Pseudomonas Fluorescens

Pseudomonads are able to form a variety of biofilms that colonize the air-liquid (A-L) interface of static liquid microcosms, and differ in matrix composition, strength, resilience and degrees of attachment to the microcosm walls. From Pseudomonas fluorescens SBW25, mutants have evolved during prolonged adaptation-evolution experiments which produce robust biofilms of the physically cohesive class at the A-L interface, and which have been well characterized. In this study we describe a novel A-L interface biofilm produced by SBW25 that is categorized as a viscous mass (VM)-class biofilm. Several metals were found to induce this biofilm in static King's B microcosms, including copper, iron, lead and manganese, and we have used iron to allow further examination of this structure. Iron was demonstrated to induce SBW25 to express cellulose, which provided the matrix of the biofilm, a weak structure that was readily destroyed by physical disturbance. This was confirmed in situ by a low (0.023-0.047 g) maximum deformation mass and relatively poor attachment as measured by crystal violet staining. Biofilm strength increased with increasing iron concentration, in contrast to attachment levels, which decreased with increasing iron. Furthermore, iron added to mature biofilms significantly increased strength, suggesting that iron also promotes interactions between cellulose fibres that increase matrix interconnectivity. Whilst weak attachment is important in maintaining the biofilm at the A-L interface, surface-interaction effects involving cellulose, which reduced surface tension bỹ 3.8 mN m "1 , may also contribute towards this localization. The fragility and viscoelastic nature of the biofilm were confirmed by controlled-stress amplitude sweep tests to characterize critical rheological parameters, which included a shear modulus of 0.75 Pa, a zero shear viscosity of 0.24 Pa s "1 and a flow point of 0.028 Pa. Growth and morphological data thus far support a non-specific metal-associated physiological, rather than mutational, origin for production of the SBW25 VM biofilm, which is an example of the versatility of bacteria to inhabit optimal niches within their environment.

On-line detection of bacterial adhesion in a shear flow gradient was measured with an engineered bioluminescent bacterium. Parallel plate flow cells showed colonization by a Pseudomonas fluorescens strain containing the Vibriofisheri (lux) bioluminescent operon. When induced by sodium salicylate, bulk phase and sessile cells were assayed quantitatively with an ammeter-photomultiplier-fiber optic system. The detection limit in a shear force gradient was 2x l05 attached celis.cm-2 on glass coupons. Light production was found to correlate with biofilm lipid synthesis on a per unit area basis (IA4C-acetate incorporation). Light correlated directly with sessile bacterial acridine orange direct count; in the range of 10 5-10 7 cells.cm-2, providing on-line detection of both biofilm and bulk phase biom jss and specific metabolic activity when induced.

Background: Pseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors' laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid.

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10 2 cells g (fresh weight) ؊1 and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10 7 to 10 8 cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.

Understanding the interaction mechanisms between plant growth-promoting rhizobacteria (PGPR), leguminous crops, and rhizobia is necessary to effectively use PGPR in increasing the biological nitrogen fixation of legumes. We determined the coinoculation effects of Bradyrhizobium japonicum A1017 and a gusA-marked strain of Pseudomonas fluorescens 2137, P. fluorescens WCS365, Azomonas agilis 125, and Azospirillum lipoferum 137 on soybean [Glycine max (L.) Merr] cv. Enrei grown under axenic conditions. The gusA-marked rhizobacteria effectively colonized the root tips and surfaces near the roots tips with a colonization rate ranging from 7.50 to 8.62 log colony forming units (cfu) gfw -1 . P. fluorescens 2137 had the highest colonization activity on soybean roots whether inoculated alone or coinoculated with B. japonicum A1017. Coinoculation of P. fluorescens 2137 and B. japonicum A1017 increased the colonization of B. japonicum A1017 on soybean roots, nodule number, and acetylene reduction activity (ARA) at 10 and 20 days after inoculation. Moreover, the addition of sterile spent medium of P. fluorescens 2137 increased the growth of B. japonicum A1017 in yeast mannitol broth (YMB), indicating that P. fluorescens 2137 may have released substances that increased the rhizobial population. The results of this study suggest that the enhanced nodulation and ARA of soybean due to the high colonization of P. fluorescens on soybean roots could depend on the production of growth-promoting substances that stimulate the growth of B. japonicum. However, coinoculation with P. fluorescens WCS365 decreased the nodule number and ARA, despite its slight stimulation of the growth of B. japonicum on the roots, indicating that coinoculation effects are strain dependent.

