Survivability and controlled release of alginate-microencapsulated Pseudomonas fluorescens VUPF506 and their effects on biocontrol of Rhizoctonia solani on potato.

Preserving the efficacy of plant probiotic bacteria in soil is a major challenge to the biological control of plant diseases. The microencapsulation technique is an important step in preserving the viability and activity of probiotics in adverse environmental conditions. The main objective of this study was to choose an appropriate coating for probiotic encapsulation. For this purpose, the survivability and controlled release of Pseudomonas fluorescens VUPF506 encapsulated with alginate (Alg) combined with whey protein concentrate (WPC), carboxymethyl cellulose (CMC), and peanut butter (PB) were evaluated. Moreover, the encapsulated cells were evaluated to control for Rhizoctonia solani in potato plants under in vivo conditions. The results showed that all tested wall material maintained more than 80% of the bacterial cells. The Alg-WPC microcapsules provided a better controlled release over two months. Interestingly, the greenhouse experiment also revealed that the treatment of potato plants with Alg-WPC microcapsules was the most effective treatment, suppressing 90% of the pathogen. The results showed that Alg-WPC is the most promising combination to improve the survivability of P. fluorescens VUPF506. Moreover, it can be used as a fertilizer due to its content of valuable amino acids.

Pseudomonas fluorescens: A Bioaugmentation Strategy for Oil-Contaminated and Nutrient-Poor Soil.

Bioremediation technology is one of the most profitable and sustainable strategies for remediating soils contaminated with hydrocarbons. This study focuses on assessing the influence of biostimulation and bioaugmentation with Pseudomonas fluorescens to contribute to the removal of total petroleum hydrocarbons (TPHs) of a soil. Laboratory studies were carried out (measurements of emitted CO2, surface tension, and residual TPH) to select the best bioaugmentation and biostimulation treatment. The sources of C, N, and P were glucose-yeast extract, NH4Cl-NaNO3, and K2HPO4-K3PO4, respectively. The effect of culture conditions on the reduction of TPH and respiratory activity was evaluated through a factorial design, 23, in a solid culture system. After 80 days of incubation, it was observed that treatments of yeast extract-NH4Cl-K2HPO4 (Y4) and glucose-NaNO3-K3PO4 (Y5) presented a higher level of TPH removal (20.91% and 20.00% degradation of TPH, respectively). Biostimulation favors the production of biosurfactants, indirectly measured by the change in surface tension in the soil extracts. The treatments Y4 and Y5 showed a lower change value of the surface tension (23.15 and 23.30 mN·m-1 at 25 °C). A positive correlation was determined between the change in surface tension and the removal of TPH; hence there was a contribution of the biosurfactants produced to the removal of hydrocarbons.

Plant Extracts Containing Saponins Affects the Stability and Biological Activity of Hempseed Oil Emulsion System.

In this study, two saponins-rich plant extracts, viz. Saponaria officinalis and Quillaja saponaria, were used as surfactants in an oil-in-water (O/W) emulsion based on hempseed oil (HSO). This study focused on a low oil phase content of 2% v/v HSO to investigate stable emulsion systems under minimum oil phase conditions. Emulsion stability was characterized by the emulsification index (EI), centrifugation tests, droplet size distribution as well as microscopic imaging. The smallest droplets recorded by dynamic light scattering (droplets size v. number), one day after the preparation of the emulsion, were around 50-120 nm depending the on use of Saponaria and Quillaja as a surfactant and corresponding to critical micelle concentration (CMC) in the range 0-2 g/L. The surface and interfacial tension of the emulsion components were studied as well. The effect of emulsions on environmental bacteria strains was also investigated. It was observed that emulsions with Saponaria officinalis extract exhibited slight toxic activity (the cell metabolic activity reduced to 80%), in contrast to Quillaja emulsion, which induced Pseudomonas fluorescens ATCC 17400 growth. The highest-stability samples were those with doubled CMC concentration. The presented results demonstrate a possible use of oil emulsions based on plant extract rich in saponins for the food industry, biomedical and cosmetics applications, and nanoemulsion preparations.

Pseudomonas fluorescens LBUM677 differentially increases plant biomass, total oil content and lipid composition in three oilseed crops.

AIMS: Pseudomonas spp. have been widely studied for their plant growth-promoting effects. However, their capacity to promote lipid accumulation in oilseed crops is not well characterized. In this study, we evaluated the effect of Pseudomonas fluorescens LBUM677 on lipid accumulation in three oilseed crops: soybean (Glycine max), canola (Brassica napus) and corn gromwell (Buglossoides arvensis), a plant of high nutraceutical interest for its accumulation of the omega-3 stearidonic acid. METHODS AND RESULTS: Pot experiments were conducted under controlled conditions where seeds were inoculated or not with LBUM677 and plants were harvested at 4, 8 and 12 weeks. A qPCR assay specifically targeting LBUM677 was used in parallel to correlate LBUM677 soil rhizosphere competency to growth promotion and seed lipid accumulation. Total oil seed content and fatty acid composition were analysed at seed maturity. Results showed that LBUM677 was able to establish itself in the rhizosphere of the three plant species at similar levels, but it differentially increased plant biomass, total oil content and lipid composition in a plant-specific manner. CONCLUSIONS: Despite some species-specific differences observed in P. fluorescens LBUM677's effect on different crops, the strain appears to be a generalist plant growth-promoting rhizobacteria of oilseed crops. SIGNIFICANCE AND IMPACT OF THE STUDY: LBUM677 shows great potential to be used as an inoculum to promote oil yield and fatty acid accumulation in oilseed crops.

