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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Molecular and chemical basis of the interaction between plant-protecting pseudomonads and their crop plants

Background and objectives

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 pest-control agents.


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Semi-quantitative evaluation of DAPG production by Pseudomonas fluorescens F113 in response to iron in a plate bioassay.


Inhibition of Fusarium oxysporum f.sp. radicis-lycopersici growth by different Pseudomonas strains on King's B agar plates. image Inhibition of Fusarium oxysporum f.sp. radicis-lycopersici growth by different Pseudomonas strains on King's B agar plates.

 

Demonstration 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. image Demonstration 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.

 

Tomato 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). image Tomato 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).

 

Approach and methodology

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.
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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.
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Conclusions

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 BIO4-CT98-0254.

 

Major publications

Carroll H., Moenne-Loccoz Y., Dowling D. and O’Gara 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 beets”.
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 O’Gara 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.
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imageResearch project
 

Contract number
BIO2-CT93-0196

Period
October 1993 – October 1996

Coordinator
B.J.J. Lugtenberg
Leiden University (NL)

Follow-up of the project
This project was continued in EC project: BIO4-CT98-0254

 
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Partners


R.A. Scheffer
Novartis Seeds B.V.
Enkhuizen (NL)

F. O'Gara
University College Cork (IE)

J. Vanderleyden
Katholieke Universiteit Leuven
Heverlee (BE)

J. Powell
Irish Sugar plc.
Carlow (IE)

 
 
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