Geneviève Défago

Biological control of soil-borne pathogens by fluorescent pseudomonads.

Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.

Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions

Pseudomonas fluorescens CHA0 suppresses black root rot of tobacco, a disease caused by the fungus Thielaviopsis basicola. Strain CHA0 excretes several metabolites with antifungal properties. The importance of one such metabolite, hydrogen cyanide, was tested in a gnotobiotic system containing an artificial, iron‐rich soil. A cyanidenegative (hcn) mutant, CHA5, constructed by a gene replacement technique, protected the tobacco plant less effectively than did the wild‐type CHA0. Complementation of strain CHA5 by the cloned wild‐type hcn+ genes restored the strains ability to suppress disease. An artificial transposon carrying the hcn+ genes of strain CHA0 (Tnhcn) was constructed and inserted into the genome of another P.fluorescens strain, P3, which naturally does not produce cyanide and gives poor plant protection. The P3::Tnhcn derivative synthesized cyanide and exhibited an improved ability to suppress disease. All bacterial strains colonized the roots similarly and did not influence significantly the survival of T.basicola in soil. We conclude that bacterial cyanide is an important but not the only factor involved in suppression of black root rot.

Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities.

Pseudomonas fluorescens strain CHA0, a root colonizing bacterium, has a broad spectrum of biocontrol activity against plant diseases. However, strain CHA0 is unable to utilize 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of plant ethylene, as a sole source of nitrogen. This suggests that CHA0 does not contain the enzyme ACC deaminase, which cleaves ACC to ammonia and alpha-ketobutyrate, and was previously shown to promote root elongation of plant seedlings treated with bacteria containing this enzyme. An ACC deaminase gene, together with its regulatory region, was transferred into P. fluorescens strains CHA0 and CHA96, a global regulatory gacA mutant of CHA0. ACC deaminase activity was expressed in both CHA0 and CHA96. Transformed strains with ACC deaminase activity increased root length of canola plants under gnotobiotic conditions, whereas strains without this activity had no effect. Introduction of ACC deaminase genes into strain CHA0 improved its ability to protect cucumber against Pythium damping-off, and potato tubers against Erwinia soft rot in small hermetically sealed containers. In contrast, ACC deaminase activity had no significant effect on the ability of CHA0 to protect tomato against Fusarium crown and root rot, and potato tubers against soft rot in large hermetically sealed containers. These results suggest that (i) ACC deaminase activity may have lowered the level of plant ethylene thereby increasing root length; (ii) the role of stress-generated plant ethylene in susceptibility or resistance depends on the host-pathogen system, and on the experimental conditions used; and (iii) the constructed strains could be developed as biosensors for the role of ethylene in plant diseases.

Salicylic Acid Biosynthetic Genes Expressed in Pseudomonas fluorescens Strain P3 Improve the Induction of Systemic Resistance in Tobacco Against Tobacco Necrosis Virus

ABSTRACT Application of salicylic acid induces systemic acquired resistance in tobacco. pchA and pchB, which encode for the biosynthesis of salicylic acid in Pseudomonas aeruginosa, were cloned into two expression vectors, and these constructs were introduced into two root-colonizing strains of P. fluorescens. Introduction of pchBA into strain P3, which does not produce salicylic acid, rendered this strain capable of salicylic acid production in vitro and significantly improved its ability to induce systemic resistance in tobacco against tobacco necrosis virus. Strain CHA0 is a well-described biocontrol agent that naturally produces salicylic acid under conditions of iron limitation. Introduction of pchBA into CHA0 increased the production of salicylic acid in vitro and in the rhizosphere of tobacco, but did not improve the ability of CHA0 to induce systemic resistance in tobacco. In addition, these genes did not improve significantly the capacity of strains P3 and CHA0 to suppress black root rot of tobacco in a gnotobiotic system.