In this work the bio-availability of amino acids for the root-colonizing Pseudomonas fluorescens strain WCS365 in the tomato rhizosphere was studied. The amino acid composition of axenically collected tomato root exudate was determined. The results show that aspartic acid, glutamic acid, isoleucine, leucine, and lysine are the major amino acid components. The concentrations of individual amino acids in the rhizosphere of gnotobiotically grown tomato plants were estimated and considered to be too low to support growth of rhizosphere micro-organisms to numbers usually found in the tomato rhizosphere. To test this experimentally, mutants of P. fluorescens WCS365 auxotrophic for the amino acids leucine, arginine, histidine, isoleucine plus valine, and tryptophan were isolated after mutagenesis with Tn5lacZ. Root tip colonization of these mutants was measured after inoculation of germinated tomato seeds and subsequent growth in a gnotobiotic quartz sand system (M. Simons, A. J. van der Bij, I. Brand, L. A. de Weger, C. A. Wijffelman, and B. J. J. Lugtenberg. 1996. Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol. Plant-Microbe Interact. 9:600-607). In contrast to the wild-type strain, none of the five amino acid auxotrophs tested was able to colonize the tomato root tip, neither alone nor after co-inoculation with the wild-type strain. However, addition of the appropriate amino acid to the system restored colonization by the auxotrophic mutants, usually to wild-type levels. Analysis of the root base showed that cells of auxotrophic mutants were still present there. The results show that, although amino acids are present in root exudate, the bio-availability of the tested amino acids is too low to support root tip colonization by auxotrophic mutants of P. fluorescens strain WCS365. The genes that are required for amino acid synthesis are therefore necessary for root colonization. Moreover, these compounds apparently play no major role as nutrients in the tomato rhizosphere.

Three fungal strains of Aspergillus niger, Trichoderma viride and Penicillium spp., were grown on four alternative plant polysaccharides as Csources, to induce enzymes able to degrade the bacterial biofilm matrix, for industrial cleaning purposes. Gum arabic and pectin were the C-sources providing supernatants with better Pseudomonas fluorescens biofilm removal ability. Comparable efficiencies, however, could be achieved with different enzymes, suggesting attacks to alternative points or structures of the biofilm matrix.

This work describes the fabrication, characterization, and biological evaluation of a thin protein-resistant poly(ethylene glycol) (PEG)-based hydrogel coating for antifouling applications. The coating was fabricated by free-radical polymerization on silanized glass and silicon and on polystyrene-covered silicon and gold. The physicochemical properties of the coating were characterized by infrared spectroscopy, ellipsometry, and contact angle measurements. In particular, the chemical stability of the coating in artificial seawater was evaluated over a six-month period. These measurements indicated that the degradation process was slow under the test conditions chosen, with the coating thickness and composition changing only marginally over the period. The settlement behavior of a broad and diverse group of marine and freshwater fouling organisms was evaluated. The tested organisms were barnacle larvae (Balanus amphitrite), algal zoospores (UlVa linza), diatoms (NaVicula perminuta), and three bacteria species (Cobetia marina, Marinobacter hydrocarbonoclasticus, and Pseudomonas fluorescens).

A total of 59 strains of Pseudomonas, isolated from meat products, were grown in micro-titer plates in a meat medium over a range of pH (5.8-7.0), a (0.97-1.00) and temperature (7-258C). Growths were performed in a meat broth with an w automated turbidimeter (Bioscreen C, Labsystem, France). The growth curves obtained in this study did not have sigmoidal shapes making it impossible to calculate growth parameters using the Gompertz equation. The medium was weakly oxygenated in the micro-titer plates and reached 0%-dissolved oxygen at the beginning of the exponential phase. Strains were separated into two groups: P. fragi and P. fluorescens. Strains of P. fragi had shorter lag times than those of P. fluorescens. The impact of such results is interesting in that these could assist to explain the succession of flora that is observed during the processing of meat: P. fluorescens is the dominant bacteria among Pseudomonas spp. at the beginning of a slaughter line and P. fragi becomes dominant during the chilling process.

