Regulation of the adhesion ofPseudomonas fluorescens cells to glass by extracellular volatile compounds

The effect of the gaseous metabolites of onePseudomonas fluorescens culture on the attachment of cells of anotherP. fluorescens culture to glass was studied. Gaseous metabolites increased the number of unattached cells by 10–30% and the mean residence time of cells attached to glass by 100%. These effects were presumably due to the yet unidentified compound, which we called volatile antiadhesin. This compound could be adsorbed by activated charcoal and HAYESEP-Q adsorbent.     

Role of distant cellular communications in regulation of Pseudomonas fluorescens adhesion

The effects of long-range interactions (LRI) and culture air on the adhesion of Pseudomonas fluorescens cells were studied. One P. fluorescens culture was found to diminish the adhesion of cells of another, glass-screened, P. fluorescens culture by 30%. This effect was interpreted to be due to penetrating LRI. Under the combined action of LRI and culture air (the latter alone reduced cell adhesion by only several percent), the amount of unattached cells increased 2- to 30-fold (on the average, by a factor of nine). Such a great reduction of cell adhesion indicated the synergistic action of LRI and culture air.     

Saturated C21-C33 hydrocarbons are involved in the self-regulation of Pseudomonas fluorescens adhesion to a glass surface

One of the two putative groups of antiadhesions was identified in Pseudomonas fluorescens by the method of gas chromatography-mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10-15 micrograms/l; the concentrations of individual HC ranged from 0.1 to 3.0 micrograms/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.     

荧光假单胞菌抗生性代谢产物合成相关基因的研究现状

荧光假单胞菌(Pseudomonas fluorescens)是一种重要的植物根际促生菌,它能够产生藤黄绿脓菌素、2,4-二乙酰基藤黄酚、硝吡咯菌素、吩嗪-1-羧酸等抗生性次级代谢产物,可抑制多种病原物,在农作物土传病害的生物防治研究中具有重要意义.总结了荧光假单胞菌中已确立的抗生性次级代谢产物的合成机制,重点阐述了相关基因的结构、功能,以及利用生物工程技术对荧光假单胞菌进行遗传操作的最新进展,同时对荧光假单胞菌在生物防治中的应用和其作为生防菌剂的前景进行了展望.     

Properties of adhesin and antiadhesin from Pseudomonas fluorescence

Some properties of the adhesion-modifying factors of Pseudomonas fluorescens are described. Adhesin, which promotes the adhesion of P. fluorescens cells, is a hydrophobic compound of a protein nature with a molecular mass of more than 10 kDa located either at the cell surface or in the medium. Anti-adhesion, which suppresses the adhesion of P. fluorescens cells, is a thermolabile hydrophobic compound of a nonprotein nature with a molecular mass of less than 3 kDa. Heating makes anti-adhesin hydrophilic. The role of adhesion and anti-adhesion in the adhesion and adaptation of P. fluorescens cells is discussed.     

Biological control of Tiarosporella phaseolina the causal agent of charcoal rot of soybean

Charcoal rot caused by Tiarosporella phaseolina (Tassi) Van der Aa is an important disease of soybean in Gorgan province of Iran. Experiments were carried out with 95 bactenal isolates that were collected from the rhizosphere of soybean plant. Among these bacteria only 50 isolates showed antagonistic effect on Tiarosporella phaseolina using dual culture test. Six highly effective bacteria were selected for subsequent studies. Based on biochemical physiological and morphological tests, isolates Pf-12 and Pf-63 were identified as Pseudomonas fluorescens, isolates B-13, B-42,B-126 and B-84 as Bacillus subtilis. The isolates of P. fluorescens produced antibiotics as well as volatile metabolites that inhibited mycelial growth of fungus. Bacillus subtilis isolates inhibited the fungal growth through volatile and non-volatile metabolites production. Only P. fluorescens isolates produced hydrogen cyanide. In greenhouse studies, the isolates B-13 and B-126 reduced 59% and 66% the intensity of charcoal rot of soybean respectively. The combinations of isolates B-13 and B-126 were also effective on reducing the intensity of disease.     

Extracellular factors affecting the adhesion of Pseudomonas fluorescens cells to glass surface

Two factors affecting the adhesion of Pseudomonas fluorescens to glass surfaces were revealed in the culture liquid (CL) of this bacterium. One of these factors, adhesin, which is responsible for cell adhesion, was found to be a protein substance located both at the cell surface and in the CL. Bacterial cells grown in rich LB medium were less adhesive than cells grown in minimal M9 medium. The adhesive capacity of cells was independent of the growth phase. The other factor, anti-adhesion (AA), which reduces cell adhesion, was found only in the CL. AA concentration in the CL increased with the culture age.     

Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.     

Isolation and characterization of a stilbene-degrading strain of Pseudomonas fluorescens, and production of antioxidant compounds by stilbene metabolism

In this study, we consider the use of hydrocarbon-degrading bacteria that degrade trans-stilbene as a novel approach for synthesizing potentially bioactive hydroxylated stilbenes. A trans-stilbene-degrading bacterium, MN2, was isolated from activated sludge through enrichment culture, and identified as Pseudomonas fluorescens using conventional techniques. Degradation of trans-stilbene by this strain yielded two metabolites that had significant antioxidant activity.     

