and chemical basis of the interaction between plant-protecting pseudomonads
and their crop plants
This project focused on the application of Pseudomonas bacteria
to prevent plant disease, i.e. to function as a biopesticide or prebiotic.
In general, a biopesticide directed against fungal pathogens should produce
an antifungal factor (AFF) and deliver this AFF along the root system
before the pathogen attacks. The latter process, root colonisation, is
often the limiting factor in biocontrol. Many agrochemicals currently
used to control fungal infestations of economically important plant species
are a threat to the environment, and therefore to our health. Interest
in biological alternatives, often utilising microbes, has therefore increased.
Indeed, microbes are known to act as biofertilisers, biopesticides and
stimulators of plant growth. Our work aims to contribute to the understanding
of microbial control of plant diseases by providing the scientific database
which is necessary to establish legislation for the safety of biological
Click to view graphic
evaluation of DAPG production by Pseudomonas fluorescens F113 in
response to iron in a plate bioassay.
of Fusarium oxysporum f.sp. radicis-lycopersici growth
by different Pseudomonas strains on King's B agar plates.
of a bioassay showing inhibition of growth of the fungus Pythium
ultimum (top) and HPLC analysis of production of the antibiotic
DAPG (bottom) by the biocontrol strain P.fluorescens F113 (left)
and its antibiotic-negative mutant strain G22. MAPG and DAPG: mono-
and di-acetylphloroglucinol, respectively.
plants inoculated with Fusarium oxysporum f.sp. radicis-lycopersici
(left) and a healthy control plant and a plant protected by P.
fluorescens strain WCS365 (far left).
The focus was on (i) the isolation and characterisation of genes involved
in the production of 2,4-diacetyl phloroglucinol (DAPG) as a model AFF,
and (ii) the isolation and characterisation of genes involved in root
colonisation. State-of-the-art molecular genetic, microbiological and
chemical techniques were used. The major test plants were sugar beet,
tomato and wheat. Biocontrol tests were carried out under microcosm and
greenhouse conditions. For the accurate measurement of root colonisation,
following inoculation of seeds, a gnotobiotic test system was developed.
In such a system the interactions between one plant and one or two different
microbes can be studied without the complexity of natural soil. This makes
results more reproducible and conclusions can be drawn faster. All interesting
results are subsequently repeated in real soil.
Main findings and outcome
We isolated several biosynthetic and regulatory genes involved in DAPG
production by P. fluorescens strain F113. Nucleotide sequence analysis
identified a biosynthetic operon containing genes with thiolase and chalcone
synthase activity, as well as a putative DAPG permease. Use of lacZ
transcriptional fusions to the chalcone synthase gene revealed that the
GacA/LemA two-component system is involved in regulation of DAPG synthesis.
In addition, environmental factors such as the carbon source and Fe3+
play a crucial role.
Competitive colonisation studies have revealed that P. fluorescens
strains WCS365 and F113 are the best colonisers of a series of tested
European biocontrol strains. Production of DAPG does not affect colonisation.
Colonisation mutants of these strains were isolated. Their analysis revealed
that the following genes and traits are involved in colonisation: (i)
a colR/colS two-component system, later shown to regulate outer
membrane permeability and therefore competition for nutrients in the rhizosphere
(the niche around the plant roots), (ii) the xerC/sss gene which
encodes a site-specific recombinase (this finding has led to the discovery
that phase variation, the ability of a bacterium to create subpopulations
with properties suitable to occupy different environmental niches, is
a very important trait in the rhizosphere), (iii) the outer membrane pore
protein OprF, and (iv) the synthesis of amino acids and vitamin B1.
We found that the biosynthetic genes of the AFF DAPG are clustered. In
principle, this creates the opportunity to improve biocontrol by overexpressing
DAPG or by transferring the DAPG biosynthetic genes to another biocontrol
strain to extend the biological control mechanisms of the recipient strain.
The regulation of DAPG production is extremely complex, suggesting that
the amount of DAPG produced in the rhizosphere may differ, depending on
plant and soil conditions. Our studies showed that many genes, possibly
even several hundred, are involved in root colonisation.
The genes characterised so far show that control plant colonisation (the
col genes) are all naturally-occurring genes. There is no indication
that these col genes reduce the safety of biopesticides. These
results revealed that DAPG and col genes are promising for improving
biocontrol properties of wild type Pseudomonas strains. Based on
these conclusions, these findings are further developed in the project
Carroll H., Moenne-Loccoz Y., Dowling D. and OGara F., Mutational
disruption of the biosynthesis genes coding for the antifungal metabolite
2,4-diacetylphloroglucinol does not influence the ecological fitness
of Pseudomonas fluorescens F113 in the rhizosphere of sugar
Appl. Environm. Microbiol., 61, 1995, p. 3002.
Dekkers L.C., Phoelich C.C., Van der Fits L. and Lugtenberg B.J.J.,
A site-specific recombinase is required for competitive root
colonization by Pseudomonas fluorescens WCS365.
Proc. Natl. Acad. Sci., 95, 1998, p. 7051.
Dunne C., Moënne-Loccoz Y., McCarthy J., Higgins P., Powell
J., Dowling D.N. and OGara F., Combining proteolytic
and phloroglucinol-producing bacteria for improved biocontrol of
Pythium-mediated damping-off of sugar beet.
Plant Pathology, 47, 1998, p. 299.
Lugtenberg B.J.J., Kravchenko L.V. and Simons M., Tomato seed
and root exudate sugars: composition, utilization by Pseudomonas
biocontrol strains and role in rhizosphere colonization.
Environ. Microbiol., 1, p. 439.
Schippers B., Scheffer R.J., Lugtenberg B.J.J. and Weisbeek P.J.,
Biocoating of seeds with plant growth-promoting rhizobacteria
to improve plant establishment.
Outlook on Agriculture, 24, 1995, p. 179.
Simons M., Van der Bij A.J., De Weger L.A., Wijffelman C.A., and
Lugtenberg B.J.J., Gnotobiotic system for studying rhizosphere
colonization by plant growth-promoting Pseudomonas bacteria.
Mol. Plant-Microbe Interact., 9, 1996, p. 600.
October 1993 October 1996
Leiden University (NL)
of the project
This project was continued in EC project: BIO4-CT98-0254
Novartis Seeds B.V.
University College Cork (IE)
Katholieke Universiteit Leuven
Irish Sugar plc.