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FEMS Microbiology Ecology 17 (199.5) 221-228
MiniReview
Colonization of the rhizosphere of crop plants by plant-beneficial
pseudomonads
Letty A. de Weger
*, Arjan J. van der Bij, Linda C. Dekkers, Marco Simons,
Care1 A. Wijffelman, Ben J.J. Lugtenberg
Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory,
Wassenaarseweg
64, 2333 AL Leiden, the Netherlands
Received 21 December 1994; revised 28 April 1995; accepted 10 May 1995
Abstract
Efficient colonization of the plant root is thought to be crucial for the plant-beneficial effect of particular Pseudomonas
strains. Since root colonization is often the limiting step for successful plant growth stimulation, this process needs
improvement. It is therefore important to acquire more information as regards (i) the conditions in the rhizosphere, and (ii)
the bacterial traits that are involved in colonization. This review discusses some recent studies that focus on these two issues.
Keywords:
Pseudomonas;
Rhizosphere colonization; Biocontrol
1. Introduction
Nowadays agriculture requires the extensive use
of chemical pesticides to protect crops against pests
and diseases. Several of these chemicals pollute our
ground water and drinking water and therefore some
governments have decided to reduce these chemical
inputs substantially. This urges the need for altemative crop protectants. One of these alternatives is the
use of biological control agents, among which are
microorganisms
that can protect the plant against
diseases. Many different microbial genera (fungi and
bacteria) have been described as potential biocontrol
agents against soil-related diseases [ 11, but especially
the group of fluorescent Pseudomonas spp. is receiving much attention. Spectacular results have been
’ Corresponding author. Tel: (31)-71-275056; Fax: (31)71275088; E-mail: [email protected]
obtained after treatment of the seeds with particular
strains, but often their performance is not consistent.
Therefore a better understanding
of the crucial steps
involved in this plant-beneficial
bacterium interaction is required for a successful commercial application of these microbes.
The mechanisms
by which Pseudomonas
spp.
exert their beneficial effect on plants can be very
diverse [1,2]. Under conditions of Fe3+ limitation,
some Pseudomonas spp. produce siderophores that
scavenge the traces of Fe3+ in the soil and thereby
deprive other microbes, including deleterious and
pathogenic ones, of this essential element. Other
Pseudomonas spp. secrete antibiotics inhibitory to
pathogens, or enzymes that degrade fungal cell walls.
Considering
these different modes of action it is
crucial that the beneficial
bacterium delivers the
active compound at the place where it is required.
When protection of the whole root is required, an
efficient colonization
of the root system is consid-
016%6496/95/$09.50 0 1995 Federation of European Microbiological Societies. AR rights reserved
SSDI 0168-6496(95)00031-3
222
L.A. de Weger et al. /FEMS
Microbiology
ered to be the best delivery system of a successful
biocontrol activity. This notion is supported by two
findings: (i) unsuccessful
field trials are often accompanied by inadequate colonization of the roots
[3], and (ii) the number of lesions in wheat roots
caused by the pathogenic fungus Gaeumannomyces
graminis var. tritici decreased with an increasing
number of plant protecting bacteria of Pseudomonas
fluorescens 2-79 on the roots [4].
To improve this limiting step in the biocontrol
performance of these bacteria, a better knowledge of
the mechanism
of root colonization
is important.
This review focuses on research that intends to increase our knowledge of root colonization.
Firstly,
we will focus on the growth conditions in the rhizosphere, since this is the site where the bacteria have
to be active in producing their biological control
compound. Secondly, bacterial traits and genes that
are involved in the colonization
process will be
discussed.
