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Molecular and chemical basis of the interaction between plant-protecting pseudomonads and their crop plants

Objectif

(1) Isolating genes and elucidating traits involved in aggressive root colonization of tomato and sugarbeet by Pseudomonas fluorescens. (2) To optimize 2,4 diacetylphloroglucinol (Phl) production for biocontrol purposes. (3) To combine adhesion, colonisation and optimal Phl production in biocontrol strains in order to improve adhesion, colonization, antagonism towards pathogenic micro-organisms, and (therefore) biocontrol properties of these improved strains under microcosm and greenhouse conditions on tomato and sugarbeet.
1. All tasks have been fulfilled. All deliverables have been produced.
2. Genes and traits involved in colonisation.
- A gnotobiotic system to study tomato root colonisation after inoculation of germinated seedlings by one or two bacterial strains has been set up.
- Competition between Pseudomonas fluorescens strains in this system showed the following order of tomato colonisation ability: WCS365 and F113 >> M114 and OE28.3 >> WCS307. The same result was found in potting soil. For attachment to wheat and tomato roots the order is OE28.3 and M114 > WCS365 and F113.
- Phl production has no effect on colonisation.
- Mutants which colonized 1E2-1E3 less than the parental stain WCS365 but which have a wild type growth rate were obtained after screening random Tn5(lacZ) mutants.
- Mutant PCL1210 has its mutation in the operon p.colR-colS-orf-222. The mutation in colS is responsible for the colonisation defect, presumably as part of a two-component ColR/ColS system in which ColS functions as the sensor and ColR as the response regulator in a pathway which is used by a so far unknown stimulus to activate a so far unknown colonisation trait. The promoter region indicates the presence of an IHF binding site.
- Mutant PCL1233 has its mutation in the operon p.lppL - lysA - dapF - orf235 - xerC - orf2381...... Mutation of xerC appeared to be responsible for the colonisation defect. xerC encodes a site-specific recombinase involved in phase variation which is often caused by an altered cell surface component. We hypothesize that P. fluorescens populations consist of at least two sub-populations of which one is best equipped for root colonisation. Mutant PCL1233 would then be `locked' in (one of) the other genetic form(s). The promoter region indicates the presence of an IHF binding site.
- Mutants auxotrophic for amino acids or vitamin B1 are severely defective in root colonisation, indicating that only minor amounts of these components are present in exudate. Mutants impaired in sugar utilisation colonise like the parent, indicating that sugars are not the major C-source in exudate.
- oprF mutants of OE28.3 are impaired in attachment to wheat and tomato roots. oprF mutants of OE28.3 and F113 are impaired in colonisation of tomato and sugarbeet roots, respectively. Genetic differences in the oprF genes of various strains were found but these were not clearly correlated with colonising ability.
- Using col mutants and introduction of col genes, we showed that colonisation is involved in biocontrol.
3. Phl production
- Biosynthetic and regulatory genes of F113 have been identified. Sequence analysis of the biosynthetic cluster identified thiolase, chalcone synthase and a putative Phl permease. Use of a lacZ transcriptional fusion to the chalcone synthase gene revealed intricate regulation through the GacA/LemA two-component system and additional responses to both carbon source and iron concentration
- Biochemical analysis of mono-acetyl-Phl (MAP) acetyl-transferase activity has demonstrated the conversion of MAP to Phl and shown that this activity is influenced by carbon source.
- Mutants within the Phl regulatory loci display identical phenotypes corresponding to a loss of Phl, casein protease and HCN synthesis. Two groups of regulatory genes were identified as gacA and lemA homologues. In addition, either of these two loci could be complemented by a third, independent region. This region was located on a 2.8 fragment encompassing a fdxA (ferredoxin I) homologue which activates the transcription of chalcone synthase in trans within gacA and lemA mutants.
4. Exudate composition
- The amino acid composition of tomato root exudate and the sugar composition of sugarbeet and tomato exudate have been determined.
- Promoters have been found which respond to bogh sugarbeet and tomato exudate. This finding opens the possibility to construct strains in which Phl synthesis only occurs in the rhizosphere.
5. Biocontrol
- F113 produces Phl which is involved in the inhibition of a range of plant-pathogenic fungi. It is a biocontrol strain in the sugarbeet - Pythium ultimum system.
- WCS365 is a biocontrol strain in the tomato-Fusarium oxysporum f.sp. radicis lycopersici system.
- A procedure was developed for coating of seeds with bacteria, using a small scale fluidised bed coater. Survival of bacteria was 1-10%. Shelf life is strain-dependent.
- A biocontrol assay for the tomato-Fusarium system was developed. Forty-six promising wild type strains were tested with yielded 8 excellent biocontrol strains.
- In this system WCS365 had biocontrol activity in all 6 tests of which the difference in 4 tests was statistically significant.
- WCS365 colonisation mutants were descreased in disease-suppressing ability, indicating - as expected but never proven - that colonisation is crucial for biocontrol.
6. Strain improvement
- Transfer of the Phl biosynthetic locus to Phl-negative strains usually leads to a Phl+ phenotype
- The non-biocontrol strain OE28.3 engineered to Phl+ has gained biocontrol activity.
- Exudate-inducible promoters have been identified and can be used to construct strains which produce Phl only when required, i.e. in the rhizosphere.
- The tomato-root colonising ability of the excellent colonizer F113 was 10-fold increased by introduction of the col-1233 locus.
- The poorly colonising non-biocontrol strain WCS307 was 100-fold improved in colonisation and became a biocontrol strain after introduction of the col-1233 locus.
- These results show that strain improvement using phl and col genes is a realistic goal.
MAIN BREAKTHROUGHS:
1. New biocontrol strains have been identified.
2. Genes involved in attachment, colonisation and Phl production have been isolated and sequenced. Their role in biocontrol has been established. The results show that strain improvement for biocontrol purpose isa a realistic goal.
3. Using overproduction, strains with improved colonisation, Phl production and biocontrol properties have been constructed.
4. Exudate components have been identified and the role of their utilisation in colonisation and biocontrol has been established.
5. Exudate-inducible promoters have been identified. They can be used to switch on Phl production only in the rhizosphere.
NUMBER OF PUBLICATION/PATENTS:- total number: 46- joint publications: 10

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LEIDEN UNIVERSITY
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64,Wassenaarseweg 64 Clusius Laboratorium
2333 AL LEIDEN
Pays-Bas

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Dernière mise à jour: 16 Août 1995

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Permalink: https://cordis.europa.eu/project/id/BIO2930196/fr

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