Zinc improves biocontrol of fusarium crown and root rot of tomato by Pseudomonas fluorescens and represses the production of pathogen metabolites inhibitory to bacterial antibiotic biosynthesis

ABSTRACT Crown and root rot of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici is an increasing problem in Europe, Israel, Japan, and North America. The biocontrol agent Pseudomonas fluorescens strain CHA0 provides only moderate control of this disease. A one-time amendment of zinc EDTA at 33 mug of Zn(2+)/ml to hydroponic nutrient solution in soilless rockwool culture did not reduce disease when used alone, but did reduce disease by 25% in the presence of CHA0. In in vitro studies with the pathogen, zinc at concentrations as low as 10 mug/ml abolished production of the phytotoxin fusaric acid, a Fusarium pathogenicity factor, and increased production of microconidia over 100-fold, but reduced total biomass. Copper EDTA at 33 mug of Cu(2+)/ml had a similar effect as zinc on the pathogen in vitro; it reduced disease when used alone, and increased the biocontrol activity of CHA0 in soilless culture. Ammonium-molybdate neither improved the biocontrol activity of CHA0 nor affected production of fusaric acid or microconidia. Strain CHA0 did not degrade fusaric acid. Fusaric acid at concentrations as low as 0.12 mug/ml repressed production by CHA0 of the antibiotic 2,4-diacetylphloroglucinol, a key factor in the biocontrol activity of this strain. Production of pyoluteorin by CHA0 was also reduced, but production of hydrogen cyanide and protease was not affected, suggesting that fusaric acid affects biosynthesis at a regulatory level downstream of gacA and apdA genes. Fusaric acid did not affect the recovery of preformed antibiotics nor did it affect bacterial growth even at concentrations as high as 200 mug/ml. When microbial meta-bolite production was measured in the rockwool bioassay, zinc amendments reduced fusaric acid production and enhanced 2,4-diacetylphloro-glucinol production. We suggest that zinc, which did not alleviate the repression of antibiotic biosynthesis by fusaric acid, improved biocontrol activity by reducing fusaric acid production by the pathogen, which resulted in increased antibiotic production by the biocontrol agent. This demonstrates that pathogens can have a direct negative impact on the mechanism(s) of biocontrol agents.

Degradation of pathogen quorum-sensing molecules by soil bacteria: a preventive and curative biological control mechanism.

Abstract The plasmid pME6863, carrying the aiiA gene from the soil bacterium Bacillus sp. A24 that encodes a lactonase enzyme able to degrade N-acyl-homoserine lactones (AHLs), was introduced into the rhizosphere isolate Pseudomonas fluorescens P3. This strain is not an effective biological control agent against plant pathogens. The transformant P. fluorescens P3/pME6863 acquired the ability to degrade AHLs. In planta, P. fluorescens P3/pME6863 significantly reduced potato soft rot caused by Erwinia carotovora and crown gall of tomato caused by Agrobacterium tumefaciens to a similar level as Bacillus sp. A24. Little or no disease reduction was observed for the wild-type strain P3 carrying the vector plasmid without aiiA. Suppression of potato soft rot was observed even when the AHL-degrading P. fluorescens P3/pME6863 was applied to tubers 2 days after the pathogen, indicating that biocontrol was not only preventive but also curative. When antagonists were applied individually with the bacterial plant pathogens, biocontrol activity of the AHL degraders was greater than that observed with several Pseudomonas 2,4-diacetylphloroglucinol-producing strains and with Pseudomonas chlororaphis PCL1391, which relies on production of phenazine antibiotic for disease suppression. Phenazine production by this well characterized biological control strain P. chlororaphis PCL1391 is regulated by AHL-mediated quorum sensing. When P. chlororaphis PCL1391 was co-inoculated with P. fluorescens P3/pME6863 in a strain mixture, the AHL degrader interfered with the normally excellent ability of the antibiotic producer to suppress tomato vascular wilt caused by Fusarium oxysporum f. sp. lycopersici. Our results demonstrate AHL degradation as a novel biocontrol mechanism, but also demonstrate the potential for non-target interactions that can interfere with the biocontrol efficacy of other strains.

Nonpathogenic Fusarium oxysporum strain Fo47 induces resistance to Fusarium wilt in tomato.

