Christoph Keel

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.

Suppression of Root Diseases byPseudomonas fluorescensCHA0: Importance of the Bacterial Secondary Metabolite 2,4-Diacetylphloroglucinol

Pseudomonas fluorescens strain CHA0 suppresses Thielaviopsis basicola-induced black root rot of tobacco and Gaeumannomyces graminis var. tritici-induced take-all of wheat. Strain CHA0 produces 2,4-diacetylphloroglucinol, a metabolite with antifungal, antibacterial, and phytotoxic activity. The role of this compound in disease suppression was tested under gnotobiotic conditions. A P. fluorescens mutant, obtained by Tn5 insertion, did not produce 2,4-diacetylphloroglucinol, showed diminished inhibition of T. basicola and of G.g. var. tritici in vitro, and had a reduced suppressive effect on tobacco black root rot and on take-all of wheat, compared with wild-type CHA0. Complementation of the mutant with an 11-kb DNA fragment from a genomic library of wild-type CHA0 largely restored production of the metabolite, inhibition of the fungal pathogens in vitro and disease suppression. The Tn5 insertion was physically mapped using a 5.8-kb complementing fragment as a probe. 2,4-Diacetylphloroglucinol was shown to be produced in the rhizosphere of wheat by strain CHA0 and by the complemented mutant, but not by the mutant defective in 2,4-diacetylphloroglucinol synthesis. These results support the importance of 2,4-diacetylphloroglucinol production by strain CHA0 in the suppression of soilborne plant pathogens in the rhizosphere.

Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria

The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb). Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P. fluorescens strains incubated under different environmental conditions for more than 1 month.

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0

In the plant‐beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two‐component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non‐exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA‐binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA‐regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strains own supernatant extract containing a quorum‐sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA‐independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4‐diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber‐Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA‐mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0.

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strains response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.

Pyoluteorin production byPseudomonas fluorescens strain CHA0 is involved in the suppression ofPythium damping-off of cress but not of cucumber

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent of various soilborne pathogens. It controls damping-off or root rot caused byPythium ultimum on cucumber, wheat and cress. Strain CHA0 synthesizes several antibiotic metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and pyoluteorin. The role of pyoluteorin in the suppression of damping-off was investigated. Two Tn5 mutants (CHA660 and CHA661) of strain CHA0 were isolated which had lost the capacity to produce pyoluteorin but still produced 2,4-diacteylphloroglucinol and HCN. These mutants still inhibitedP. ultimum on malt agar (which favours the production of 2,4-diacetylphloroglucinol) but had partially lost the ability to inhibit this pathogen on Kings B agar (which favours the production of pyoluteorin). The two pyoluteorin-negative mutants showed a reduced capacity to suppress damping-off of cress caused byP. ultimum but were as effective in the protection of cucumber against this pathogen as the wild-type strain.These results indicate that, depending on the plant, pyoluteorin production plays a role in the suppression of damping-off by strain CHA0 without being a major mechanism in disease suppression. We suggest that the contribution of pyoluteorin to the biocontrol activity of strain CHA0 is determined by the quantity of this antibiotic produced in the rhizosphere, which might depend on the root exudates of the host plant.

Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation.

Root diseases caused by fungal pathogens can be suppressed by certain rhizobacteria that effectively colonize the roots and produce extracellular antifungal compounds. To be effective, biocontrol bacteria need to be present at sufficiently high cell densities. These conditions favor the operation of positive feedback mechanisms that control the production of antifungal compounds in biocontrol strains of fluorescent pseudomonads, via both transcriptional and post-transcriptional mechanisms.

Characterisation of microbial communities colonising the hyphal surfaces of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil fungi that are intimately associated with the roots of the majority of land plants. They colonise the interior of the roots and the hyphae extend into the soil. It is well known that bacterial colonisation of the rhizosphere can be crucial for many pathogenic as well as symbiotic plant–microbe interactions. However, although bacteria colonising the extraradical AMF hyphae (the hyphosphere) might be equally important for AMF symbiosis, little is known regarding which bacterial species would colonise AMF hyphae. In this study, we investigated which bacterial communities might be associated with AMF hyphae. As bacterial-hyphal attachment is extremely difficult to study in situ, we designed a system to grow AMF hyphae of Glomus intraradices and Glomus proliferum and studied which bacteria separated from an agricultural soil specifically attach to the hyphae. Characterisation of attached and non-attached bacterial communities was performed using terminal restriction fragment length polymorphism and clone library sequencing of 16S ribosomal RNA (rRNA) gene fragments. For all experiments, the composition of hyphal attached bacterial communities was different from the non-attached communities, and was also different from bacterial communities that had attached to glass wool (a non-living substratum). Analysis of amplified 16S rRNA genes indicated that in particular bacteria from the family of Oxalobacteraceae were highly abundant on AMF hyphae, suggesting that they may have developed specific interactions with the fungi.

The sigma factor AlgU (AlgT) controls exopolysaccharide production and tolerance towards desiccation and osmotic stress in the biocontrol agent Pseudomonas fluorescens CHA0.

ABSTRACT A variety of stress situations may affect the activity and survival of plant-beneficial pseudomonads added to soil to control root diseases. This study focused on the roles of the sigma factor AlgU (synonyms, AlgT, RpoE, and ς22) and the anti-sigma factor MucA in stress adaptation of the biocontrol agent Pseudomonas fluorescens CHA0. ThealgU-mucA-mucB gene cluster of strain CHA0 was similar to that of the pathogensPseudomonas aeruginosa and Pseudomonas syringae. Strain CHA0 is naturally nonmucoid, whereas amucA deletion mutant oralgU-overexpressing strains were highly mucoid due to exopolysaccharide overproduction. Mucoidy strictly depended on the global regulator GacA. An algU deletion mutant was significantly more sensitive to osmotic stress than the wild-type CHA0 strain and the mucA mutant were. Expression of analgU′-′lacZ reporter fusion was induced severalfold in the wild type and in themucA mutant upon exposure to osmotic stress, whereas a lower, noninducible level of expression was observed in thealgU mutant. Overexpression of algU did not enhance tolerance towards osmotic stress. AlgU was found to be essential for tolerance of P. fluorescens towards desiccation stress in a sterile vermiculite-sand mixture and in a natural sandy loam soil. The size of the population of thealgU mutant declined much more rapidly than the size of the wild-type population at soil water contents below 5%. In contrast to its role in pathogenic pseudomonads, AlgU did not contribute to tolerance of P. fluorescens towards oxidative and heat stress. In conclusion, AlgU is a crucial determinant in the adaptation of P. fluorescens to dry conditions and hyperosmolarity, two major stress factors that limit bacterial survival in the environment.

Role of Gluconic Acid Production in the Regulation of Biocontrol Traits of Pseudomonas fluorescens CHA0

ABSTRACT The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Δgcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.