Xiphinema index is the most aggressive root parasite affecting vines in Chile, controlled mainly chemically. The aim of this study was to identify rhizobacteria in grapevine roots growing in infested soils and to determine if some of these affect the parasitism caused by the nematode. Rhizobacteria of ungrafted grapevine cultivars from 11 vineyards were isolated using TSBA and identified with FAMEs analysis. The antagonistic effect to X. index of those from soils 1 to 4 was assessed in 500 ml pots treating 2 months old in vitro plants with a bacterial suspension containing 1×10 6 c.f.u./ml and 400 nematodes, determining after 4 months growth nematode populations and root damages. Also culture filtrates were assessed. 400 isolates in 25 genera were obtained from the 11 vineyards. The most frequent species were Pseudomo-nas putida (35.1%), Escherichia coli (7.6%) and Pseudomonas fluorescens (6.1%). Isolates of soils 1 to 4, show that Bacillus megaterium, B. brevis, Pseudomonas corrugata, P. savastanoi, Stenotrophomonas maltophilia and Serratia plymuthica reduced root damage and suppressed populations, meanwhile strains of B. brevis and Comamonas acidovorans increased plant growth but did not control nematodes. It is concluded that these isolates may be useful in biological control programmes in vineyards. Keywords Rhizobacteria. Vitis vinifera. Biological control. Plant-parasitic nematodes. Roots. Xiphinema index Abbreviations GFLV Grape fan leaf virus TSBA Tryptic soy broth agar FAMEs Fatty acids methyl esters MIS Microbial identification system RDA Redundancy analysis ANOVA Analysis of variance SIs Similarity indices PGPR Plant growth promoting rhizobacteria ACC 1-Aminocyclopropane-1-carboxylate

The production of a biosurfactant by Pseudomonas fluorescens Migula 1895-DSMZ was studied. Cultures conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined at constant temperature and pH, with the aim of increasing productivity in the process. Biosurfactant synthesis was followed by measuring surface tension and emulsifying index E24. The best results were obtained when using olive oil and ammonium nitrate as carbon and nitrogen sources respectively with a C:N ratio of 10. The production of biosurfactant was growth associated as indicated by the growth and biosurfactant production kinetics. The surface tension was reduced to below 32 dyne/cm and with emulsification index E24 of 65% in 36 to 48 h. The properties of biosurfactant that was separated by acetone precipitation were investigated. The biosurfactant was a rhmnolipid-type in nature as a positive rhamnose assay was tested positive. It had a good foaming and emulsifying and antimicrobial activities. It showed stability during exposure to high temperatures (up to 120°C for 15 min), high salinity (10% Nacl) and a wide range of pH. Analysis by infrared spectroscopy (FTIR) and HPLC were also performed.

The microbial populations associated with the different sections of broccoli harvested from three locations in Australia were studied during storage at 5, 15 and 208C. Bacterial and yeast populations associated with the outer florets and cut surfaces of the stem were generally 10-fold or more higher than those associated with inner florets or non-cut stems, respectively. The predominating bacterial species varied with the origin of the broccoli. Pseudomonas fluorescens, Ps. 5 7 corrugata and Ps. viridiflava predominated at populations of 10 -10 cfu / g on broccoli harvested from Victoria, Ps. 3 6 fluorescens, Ps. mendocina and Ps. fragii and Arthrobacter spp. (10 -10 cfu / g) were prevalent on broccoli harvested from Queensland. Broccoli harvested from New South Wales exhibited a predominance of Ps. fluorescens, Arthrobacter spp. and 3 5

Pseudomonas fluorescens strain Pf1, inhibitory to the growth of the rice blast pathogen Pyricularia oryzae in vitro, was developed as a talc-based powder formulation. When rice seeds were treated with this formulation, the bacteria spread to roots, stems and leaves of the plants and protected against leaf infection by P. oryzae. When applied as a foliar spray, the bacteria survived on the leaves. The powder formulation controlled leaf blast under greenhouse conditions. In tests as a seed treatment and foliar spray in four field trials it effectively controlled the disease and increased grain yield.

