Isolation of an n-alkylated benzylamine derivative from Pseudomonas putida BTP1 as elicitor of induced systemic resistance in bean

in Molecular Plant-Microbe Interactions (2005), 18(6), 562-569

Root treatment of Phaseolus vulgaris with the nonpathogenic Pseudomonas putida BTP1 led to significant reduction of the disease caused by the pathogen Botrytis cinerea on leaves. The molecular determinant ... [more ▼]

Root treatment of Phaseolus vulgaris with the nonpathogenic Pseudomonas putida BTP1 led to significant reduction of the disease caused by the pathogen Botrytis cinerea on leaves. The molecular determinant of P putida BTP1 mainly responsible for the induced systemic resistance (ISR) was isolated from cell-free culture fluid after growth of the strain in the iron-poor casamino acid medium. Mass spectrometry analyses performed on both the bacterial product and synthetic analogues revealed a polyalkylated benzylamine structure, with the quaternary ammonium substituted by methyl, ethyl, and C-13 aliphatic groups responsible for the relative hydrophobicity of the molecule. The specific involvement of the N-alkylated benzylamine derivative (NABD) in ISR elicitation was first evidenced by testing the purified compound that mimicked the protective effect afforded by crude supernatant samples. The evidence was supported by the loss of elicitor activity of mutants impaired in NABD biosynthesis. Our experiments also showed that other iron-regulated metabolites secreted by the strain are not involved in ISR stimulation. Thus, these results indicate a wider variety of Pseudomonas determinants for ISR than reported to date. [less ▲]

Stimulation of the lipoxygenase pathway is associated with systemic resistance induced in bean by a nonpathogenic Pseudomonas strain

in Molecular Plant-Microbe Interactions (2004), 17(9), 1009-1018

Systemic defense reactions induced in bean by the nonpathogenic Pseudomonas putida BTP1 strain reduced disease caused by Botrytis cinerea. Phenylalanine ammonialyase activity and the level of endogenous ... [more ▼]

Systemic defense reactions induced in bean by the nonpathogenic Pseudomonas putida BTP1 strain reduced disease caused by Botrytis cinerea. Phenylalanine ammonialyase activity and the level of endogenous free sallicylic acid were compared in plant growth-promoting rhizobacteria-treated versus control plants, but no significant differences were detected. Furthermore, no enhanced fungitoxicity was detected in methanolic leaf extracts, suggesting that accumulation of bean phytoalexins was not part of the stimulated defense mechanisms. However, BTP1-inoculated plants showed increased levels of both linoleic and linolenic acids. On this basis, we further investigated whether the lipoxygenase pathway, leading to antifungal phytooxylipins, could have been stimulated. Two key enzymatic activities of this metabolic route, namely lipoxygenase and hydroperoxidelyase, were significantly stimulated during the first four days after challenging BTP1-treated plants with the pathogen. This was observed in parallel with a more rapid consumption of the respective substrates of these enzymes, as revealed by measurements of endogenous concentrations of linolenic acid and their hydroperoxide derivatives. Moreover, headspace-gas chromatography analyses showed significantly higher concentrations of the fungitoxic final product Z-3-hexenal in leaves from BTP1-inoculated beans as compared with control plants. Taken together, these results strongly suggest that the oxylipin pathway can be associated with enhanced disease resistance induced in bean plants by nonpathogenic rhizobacteria. [less ▲]

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

in Journal of Applied Microbiology (2004), 96(5), 1151-1160

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in ... [more ▼]

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits. [less ▲]

The lipoxygenase pathway as defence mechanism induced in bean by a non-pathogenic fluorescent Pseudomonas strain.

Conference (2003)

Fluorescent Pseudomonas mainly produce the dihydro form of pyoverdine at low specific growth rate.

in Letters in Applied Microbiology (2003), 36(5), 259-62

AIMS: To analyse the influence of cell growth rate and iron concentration on the production of pyoverdines (PVDs) and of their reduced dihydro forms by three fluorescent Pseudomonas strains (P. putida ... [more ▼]

AIMS: To analyse the influence of cell growth rate and iron concentration on the production of pyoverdines (PVDs) and of their reduced dihydro forms by three fluorescent Pseudomonas strains (P. putida BTP16, P. fluorescens BTP7 and P. aeruginosa 7NSK2). METHODS: PVD and dihydropyoverdine (DHPVD) productions were determined by LC ESI-MS and spectrophotometry during batch and chemostat culture at different dilution rates. SIGNIFICANCE: The relatively high PVD-to-DHPVD ratio (0.57) observed in pH-controlled batch cultures suggested that a base-catalysed chemical oxidation of the dihydroform is not the prime mechanism involved in generating PVDs. Interestingly, in chemostat cultures the PVD-to-DHPVD ratio was significantly reduced at low specific growth rate. Our results suggest that the oxidation of DHPVD to PVD is catalysed by an iron-dependent enzymatic reaction rather than a chemical oxidation. [less ▲]

Unusual traits of the pyoverdin-mediated iron acquisition system in Pseudomonas putida strain BTP1.

in Biometals (2002), 15(1), 1-13

Fluorescent Pseudomonas species are characterized by the production of pyoverdin-type siderophores for Fe3+ acquisition in iron-limited environments. Since it produces a structurally specific pyoverdin ... [more ▼]