Volatiles of Pseudomonas aeruginosa and related species by automated headspace concentration--gas chromatography

The volatile metabolites of three strains of Pseudomonas aeruginosa and one strain each of Pseudomonas cepacia, Pseudomonas maltophilia, Pseudomonas fluorescens, and Pseudomonas putida were analyzed using an automated headspace concentrator incorporating a gas chromatograph. The procedure does not require sample preparation and automates the entire analytical sequence to yield reproducible profiles of volatile constituents. Gas chromatographic profiles of the volatile metabolites of each species were obtained using a 20-min concentration period and two fused silica capillary columns of different polarities. The production of headspace metabolites from trypticase soy broth was studied in relationship to culture incubation time and initial cell concentration. The volatiles identified after 24 h incubation consisted of 1-butanol, isopentanol, toluene, 1-undecene, 2-butanone, 2-heptanone, 2-nonanone, and 2-undecanone. Sufficient amounts of specific metabolites were produced after 5 h incubation to provide information of possible diagnostic value. In particular, all P. aeruginosa strains produced a distinctive series of 1-undecene and methyl ketones after 5 h incubation of media inoculated to provide 2 X 10(6) cells/mL. The results indicate that when growth and analytical conditions are held constant, P. aeruginosa and related species produce characteristic profiles of headspace metabolites. Since conventional bacteriological tests require 24 h or more for the identification of these pseudomonads, automated volatile analysis could provide an alternative means for the rapid detection of these bacteria.     

Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improves biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: To determine the influence of soil-borne fungus Trichoderma harzianum on the biocontrol performance of Pseudomonas fluorescens strain CHA0 and its 2,4-diacetylphloroglucinol (DAPG) overproducing derivative CHA0/pME3424 against Meloidogyne javanica. METHODS AND RESULTS: Amendment of the culture filtrate (CF) or methanol extract of the CF of a T. harzianum strain Th6 to P. fluorescens growth medium enhanced the production of nematicidal compound(s) by bacterial inoculants in vitro. In addition, bacteria overwhelmingly expressed phl'-'lacZ reporter gene when the medium was amended with CF of T. harzianum. Pseudomonas fluorescens and T. harzianum applied together in unsterilized sandy loam soil caused greater reduction in nematode population densities in tomato roots. CONCLUSIONS: Trichoderma harzianum improves root-knot nematode biocontrol by the antagonistic rhizobacterium P. fluorescens both in vitro and under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic effect of T. harzianum on the production of nematicidal compound(s) critical in biocontrol may improve the efficacy of biocontrol bacteria against plant-parasitic nematodes. Considering the inconsistent performance of the biocontrol agents under field conditions, application of a mixture of compatible T. harzianum and P. fluorescens would more closely mimic the natural situation and might broaden the spectrum of biocontrol activity with enhanced efficacy and reliability of control.     

Enhancement of plant stem growth by flocculation of the antibiotic-producing bacterium, Pseudomonas fluorescens S272, on the roots

The antibiotic-producing bacterium, Pseudomonas fluorescens, is assumed to be important in protecting plants from soilborne diseases. S. fluorescens S272, a hyper-producing strain of pyoluteorin (PT) and 2,4-diacetylphloroglucinol (DG), had previously been isolated from soil. The present paper reported that the growth of water-cultivated Kaiware radish was promoted to 120-140% of its normal level by the coaddition of an S272 culture broth (0.01-1% v/v) and a polysaccharide flocculant (1-100 ppm) from Klebsiella pneumoniae H12. Tight adhesion of S272 cells to the root tissue was microscopically observed. The growth promotion is assumed to have been caused by antibiotic effects for the following two reasons: 1) PT (4 mg/l) and DG (24 mg/l) addition to a radish culture enhanced stem growth to 130% of the normal level; 2) a culture solution containing the S272 culture broth (0.01-1% v/v) markedly inhibited the decomposition of hypersensitive chrysanthemum leaves. A soil-cultivation experiment with Gomphrena globosa under natural conditions also exhibited enhanced stem length (160%) by coaddition of the S272 culture broth and H12 polysaccharide. These results suggest that polysaccharide-enhanced adhesion of P. fluorescens S272 cells might be useful for promoting plant growth through the increased antibiotic effect.     

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.     

Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens.

Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry. The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies. Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere.     

Siderophore production by using free and immobilized cells of two pseudomonads cultivated in a medium enriched with Fe and/or toxic metals (Cr, Hg, Pb)

Pseudomonads are serious candidates for siderophore production applied to toxic metal (TM) solubilization. The bioaugmentation of contaminated soils by these TM-solubilizing bacteria combined with phytoextraction is an emerging clean-up technology. Unfortunately, siderophore synthesis may be drastically reduced by soluble iron in soils and bacteria can suffer from TM toxicity. In this study, we compared siderophore production by Pseudomonas aeruginosa and Pseudomonas fluorescens by using free and immobilized cells in Ca-alginate beads incubated in a medium containing Fe and/or TM (mixture of Cr, Hg, and Pb in concentrations which represented the soluble fraction of a contaminated agricultural soil). Free cell growth was stimulated by Fe, whatever the microorganism, the inoculum size and the presence or not of TM might have been. P. aeruginosa was less sensitive to TM than P. fluorescens. By comparison with free cells, immobilization with the high inoculum size showed less sensitivity to TM most probably because of lower metal diffusion in beads. Indeed, a maximum of 99.1% of Cr, 57.4% of Hg, and 99.6% of Pb were adsorbed onto beads. The addition of iron in the culture medium reduced significantly siderophore production of free cells while it led only to a low decrease with their immobilized counterparts, in particular with P. aeruginosa. In culture medium enriched with Fe and/or TM, siderophore-specific production of immobilized cells was higher than for free cells.     

Protozoan growth rates on secondary-metabolite-producing Pseudomonas spp. correlate with high-level protozoan taxonomy

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090(T) and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.     

Transposon Tn5 mutagenesis of Pseudomonas fluorescens to isolate mutants deficient in antibacterial activity

Abstract Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn 5 -GM into the chromosome of P. fluorescens was achieved by heat ttreatment of the transformed cells at 42°C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens . One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis . These results introduce Tn 5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens .     

Biological control of apple blue mold with Pseudomonas fluorescens

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.