The antimicrobial activity of Eucalyptus globulus essential oil was evaluated against 14 food spoilage microorganisms in liquid and vapour phase using agar dilution/well diffusion method and disc volatilisation method, respectively. The minimum inhibitory concentration (MIC) varied from 2.25 to 9 mg/ml for bacterial and fungal strains, and from 1.13 to 2.25 mg/ml for yeast strains. Significantly higher antimicrobial activity was observed in the vapour phase. The chemical composition of E. globulus in liquid as well as in the vapour phase was determined by gas chromatography (GC), gas chromatography/mass spectrometry (GC-MS) and solid phase micro extraction-gas chromatography mass spectrometry (SPME GC-MS), respectively. The dominant compounds in E. globulus oil were 1,8-cineole (45.4%), limonene (17.8%), p-cymene (9.5%), c-terpinene (8.8%), a-pinene (4.2%) and a-terpineol (3.4%), while in the vapour, 1,8-cineole (34.6%), limonene (29.9%), p-cymene (10.5%), c-terpinene (7.4%), a-pinene (4.0%) and a-phellandrene (2.4%) were identified.

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas £uorescens ATCC 33231 to characterize quantitatively the e¡ects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain±heart infusion broth (BHI), using a 3636362 complete factorial design to assess the impact of temperature (4,12,198C), initial pH (5Á0, 6Á0, 7Á0), and sodium chloride content (5, 25, 45 g l 71) on the interaction between the two microorganisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by ¢tting the data to the Gompertz equation, and the derived growth kinetics were compared. When P. £uorescens did in£uence the growth of L. monocytogenes, the primary e¡ect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (48C) and sodium chloride levels (5 and 25 g l 71). Slight increases (51Á0 log cfu ml 71) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. £uorescens at higher temperatures (12 and 198C) and sodium chloride levels (25 and 45 g l 71) when the pH was 5Á0.The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the micro£ora is a¡ected by environmental and food characteristics such as storage temperature, pH, and water activity.

Natural antimicrobial compounds perform their action mainly against cell membranes. The aim of this work was to evaluate the interaction, meant as a mechanism of action, of essential oil antimicrobial compounds with the microbial cell envelope. The lipid profiles of Escherichia coli O157:H7, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas fluorescens, and Brochothrix thermosphacta cells treated with thymol, carvacrol, limonene, eugenol, and cinnamaldehyde have been analyzed by gas chromatography. In line with the fatty acids analysis, the treated cells were also observed by scanning electron microscopy (SEM) to evaluate structural alterations. The overall results showed a strong decrease of the unsaturated fatty acids (UFAs) for the treated cells; in particular, the C18:2trans and C18:3cis underwent a notable reduction contributing to the total UFA decreases, while the saturated fatty acid C17:0 raised the highest concentration in cinnamaldehyde-treated cells. SEM images showed that the used antimicrobial compounds quickly exerted their antimicrobial activities, determining structural alterations of the cell envelope.

Background: Antibodies directed against Saccharomyces cerevisiae (ASCA), perinuclear components of neutrophils (pANCA), and porin protein C of Escherichia coli (anti-OmpC) are reported to be associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Since limited data are available from Eastern Europe, we assessed the above antibodies in Hungarian IBD patients.

Siderophores produced by rhizosphere bacteria may enhance plant growth by increasing the availability of Fe near the root or by inhibiting the colonization of roots by plant pathogens or other harmful bacteria. To examine the populations of siderophore-producing bacteria colonizing the roots of two grass species that differed in their susceptibility to Fe deficiency, we inoculated serial dilutions of root samples onto chrome azurol S (CAS) agar and several other selective and non-selective culture meida. CAS agar effectively differentiated bacteria that were capable of excreting large amounts of siderophore, but the composition of the medium limited its usefulness for ecological studies. A large proportion (71–79%) of the bacterial population that grew on a non-selective medium (tryptic soy agar) failed to grow on CAS agar, and several isolates that showed no sign of siderophore production on CAS agar produced siderophore in liquid culture. Similar populations of siderophore-producing bacteria were observed on roots of St. Augustine grass, which frequently exhibits Fe chlorosis, and bermuda grass, which does not. Roots of both grasses were colonized by bacteria that produced siderophore in vitro at concentrations ranging from 100 to 230 μM. The CAS assay solution was also used to compare siderophore production by Pseudomonas fluorescens Q6, an isolate from bermuda grass, and by P. putida B 10, a plant growth-promoting pseudomonad. P. fluorescens Q6 produced 2.4 times more siderophore in vitro than P. putida B 10.