2. Growth conditions in the rhizosphere
The rhizosphere is an ecological niche which is
mainly a black box. Hardly anything is known about
the availability of nutrients (organic or inorganic),
the dangers which must be survived (e.g. predation,
harmful compounds) or the interactions with other
rhizosphere organisms. The deficiency in our knowledge on the growth conditions in the rhizosphere is
illustrated by the fact that only 1 to 10% of the
microorganisms
present in the rhizosphere can be
cultured on laboratory media [5]. The classical methods (e.g. chemical methods) have helped us to determine the amount of various nutrients like nitrate,
phosphate, or iron. However, they did not demonstrate the proportion of these nutrients that are really
available for microbes growing in the rhizosphere.
Novel molecular techniques, which can be used for
measurements in situ should help us to increase our
knowledge on this ecological niche.
The in situ determinations
on the availability of
nutrients have been studied in different ways. Using
a microelectrode
the oxygen gradient in the rhizosphere of barley was measured [6]. The oxygen
concentration
in bulk soil was much higher than in
the rhizosphere soil, due to the high microbial respi-
Ecology I7 (1995) 221-228
ration in the rhizosphere and the oxygen uptake by
the root. Furthermore the measurements showed that
the rhizosphere can easily develop very low oxygen
concentrations
and even complete anoxia.
Employment of the denitrifying activity of bacteria appeared to be very helpful in measuring two
different forms of nitrogen, nitrate and nitrite, in the
rhizosphere. Besides nitrate fertilizer which is often
added to agricultural soils, nitrification activity is the
only other source of nitrate. Nitrite is usually present
in small amounts in natural soils, probably because it
is an intermediate in both nitrification and denitrification. A very sensitive bioassay was developed,
employing
denitrifying
Pseudomonas
aeruginosa
cells [7]. A mutant strain was used that was unable to
reduce nitrate, but still able to convert nitrite into
N,O, which can be detected using gas chromatography. The remaining nitrate in the samples can subsequently be determined after addition of wild-type P.
aeruginosa cells capable of reducing nitrate to N,O.
This bioassay was used to measure the nitrate and
nitrite pools in 10 mg soil samples taken along a
microgradient from various parts of the rhizosphere
of field-grown barley plants. The relative amounts of
nitrate and nitrite in the rhizosphere were higher than
in bulk soil, indicating a net accumulation of these
two compounds from nitrification activity at the root
surface. Wetting of the soil reduced the amount of
nitrate in the rhizosphere, probably due to a decrease
in the O2 concentration in the rhizosphere, which in
turn may favour denitrifying activities [7].
For measuring the bioavailability
of phosphate
and iron in the soil and in the rhizosphere so-called
reporter bacteria were constructed, which can report
whether growth conditions in situ are limiting for a
certain compound [8,9]. Phosphate reporter bacteria
were constructed which respond to phosphate-limiting conditions by the production of P-galactosidase
[8]. The level of this enzyme can easily be quantified
using
the Miller
test employing
ONPG
(Onitrophenyl-/3-galactopyranoside)
as the substrate.
When phosphate reporter cells were growing under
gnotobiotic conditions in the rhizosphere of tomato,
potato or radish plants or in the bulk soil, they
sensed phosphate limitation as judged from the significant
increase in /3-galactosidase
activity [s].
Iron-reporter bacteria were constructed using another
reporter protein, InaZ, which catalyses ice formation
LA. de Weger et al. / FEMS Microbiology
at - 2 to - 10°C [9]. When these cells experience
iron-limitation
they start to produce the InaZ-protein,
which can be quantified by a droplet freezing assay.
Using this reporter system it was shown that Pseudomonas cells growing in the rhizosphere of bean
experience a moderate but not very severe iron limitation. Comparing the two reporter systems discussed
here, it is evident that the InaZ-based system is more
sensitive than pgalactosidase.
Therefore, the former
system will be better suited for situations where low
transcriptional
activity of the promoter of interest is
to be expected or when only low numbers of bacteria
can be reisolated from the microniche.
However,
when enough cells can be recovered from the environment and when transcriptional
activity is high,
/3-galactosidase is a very fast and easy reporter system for whose detection no special equipment is
required.