Fluorescent Pseudomonas species are characterized by the production of pyoverdin-type siderophores for Fe3+ acquisition in iron-limited environments. Since it produces a structurally specific pyoverdin, Pseudomonas putida strain BTP1 could represent a valuable tool in an attempt to correlate the structural features of these compounds with some specificity in their two main properties i.e. affinity for iron and recognition rate by other Pseudomonas strains. An uncommonly high affinity for iron of the pyoverdin synthetized by P. putida BTP1 was observed by comparing both the apparent stability constant and the decomplexation kinetic of its ferric complex with those of ferripyoverdins from other strains. On another hand, results from growth stimulation experiments and labeled ferripyoverdin uptake assays highlighted the very low recognition rate of BTP1 isopyoverdins by membrane receptors of foreign strains. By contrast, P. putida BTP1 was able to utilize a broad spectrum of structurally unrelated exogenous pyoverdins by means of multiple receptors that are likely constitutively expressed in its outer membrane. The unusual traits of its pyoverdin-mediated iron acquisition system should contribute to enlarge the ecological competence of Pseudomonas putida BTP1 in terms of colonization and persistence in the rhizosphere. [less ▲]

Dihydropyoverdine production by Pseudomonas putida in transient state and steady-state cultivations.

Poster (2001, September)

Specificity of the pyoverdine-mediated iron transport in Pseudomonas putida BTP1

Poster (2001, September)

Influence of moderate temperatures on myristoyl-CoA metabolism and acyl-CoA thioesterase activity in the psychrophilic antarctic yeast Rhodotorula aurantiaca.

in Journal of Biological Chemistry (2001), 276(16), 12691-6

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast ... [more ▼]

The inability of psychrophilic microorganisms to grow at moderate temperatures (>20 degrees C) presently represents an unresolved thermodynamic paradox. Here we report for the psychrophilic yeast Rhodotorula aurantiaca A19, isolated from Antarctic ice, that the inability to grow at temperatures close to 20 degrees C is associated with profound alterations in cell morphology and integrity. High performance liquid chromatography analysis of the intracellular acyl-CoA esters revealed an abnormal accumulation of myristoyl-CoA (C14-CoA) in cells cultivated close to the nonpermissive temperature. Its concentration (500 microm) was found to be 28-fold higher than in cells cultivated at 0 degrees C. If one considers its ability to disrupt membrane bilayers and to inhibit many cellular enzymes and functions, intracellular myristoyl-CoA accumulation in the psychrophile R. aurantiaca represents one of the principal causes of growth arrest at moderate temperatures. Intracellular acyl-CoA concentrations are believed to be regulated by thioesterase activity. Thus in an attempt to explore the mechanism by which temperature disrupts myristoyl-CoA metabolism, we isolated and characterized a long chain acyl-CoA thioesterase. The monomeric 80-kDa thioesterase from the psychrophilic yeast shows a very strong specificity for myristoyl-CoA. The affinity for substrate and the catalytic efficiency of the thioesterase are optimal below 5 degrees C (temperatures habitually experienced by the strain) and dramatically decrease with increasing temperature. The loss of affinity for substrate is related to the intracellular increase of myristoyl-CoA concentration. Our observations reveal one of the probable mechanisms by which temperature fixes the limit of growth for this psychrophilic yeast. [less ▲]

Induction of phenolic compounds in two cultivars of cucumber by treatment of healthy and powdery mildew-infected plants with extracts of Reynoutria sachalinensis

in Journal of Chemical Ecology (2000), 26

Accumulation of phenolic compounds (p-coumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester) was followed in susceptible (Mustang) and tolerant (Flamingo) cucumber (Cucumis sativus ... [more ▼]

Accumulation of phenolic compounds (p-coumaric, caffeic, and ferulic acids and p-coumaric acid methyl ester) was followed in susceptible (Mustang) and tolerant (Flamingo) cucumber (Cucumis sativus) cultivars. The objective was to determine whether these compounds played a role in resistance against powdery mildew following a prophylactic treatment with Milsana (leaf extracts from the giant knot weed Reynoutria sachalinensis, polygonaceae). This treatment significantly reduced the incidence of powdery mildew in both cultivars. Phenolic compounds were extracted from leaves. In the hydrolyzed fraction containing phenolic aglycones, levels of p-coumaric, caffeic, and ferulic acids and of p-coumaric acid methyl ester increased in all treatments (with leaf extracts of R. Sachalinensis, powdery mildew, or both) except the control, one or two days after treatment. In the fraction containing free phenolics, from the tested compounds, only ferulic acid showed an increase in cv. Flamingo (tolerant), and was particularly evident following treatments. On the other hand, the amounts of hydroxycinnamic acids increased rapidly in the two cultivars following Milsana treatment, suggesting their role in disease reduction. All compounds showed antifungal activity when tetsed against common pathogens of cucumber (Botrytis cinerea, Pythium ultimum, and P. aphanidermatum), but in general methyl esters were more fungitoxic than their corresponding free acids. This study suggests that cucumber is able to release antifungal compounds that are instrumental in repressing powdery mildew infection. This response is seemingly independent from the level of genetic resistance associated with each cultivar. [less ▲]