The aim of this study was to speed up the composting process of municipal solid wastes, piles of material were inoculated with a mixed of endogenous microorganism. The bacteria identified were Bacillus subtillis and Pseudomonas fluorescens and one fungus, Aspergillus fumigatus. An inoculant was prepared in a concentration of 1x10 7 UFC/ml for each microorganism and was applied through aspersion, 2 L m -3 . The following parameters were evaluated: Physical aspect, temperature, moisture content, pH, C:N ratio, organic matter, electric conductivity and cation exchange capacity. The results showed that inoculated piles reached the stability and maturity, four weeks earlier than the control pile without inoculation. These results suggested that the inoculant was useful to accelerate the composting process in the urban wastes.

The complete genomic sequences of several Pseudomonas spp. that inhabit the rhizosphere are now available, providing a new opportunity to advance knowledge of plant growth-promoting rhizobacteria (PGPR) through genomics. Among these is the biological control bacterium Pseudomonas fluorescens Pf-5. Nearly 6% of the 7.07 Mb genome of Pf-5 is devoted to the biosynthesis of secondary metabolites, including antibiotics toxic to soilborne fungi and Oomycetes that infect plant roots, and two siderophores involved in iron acquisition. Three orphan gene clusters, for which the encoded natural product was unknown, also were identified in the genome of Pf-5. The product synthesized from one of the orphan gene clusters was identified recently using a new 'genomisotopic approach', which employs a combination of genomic sequence analysis and isotope guided fractionation. Application of the genomisotopic approach to one orphan gene cluster in Pf-5 resulted in the discovery of orfamide A, founder of a new group of bioactive cyclic lipopep-tides with a putative role in biological control of plant disease.

Bacterial populations inhabiting pharmaceutical grade water systems were investigated over a fifteen year period. The systems analyzed were mains water, purified and Water-for-Injection (WFI). Samples of water were tested by membrane filtration and the samples cultured using R2A agar. Culture based methods and phenotypic identification methods were used to characterize the isolates. The research was undertaken to produce an in-depth study of the microbiota of pharmaceutical grade water systems. The results presented act as a benchmark for industrial and pharmaceutical microbiologists to review comparable systems against, as well as to present a review of the typical culturable microorganisms recoverable from pharmaceutical water systems.

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.

In order to fill the knowledge gaps between lab/pilot-scale and full-scale operations and contribute basic information to long-term and stable operation of large-scale membrane bioreactors (MBRs), a systematic investigation was first conducted focused on soluble microbial products (SMPs) in 10 large-scale MBRs (capacity over 10,000 m 3 /d) for municipal wastewater treatment distributed in 4 different areas of China from fall to winter. The majority of these MBR plants had been operated stably for at least 1 year. Fundamental properties of SMPs were investigated, including composition, molecular weight distribution, hydrophobicity, fluorescent characteristics and fouling potential. The results showed that the concentration of SMPs ranged roughly from 5 to 25 mg TOC/L, with the major component being polysaccharides (ca. 3-18 mg/L) followed by humic substances (ca. 2-10 mg/L); while the protein concentration was relatively low (o5 mg/L). The SMPs presented a broad molecular weight distribution from smaller than 1 kDa to over 100 kDa. About half of the SMPs were hydrophilic substances mainly contributed by polysaccharides; humic substances were concentrated in hydrophobic fractions while proteins showed a relatively wide distribution. The fluorescent properties were found to be affected appreciably by the influent quality. The batch fouling tests indicated that the initial fouling rate correlated significantly with SMPs concentrations, which was particularly the case for hydrophilic and large-molecular-weight fractions. These findings may contribute to better understanding of membrane fouling in engineering conditions and assist in long-term and stable operation of full-scale MBRs.