Plant roots can lose a large portion (up to 31%) of
their assimilates, mainly in the form of amino acids,
organic acids and sugars [lo]. However, using HPLC
analysis, only small amounts of amino acids including histidine, leucine and isoleucine could be detected in sterile tomato root exudate (M. Simons et
al., unpublished
results). Auxotrophic
mutants impaired in the synthesis of these amino acids were
used to study whether tomato roots are able to
supply these auxotrophs with a sufficient amount of
the relevant amino acid to support colonization.
In
contrast to the parent strain the mutants appeared to
be unable to colonize the root tips of tomato. Addition of the relevant amino acid to the sand in which
the plants were growing restored the colonization
ability of the mutants (M. Simons et al., unpublished
results). These results show that these mutants are
able to colonize tomato roots provided that the required amino acid is present, which apparently cannot be supplied by the tomato roots in amounts
sufficient
to support colonization
of these auxotrophs.
Predation is one of the hazards that bacteria in the
rhizosphere can encounter. This may either be predation by protozoa or by bacteriophages. The presence
of a bacteriophage
in the soil specific for a given
strain significantly
reduces the numbers of cells of
this strain present in the rhizosphere [ll]. Attachment to soil particles may protect the cells from
predation by protozoa [12]. Using Zux marked bacte-
Ecology 17 (1995) 221-228
223
ria it was nicely demonstrated that cells present in
the small pores of soil particles were better protected
from protozoan predation than the cells in the larger
pores (J. Presser, personal communication).
Recently a new class of signalling molecules have
been discovered in a wide range of Gram-negative
bacteria, which enable the bacterial cells to monitor
the growth of its population [13]. When the bacterial
population reaches high densities, these signalling
molecules with a common structure (N-acyl homoserine lactones = acyl-HSLs) mediate the induction of new sets of genes responsible for e.g. bioluminescence
in Vibrio fischeri [14], antibiotic and
exoenzyme production in Erwinia carotovora [ 151,
and Ti-plasmid conjugation in Agrobacterium tumefaciens [16]. Also the phenazine production in the
biocontrol
bacterium
Pseudomonas
aureofaciens
strain 30-84 appeared to be quorum-regulated
[17].
This stresses again the relevance of an efficient
colonization of the plant root which must lead to the
formation of microcolonies in which the cell density
is high enough to induce the production of this
pathogen-inhibiting
antibiotic. Interestingly,
certain
bacteria can respond to the signalling molecules of
other bacterial species. Using an Agrobacterium
tumefaciens indicator strain with a 1acZ gene introduced into the acyl-HSL responsive tra operon [16],
a series of 25 different Pseudomonas
root isolates
and a Rhizobium leguminosarum
strain have been
tested for their ability to induce the A. tumefaciens
tra operon. One biocontrol
Pseudomonas
putida
strain, strain WCS358, and the R. leguminosarum
strain appeared to produce signalling
molecules
which were recognized by the A. tumefaciens induction machinery (de Weger, unpublished results). This
inter-species
signalling
challenges
our traditional
view of microbial life in the rhizosphere. Rather than
an independent entity, bacterial cells may communicate with many of its neighbouring cells either of its
own kind or of other species [13].
3. Bacterial traits involved in colonization
We hypothesised
that structures located at the
outer surface of the cell could be important for the
interaction with the root, and therefore for colonization. By comparing the colonization abilities of mu-
224
L.A. de Weger et al. / FEMS Microbiology
tants deficient in particular structures with that of the
parent strain, the role of these structures in colonization could be determined. Flagellaless mutants of P.
fluorescens strain WCS374 appeared to be impaired
in the colonization of the deeper root parts of potato
plants [18]. In contrast Howie et al. [19] did not
observe any difference between nonmotile mutants
and the parent strain in the colonization
of wheat
roots. These results may be due to differences in
experimental
design
(e.g. plant species,
Pseudomonas strain, soil type).