Persistence of Pseudomonas fluorescens LBUM677 in the rhizosphere of corn gromwell (Buglossoides arvensis) under field conditions and its impact on seed oil and stearidonic acid bioaccumulation.

AIMS: The aim of this study was to evaluate the persistence of Pseudomonas fluorescens LBUM677 in the rhizosphere of Buglossoides arvensis under agricultural field conditions and determine if B. arvensis intraspecific genetic variations affect the capacity of LBUM677 to colonize its rhizosphere and increase seed oil and stearidonic acid (SDA) accumulation. METHODS AND RESULTS: Two field experiments were performed to: (i) study the persistence of various concentrations of LBUM677 inoculated in the rhizosphere of B. arvensis and determine a minimum inoculation threshold required to maximize biological activity; and (ii) study the impact of B. arvensis intraspecific genetic variations on LBUM677 rhizosphere colonization and seed oil and SDA accumulation. In order to track LBUM677 populations in soil over time, a specific quantitative polymerase chain reaction assay was developed. Inoculation with a minimum of 109 LBUM677 bacterial cells per plant was determined as a threshold to promote maximum B. arvensis rhizosphere colonization and seed oil and SDA accumulation. Buglossoides arvensis intraspecific genetic variations had an impact on rhizosphere colonization, B. arvensis seed oil and SDA accumulation, where two cultivars benefited more than others from LBUM677 inoculation. CONCLUSIONS: LBUM677 can colonize the rhizosphere and increase seed oil and SDA yields in B. arvensis plants in a cultivar-dependant manner. SIGNIFICANCE AND IMPACT OF THE STUDY: LBUM677 shows potential to be used as a biofertilizer to specifically increase seed oil and SDA yields in B. arvensis. This will in turn promote the development of an economically viable agricultural-based approach as an alternative for producing high-quality polyunsaturated fatty acids.

Co-culture development and bioformulation efficacy of psychrotrophic PGPRs to promote growth and development of Pea (Pisum sativum) plant.

Numerous microbes reside in the rhizosphere having plant growth promoting activity, and enhancing the property by increasing plant yield. Plant growth promoting rhizobacteria (PGPR) has gradually increased in agriculture and offers an attractive way to replace chemical fertilizers, pesticides and supplements. Soil was collected from the rhizosphere of an agricultural farm and the psychrotrophic bacterial strains STA3 (KY888133) and RM2 (KY888134) were successfully isolated, and screened on the basis of phosphate solubilization. Further characterization was carried out by morphological, biochemical, and 16S rDNA characterization methods. The unique nature of psychrotrophic Pentoea ananatis and a suitable combination with Pseudomonas fluorescens regarding plant growth promotion activity has not been studied before to our knowledge. An assessment of various parameters of plant growth promoting activity, such as IAA, phosphate solubilization, bio-control activity, HCN and siderophore production, has been carried out. Both strains were found to be positive in various parameters except HCN and Biocontrol activity, which were positive only for the strain RM2. Also, shelf life and their efficacy was determined before and after formulation. A great consistency was observed in all the cultures, even after 70 days of storage under bio-formulation at room temperature, while in the case of the co-culture CPP-2, the cfu ml-1 was greater, followed by RM2 and STA3. Moreover, the growth indices of the pea plant were found to be better in the co-culture CPP-2 compared with individual strains, followed by RM2 and STA3. Thus, the study suggests that the co-culture CPP-2 has a great potential for plant growth promotion as compared with individual strains followed by RM2 and STA3.

Biocontrol of Potato Common Scab is Associated with High Pseudomonas fluorescens LBUM223 Populations and Phenazine-1-Carboxylic Acid Biosynthetic Transcript Accumulation in the Potato Geocaulosphere.

Pseudomonads are often used as biocontrol agents because they display a broad range of mechanisms to control diseases. Common scab of potato, caused by Streptomyces scabies, was previously reported to be controlled by Pseudomonas fluorescens LBUM223 through phenazine-1-carboxylic acid (PCA) production. In this study, we aimed at characterizing the population dynamics of LBUM223 and the expression of phzC, a key gene involved in the biosynthesis of PCA, in the rhizosphere and geocaulosphere of potato plants grown under controlled and field conditions. Results obtained from controlled experiments showed that soil populations of LBUM223 significantly declined over a 15-week period. However, at week 15, the presence of S. scabies in the geocaulosphere was associated with significantly higher populations of LBUM223 than when the pathogen was absent. It also led to the detection of significantly higher phzC gene transcript numbers. Under field conditions, soil populations of LBUM223 followed a similar decline in time when a single inoculation was applied in spring but remained stable when reinoculated biweekly, which also led to greater phzC gene transcripts accumulation. Taken together, our findings suggest that LBUM223 must colonize the potato geocaulosphere at high levels (10(7) bacteria/g of soil) in order to achieve biocontrol of common scab through increased PCA production.