Pseudomonas fluorescens SBW25 is a Gram-negative bacterium that grows in close association with plants. In common with a broad range of functionally similar bacteria it plays an important role in the turnover of organic matter and certain isolates can promote plant growth. Despite its environmental significance, the causes of its ecological success are poorly understood. Here we describe the development and application of a simple promoter trapping strategy (IVET) to identify P. fluorescens SBW25 genes showing elevated levels of expression in the sugar beet rhizosphere. A total of 25 rhizosphere-induced ( rhi ) fusions are reported with predicted roles in nutrient acquisition, stress responses, biosynthesis of phytohormones and antibiotics. One rhi fusion is to wss , an operon encoding an acetylated cellulose polymer. A mutant carrying a defective wss locus was competitively compromised (relative to the wild type) in the rhizosphere and in the phyllosphere, but not in bulk soil. The rhizosphere-induced wss locus therefore contributes to the ecological performance of SBW25 in the plant environment and supports our conjecture that genes inactive in the laboratory environment, but active in the wild, are likely to be determinants of fitness in natural environments.

The immune response to mixed whole cell antigens of Aeromonas hydrophila, Edwardsiella tarda and Pseudomonas fluorescens, the common Gram negative bacterial pathogens associated with diseases of Indian major carps were evaluated for their efficacy in triggering antibody responses in rohu, Labeo rohita (Ham.). The rohu yearlings were either immunized with antigens from single bacterial strain, A. hydrophila, E. tarda and P. fluorescens or a combination of all three. An antibody response was detected at 1st week post immunization that rose significantly (p<0.05) at 4th week post immunization in all the immunized groups. The antibody level started declining after 8th week but persisted up to 10th week post immunization in all the immunized groups. Similarly, no significant difference (p>0.05) in the antibody level was found between groups immunized with single and mixed bacterial antigens. Moreover, the use of mixed bacterial antigens did not jeopardize the specific immune response to the vaccine components. Upon challenge with single pathogen, a high relative percent survival was recorded in the group immunized with mixed bacterial antigens and was comparable to those fish immunized with the single bacteria.

The probiotic activity of two bacteria (Bacillus subtilis and Lactobacillus acidophilus) was evaluated by its effect on the immune response of Nile tilapia (Oreochromis niloticus), beside its protective effect against challenge infections. Furthermore, their in-vitro inhibitory activity was evaluated.

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.

Endophytic actinomycetes from Azadirachta indica A. Juss. were screened and evaluated for their anti-microbial activity against an array of pathogenic fungi and bacteria. A total of 55 separate isolates were obtained from 20 plants, and 60% of these showed inhibitory activity against one or more pathogenic fungi and bacteria. Actinomycetes were most commonly recovered from roots (54.5% of all isolates), followed by stems (23.6%), and leaves (21.8%). The dominant genus was Streptomyces (49.09% of all isolates), while Streptosporangium (14.5%), Microbispora (10.9%), Streptoverticillium (5.5%), Sacchromonospora sp. (5.5%), and Nocardia (3.6%) were also recovered. Streptomyces isolates AzR 006, 011, and 031 (all from roots) had acute activity against Pseudomonas fluorescens, while AzR027, 032, and 051 (also all from roots) showed activity against Escherichia coli. Meanwhile, an isolate of Nocardia sp. from leaves (AzL025) showed antagonism against Bacillus subtilis. Overall, 32 of the 55 were found to have broad spectrum significant antimicrobial activity, while about 4% of them showed strong and acute inhibition to pathogenic fungi and bacteria. Isolates of Streptomyces AzR031, 008, and 047, Nocardia sp. AzL025, and Streptosporangium sp. AzR 021 and 048 are of particular interest because they showed significant antagonistic activity against root pathogens, including Pythium and Phytophthora sp. Thus, many of the isolates recovered from A. indica in this study may be used in developing potential bio-control agents against a range of pathogenic fungi and bacteria and in the production of novel natural antimicrobial compounds. These results not only further our understanding of plant–microbe interactions but also indicate that there is an untapped resource of endophytic microorganisms that could be exploited in the biotechnological, medicinal, and agricultural industries.