De Mot et al. [20] showed that mutants of P.
fluorescens strain OE28.3 which are deficient in the
major outer membrane protein OprF are impaired in
their adhesion to wheat roots. Pili, filamentous, proteinaceous extrusions, have been found to be involved in the adhesion of many plant pathogenic
bacteria to plant surfaces, e.g. Erwinia spp., Pseudomonas syringae [21]. Vesper showed that also in
the plant growth-promoting
P. fluorescens
strain
2-79 the presence of pili was correlated with attachment to corn roots [22]. In neither of these studies
the question whether attachment is involved in colonization is addressed and this remains a matter of
debate. Some studies do find a correlation between
the abilities to attach to and to colonize the root [23],
while in other studies such a correlation was not
found [24].
Polysaccharides
at the bacterial cell surface are
involved in the Rhizobium-legume
interaction [25].
Two polysaccharides
at the surface of plant-beneficial Pseudomonas strains have been studied for their
role in root colonization. Mutants of P. jluorescens
WCS374 and P. putida WCS358 that lacked the
0-antigenic
side chain of their lipopolysaccharide
were impaired in the colonization
of the deeper
potato root parts [24]. This reduced colonization
ability appeared neither to be due to reduced binding
to the plant root, nor to poor survival of these
mutants. Apparently the 0-antigenic
side chain is
relevant for the distribution of the cells along the
root system. In contrast to the O-antigen, a polysaccharide located at the cell surface of P. putida strain
WCS358, which shares characteristics with the K-antigens present in Escherichia cob, appeared not to
be relevant for colonization (de Weger et al., submitted for publication).
The major factors responsible for the stimulation
Ecology 17 (1995) 221-228
of microorganisms
in the rhizosphere are the excretion of organic compounds and the sloughing off of
root hairs and epidermal cells 1261. The ability to
utilize these various nutrients is assumed to play an
important role in colonization. Cells which can utilize many major rhizosphere nutrients efficiently may
therefore have an advantage over cells that are fastidious. On the other hand a complete dependence on
specific rhizosphere nutrients seemed to be a disadvantage since it appeared that amino acid auxotrophs
of P. fluorescens WCS365, in contrast to the wildtype, were unable to colonize the tomato rhizosphere
unless the required amino acid was added to the
medium (see also above, M. Simons et al., unpublished). So, to be competitive in the rhizosphere the
cells have to be able to synthesise their own amino
acids.
Another aspect of life in the rhizosphere is that
the cells will have to be able to compete with the
indigenous
microorganisms
for the available resources in the rhizosphere. Production of compounds
that reduce the growth of competing colleagues are
likely to be favourable for effective colonization of
the rhizosphere. Siderophores
play a role in the
antagonistic activity of certain Pseudomonas strains
(see above), but they can also be relevant for the
establishment
of the cells in the rhizosphere.
P.
putida strain WCS358 is a unique strain in that it
cannot only utilize its own iron-siderophore
but also
the iron-siderophores
of a diversity of other strains,
for example that of strain P. fluorescens
strain
WCS374 [27]. When a siderophore-negative
mutant
of strain WCS358 is mixed through soil, its cell
numbers in the rhizosphere are increased by the
presence of wild-type (siderophore-producing)
cells
of WCS358 as well as those of WCS374. In contrast,
the rhizosphere population of a siderophore-negative
mutant
of WCS374,
which cannot
utilize the
siderophore of WCS358 [27] is increased by the
presence of its own wild-type cells but reduced by
the presence of cells of strain WCS358 [28]. This
result shows that the production of siderophores and
the ability to utilize the iron-siderophore
complexes
of other microorganisms
is an important trait for the
establishment
of cells in the rhizosphere.
Many
Pseudomonas
spp. produce antibiotics which can
play a role in the competition with other rhizosphere
microorganisms.
