantonsschule Stadelhof en, Zurich
1978-1986
Primary
and
Zurich
Défago.
by permanent appointment
"AG für Natur und Landschaft
Technology
in the group of Nature and
Sciences,
year
secondary
Landscape Conservation,
ETHZ
USA, High School degree
school in Richterswil ZH
107
ETHZ