Nonpathogenic Fusarium oxysporum strain Fo47 controls the incidence of Fusarium wilt. Four bioassays in which a strain of the pathogen F. oxysporum f. sp. lycopersici and Fo47 were not in direct contact were developed to evaluate whether Fo47 could induce resistance to Fusarium wilt in tomato plants. Fo47 and the pathogen were separated either physically or in time. Bio-assays were carried out under hydroponic conditions (two bioassays), in potting mix, or in autoclaved soil. Strain Fo47 protected tomato against Fusarium wilt in all four bioassays. Inoculation with Fo47 increased chitinase, β-1,3-glucanase, and β-1,4-glucosidase activity in plants, confirming the ability of Fo47 to induce resistance in tomato. This report is the first to demonstrate that a nonpathogenic strain of F. oxysporum can induce resistance to Fusarium wilt in tomato plants. This result has important practical implications for biocontrol of tomato diseases under commercial conditions.

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

ABSTRACT The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA+) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA−) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.

Biocontrol of soil-borne fungal plant diseases by 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads with different restriction profiles of amplified 16S rDNA

Fluorescent pseudomonads producing the antimicrobial compound 2,4-diacetylphloroglucinol (Phl) are being studied extensively for use as biocontrol agents of soil-borne fungal diseases. Some of them can produce pyoluteorin (Plt) in addition to Phl, whereas others synthesise only Phl. Here, a collection of seven Phl+ Plt- pseudomonads, seven Phl+ Plt+ pseudomonads and seven Phl- biocontrol pseudomonads were compared for protection of plant roots against fungal pathogens. The seven Phl+ Plt+ pseudomonads were identical by restriction analysis of amplified spacer ribosomal DNA (spacer ARDRA), whereas the Phl+ Plt- pseudomonads and especially the Phl- biocontrol pseudomonads were quite diverse by spacer ARDRA. Collectively, the Phl+ Plt- pseudomonads proved superior to the Phl+ Plt+ pseudomonads and the Phl- biocontrol pseudomonads for protection of tomato against Fusarium crown and root rot (in rockwool microcosms) or cucumber against Pythium damping-off (in non-sterile soil microcosms). There was no correlation between protection in vivo and inhibition of the corresponding fungal pathogen on plates. However, there was a significant correlation between the amount of Phl produced on plates and protection of tomato against Fusarium crown and root rot, but not with protection of cucumber against Pythium damping-off. Interestingly, the minority of strains unable to produce HCN, an extracellular protease, or both, were among those unable to protect plants in both pathosystems. A seedling assay was developed to compare pseudomonads for suppression of Fusarium crown and root rot in vitro, and a significant correlation was found between disease severity in vitro and in vivo. Overall, results suggest that promising biocontrol pseudomonads may be identified based on the ability to produce Phl and/or specific ARDRA-based fingerprints.

Phylogeny of HCN synthase-encoding hcnBC genes in biocontrol fluorescent pseudomonads and its relationship with host plant species and HCN synthesis ability.

Hydrogen cyanide (HCN) is a broad-spectrum antimicrobial compound involved in biological control of root diseases by many plant-associated fluorescent pseudomonads. The HCN synthase is encoded by three biosynthetic genes (hcnA, hcnB, and hcnC), but little is known about the diversity of these genes in fluorescent Pseudomonas spp. and in other bacteria. Here, the partial hcnBC sequence was determined for a worldwide collection of biocontrol fluorescent Pseudomonas spp. Phylogenies based on hcnBC and deduced protein sequences revealed four main bacterial groups, but topological incongruences were found between hcnBC and rrs-based phylogenies, suggesting past lateral transfer of hcnBC among saprophytic root-colonizing pseudomonads. Three of the four groups included isolates from different countries and host plants. Yet, these groups corresponded to distinct, ecologically-adapted populations of HCN-producing biocontrol fluorescent pseudomonads, as indicated by high hcnBC distinctness ratio values and the differences in production levels of HCN in vitro found between groups. This is in accordance with previous results on catabolic properties and biocontrol abilities of these strains. HCN synthase gene diversity may thus reflect the adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations were found between HCN production in vitro and plant protection in the cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici pathosystems.