When a mutant of P. fluorescens
LA. de Weger et al. / FEMS Microbiology
strain 2-79, deficient in the synthesis of the antibiotic, is studied for its wheat root colonization ability,
the mutant was present at population levels comparable to those of the parent strain [29]. However, when
the mutant and parent strain were compared after
five successive 20-day plant-harvest cycles of wheat
in the same soil, the mutant strain declined more
rapidly in population size than the wild-type strain
[30]. This indicates that the presence of antibiotic
production can be a relevant factor for growth and
survival in the rhizosphere.
Another approach which may lead to the discovery of new unexpected traits involved in colonization
is the direct isolation of mutants defective in colonization. The impaired functions in these mutants
will teach us more about novel traits involved in
colonization.
Using P. fluorescens strain WCS365
with excellent root colonizing properties, we have
screened approximately
900 mutants on individual
potato, tomato or wheat plants in a gnotobiotic colonization assay (Fig. 1) for their colonization ability
in competition with the parental strain. We isolated
approximately
20 mutants which show an impaired
colonization
phenotype on either of these plants.
Among them were auxotrophic mutants, mutants impaired in motility, mutants lacking the 0-antigenic
side chain of LPS and mutants with a reduced growth
rate. Three mutants were found that did not show
any known defects. These mutants are the most
interesting since they can lead to new colonization
traits after cloning and screening of the corresponding wild-type genes.
4. Future prospects
The current efforts to understand the mechanisms
of plant growth promotion by Pseudomonas
spp.
will teach us which steps in this process are inefficient. Future research will have to focus on the
regulation of crucial genes, like genes for antibiotic
synthesis or genes involved in colonization.
Since
environmental
factors in the rhizosphere may influence the expression of these genes and thus influence
the biocontrol activities of the strains, we will have
to increase our knowledge on the growth conditions
in the rhizosphere. Once we know these conditions
we may be able to construct more efficient biocon-
Ecology 17 (I 995) 221-228
wildtype :
mutant = 1
1
Fig. 1. Schematic representation of the colonization assay used to
screen colonization-negative
mutants. Sterile plants are inoculated
with a 1:l mixture of the wild-type and the In&marked
mutant.
After one week of growth the ratio of wild-type and mutant cells
on the root tips is determined on plates containing X-gal (5.
bromo-4-chloro-3-indolyl-P-r>galactoside).
trol bacteria for instance by manipulating
antibiotic
production with respect to timing and site. Furthermore, a better understanding
of the rhizosphere conditions may allow us to mimic the rhizosphere conditions in the laboratory such that non-culturable
microorganisms, which may comprise 90% to 99% of
the bacterial population present in the rhizosphere,
can be cultured. This would allow us to study this
anonymous group of bacteria.
Several bacterial traits have been identified as
being relevant for efficient root colonization,
i.e.
presence of flagella, presence of the 0-antigenic side
chain, and the ability to synthesise amino acids.
Other traits reported to be relevant for the ability to
compete with other rhizosphere microorganisms
are
226
L.A. de Weger et al. / FEMS Microbiology
e.g. production of antibiotics or siderophores and the
ability to utilize siderophores of other rhizosphere
bacteria. The present knowledge can be used by
companies involved in the development of inoculants
to improve strains by genetic engineering
or for
screening of isolates on these particular traits.
Efficient colonization
of plant roots can also be
employed in other areas of research e.g. for bioremediation of polluted soil by employing bacterial colonizers of a plant with an extensive root system.
Furthermore, the relevance of colonization
and microcolony formation is stressed by the newly discovered quorum-sensing
signal molecules (acyl-HSL’s).
These molecules mediate production of antibiotics
and secretion of exoenzymes only when cell numbers are high. Therefore the establishment
of high
populations on the root is not only relevant for the
delivery of the biocontrol compound but even already for its synthesis. Disclosure of the complex
network of interactions which comprises life in the
rhizosphere can only be reached by a multidisciplinary approach.
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