Rhizosphere and endophytic bacterial isolates from the roots and corms of banana were tested for their biocontrol efficiency against Banana bunchy top virus (BBTV). Molecular characterization using RAPD and microsatellite markers revealed genomic variability in the endophytic Pseudomonas and Bacillus isolates. Bio-formulations of mixtures of the rhizobacterial isolate Pseudomonas fluorescens (Pf1) and endophytic Bacillus spp. (EPB22) were effective in reducing the incidence of BBTV under green-house (80%) and field conditions (52%). Reduction in virus titer (0.64) was noticed in the plants treated with compatible mixtures of rhizobacterial and endophytic bacterial isolates as evidenced by ELISA, in comparison to control plants (1.69). In addition to disease control, a significant increase in the yield (53.33%) was noticed in the bacterized plants when compared to the control plants. Pathogenesis-related (PR) proteins, chitinase and b-1,3-glucanase and defense-related proteins, peroxidase, polyphenol oxidase, phenylalanine ammonialyase and phenolic compounds were significantly activated in the bacterized plants, thus inducing resistance against bunchy top virus. Populations of endophytic bacteria also remained high and stable throughout the growing period. Thus, application of mixtures of rhizosphere and endophytic bacteria increases yield and has a potential role in inducing resistance against Banana bunchy top virus.

Purpose -The purpose of this paper is to examine pure culture isolates from traditionally fermented product of African oil bean seeds (ugba) with a view to identifying the appropriate starters. Design/methodology/approach -The isolates were purified and characterized, and used singly and in combination to ferment freshly prepared oil bean seed slices for 72 h at 37 C. The pH and microbial load of fermenting beans were monitored daily, and organoleptic tests using semi-trained panelists were employed to assess the fermented products based on quality attributes of flavour, appearance, texture and overall acceptability. Findings -The isolates included Bacillus subtilis, Bacillus licheniformis and Pseudomonas fluorescens. Total viable counts ranged from 1.3 Â 10 7 -9.3 Â 10 9 cfu/g, 7.0 Â 10 6 -4.0 Â 10 9 cfu/g, and 3.0 Â 10 6 -1.5 Â 10 9 cfu/g, respectively for the three isolates; while counts for the mixed culture fermentation ranged from 7.0 Â 10 6 -1.72 Â 10 10 cfu/g. The mixed culture fermentation gave the best rated product, indicating a synergy among the isolates. The pH values increased steadily in the samples fermented by Bacillus sp. and the mixed culture organisms, showing that the production of ugba follows alkaline fermentation pattern.

Arbuscular mycorrhizal (AM) fungi and plant growth-promoting bacteria (PGPB) can increase the growth and yield of major crops, and improve the quality of fruits and leaves. However, little is known about their impact on seed composition. Plants were inoculated with AM fungi and/or the bacterial strain Pseudomonas fluorescens Pf4 and harvested after 7 months of growth in open-field conditions. Plant growth parameters were measured (biomass, length and circumference of spikes, number of grains per cob, grain yield, and grain size) and protein, lipid, and starch content in grains were determined. Plant growth and yield were increased by inoculation with the microorganisms. Moreover, spikes and grains of inoculated plants were bigger than those produced by uninoculated plants. Regarding grain composition, the bacterial strain increased grain starch content, especially the digestible components, whereas AM fungi-enhanced protein, especially zein, content. Plant inoculation with the fluorescent pseudomonad and mycorrhizal fungi resulted in additive effects on grain composition. Overall, results showed that the bacterial strain and the AM fungi promoted maize growth cultivated in field conditions and differentially affected the grain nutritional content. Consequently, targeted plant inoculation with beneficial microorganisms can lead to commodities fulfilling consumer and industrial requirements.

Terpenoids are important compounds for the fragrance industry, and recently, biocatalytic methods have been developed to produce them from cheap monoterpenes, such as R-pinene oxide. The biotransformation of R-pinene oxide using resting cells of Pseudomonas fluorescens NCIMB 11671 produces isonovalal (cis-2-methyl-5-isopropylhexa-2,5-dienal), which is a fragrance. However, this biotransformation has technical problems including the following: R-pinene oxide undergoes autoxidation in water and light; it is hydrophobic and relatively toxic to the biocatalyst; and it suffers from product inhibition. Therefore, removal of isonovalal as it is formed should reduce its toxicity and increase volumetric productivity and production yield. Aqueousorganic two-phase (AOTP) systems can be used in the biotransformation of hydrophobic substrates and can protect biocatalysts from toxic substrates and products. However, the formation of stable emulsions makes further downstream processing and continuous operation difficult. One solution to these problems is to use a solid-phase membrane between the aqueous and organic phases in a Membrane Bioreactor for Biotransformation (MBB). Since there are no data in the literature on the behavior of terpenes and terpenoids with solid-phase membranes, or their mass transfer behavior, the objective of this work was to measure these parameters for a wide range of compounds so as to be able to design an MBB, and to gain a greater understanding of their behavior in these types of systems. Organic/aqueous (P aq org ) and membrane/aqueous (P aq mem ) partition coefficients were measured first, and subsequently used to quantify the overall mass transfer coefficients (k ov ). The overall mass transfer coefficient (k ov ) of R-pinene oxide through the membrane was found to be 2.5 × 10 -5 m‚s -1 using thicknesses of both 250 and 1000 µm. Extraction kinetics were successfully described using a resistance-in-series model and were controlled by the aqueous boundary layer and/or membrane resistance (k mem ), while organic film resistance played an insignificant role. Aqueous film resistance (k aq ) was found to be the limiting step for mono-and diterpenes, and the effect of the hydrodynamics on k ov was successfully predicted using a Wilson plot. However, the extraction kinetics of larger terpenes, such as steroids, were influenced solely by the k mem , suggesting that membrane diffusivity also depended on the size of the permeating molecule. Finally, the influence of other terpene byproducts on the flux of R-pinene oxide was investigated and found to decrease the flux into the organic phase by up to 10%.

Acquisition of nutrients by plants is primarily dependent on root growth and bioavailability of nutrients in the rooting medium. Most of the beneficial bacteria enhance root growth, but their effectiveness could be influenced by the nutrient status around the roots. In this study, two 1-aminocyclopropane-1-carboxylate (ACC)-deaminase containing plant-growth-promoting rhizobacteria (PGPR), Pseudomonas fluorescens and P. fluorescens biotype F were tested for their effect on growth, yield, and nutrient use efficiency of wheat under simultaneously varying levels of all the three major nutrients N, P, and K (at 0%, 25%, 50%, 75%, and 100% of recommended doses). Results of pot and field trials revealed that the efficacy of these strains for improving growth and yield of wheat reduced with the increasing rates of NPK added to the soil. In most of the cases, significant negative linear correlations were recorded between percentage increases in growth and yield parameters of wheat caused by inoculation and increasing levels of applied NPK fertilizers. It is highly likely that under low fertilizer application, the ACC-deaminase activity of PGPR might have caused reduction in the synthesis of stress (nutrient)-induced inhibitory levels of ethylene in the roots through ACC hydrolysis into NH3 and α-ketobutyrate. The results of this study imply that these Pseudomonads could be employed in combination with appropriate doses of fertilizers for better plant growth and savings of fertilizers.

Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracel...

Agricultural soils in Iran are predominantly calcareous with very low plant available phosphorus (P) content. In addition to their beneficial N 2 -fixing activity with legumes, rhizobia can improve plant P nutrition by mobilizing inorganic and organic P. Isolates from different cross-inoculation groups of rhizobia, obtained from Iranian soils were tested for their ability to dissolve inorganic and organic phosphate. From a total of 446 rhizobial isolates tested for P solubilization by the formation of visible dissolution halos on agar plates, 198 (44%) and 341(76%) of the isolates, solubilized Ca 3 (PO 4 ) 2 (TCP) and inositol hexaphosphate (IHP), respectively. In the liquid Sperber TCP medium, phosphate-solubilizing bacteria (Bacillus sp. and Pseudomonas fluorescens) used as positive controls released an average of 268.6 mg L )1 of P after 360 h incubation. This amount was significantly (P < 0.05) higher than those observed with all rhizobia tested. The group of Rhizobium leguminosarum bv. viciae mobilized in liquid TCP Sperber medium significantly (P < 0.05) more P (197.1 mg L )1 in 360 h) than other rhizobia tested,. This group also showed the highest dissolution halo on the TCP solid Sperber medium. The release of soluble P was significantly correlated with a drop in the pH of the culture filtrates indicating the importance of acid production in the mobilization process. None of the 70 bradyrhizobial isolates tested was able to solubilize TCP. These results indicate that many rhizobia isolated from soils in Iran are able to mobilize P from organic and inorganic sources and this beneficial effect should be tested with crops grown in Iran.