Evaluation of economically feasible, natural plant extract-based microbiological media for producing biomass of the dry rot biocontrol strain Pseudomonas fluorescens P22Y05 in liquid culture.

The production of microbial biomass in liquid media often represents an indispensable step in the research and development of bacterial and fungal strains. Costs of commercially prepared nutrient media or purified media components, however, can represent a significant hurdle to conducting research in locations where obtaining these products is difficult. A less expensive option for providing components essential to microbial growth in liquid culture is the use of extracts of fresh or dried plant products obtained by using hot water extraction techniques. A total of 13 plant extract-based media were prepared from a variety of plant fruits, pods or seeds of plant species including Allium cepa (red onion bulb), Phaseolus vulgaris (green bean pods), and Lens culinaris (lentil seeds). In shake flask tests, cell production by potato dry rot antagonist Pseudomonas fluorescens P22Y05 in plant extract-based media was generally statistically indistinguishable from that in commercially produced tryptic soy broth and nutrient broth as measured by optical density and colony forming units/ml produced (P ≤ 0.05, Fisher's protected LSD). The efficacy of biomass produced in the best plant extract-based media or commercial media was equivalent in reducing Fusarium dry rot by 50-96% compared to controls. In studies using a high-throughput microbioreactor, logarithmic growth of P22Y05 in plant extract-based media initiated in 3-5 h in most cases but specific growth rate and the time of maximum OD varied as did the maximum pH obtained in media. Nutrient analysis of selected media before and after cell growth indicated that nitrogen in the form of NH4 accumulated in culture supernatants, possibly due to unbalanced growth conditions brought on by a scarcity of simple sugars in the media tested. The potential of plant extract-based media to economically produce biomass of microbes active in reducing plant disease is considerable and deserves further research.

Pseudomonas fluorescens pirates both ferrioxamine and ferricoelichelin siderophores from Streptomyces ambofaciens.

Iron is essential in many biological processes. However, its bioavailability is reduced in aerobic environments, such as soil. To overcome this limitation, microorganisms have developed different strategies, such as iron chelation by siderophores. Some bacteria have even gained the ability to detect and utilize xenosiderophores, i.e., siderophores produced by other organisms. We illustrate an example of such an interaction between two soil bacteria, Pseudomonas fluorescens strain BBc6R8 and Streptomyces ambofaciens ATCC 23877, which produce the siderophores pyoverdine and enantiopyochelin and the siderophores desferrioxamines B and E and coelichelin, respectively. During pairwise cultures on iron-limiting agar medium, no induction of siderophore synthesis by P. fluorescens BBc6R8 was observed in the presence of S. ambofaciens ATCC 23877. Cocultures with a Streptomyces mutant strain that produced either coelichelin or desferrioxamines, as well as culture in a medium supplemented with desferrioxamine B, resulted in the absence of pyoverdine production; however, culture with a double mutant deficient in desferrioxamines and coelichelin production did not. This strongly suggests that P. fluorescens BBbc6R8 utilizes the ferrioxamines and ferricoelichelin produced by S. ambofaciens as xenosiderophores and therefore no longer activates the production of its own siderophores. A screening of a library of P. fluorescens BBc6R8 mutants highlighted the involvement of the TonB-dependent receptor FoxA in this process: the expression of foxA and genes involved in the regulation of its biosynthesis was induced in the presence of S. ambofaciens. In a competitive environment, such as soil, siderophore piracy could well be one of the driving forces that determine the outcome of microbial competition.

[Effects of actinomycetes agent on ginseng growth and rhizosphere soil microflora].

Taking the ginseng in Xiao Xing' an Mountains of Northeast China as test object, this paper studied the effects of applying Streptomyces pactum (Act12) on ginseng growth and on the soil microflora in root zone and root surface. After treated with Act12, the yield and quality of ginseng' s medicinal part improved, the induced enzyme activities in leaves and the root activity increased, and the numbers and proportions of soil bacteria and actinomycetes increased significantly while those of soil fungi decreased. Compared with the control, the soil microflora in treatment Act12 changed. The numbers of the dominant bacteria Pseudomonas fluorescens, Pseudomonas koreensis, and Microbacterium oxydans were much higher in root zone soil and root surface soil, and the pathogen Plectosphaerella cucumerina decreased in root zone soil and disappeared in root surface soil. These results suggested that the addition of Act12 could improve the soil microflora, enhance the resistance and root activity of ginseng plant, and increase the ginseng yield and its quality.

Combined inoculation of Pseudomonas fluorescens and Trichoderma harzianum for enhancing plant growth of vanilla (Vanilla planifolia).

This study was conducted to evaluate the plant growth promoting efficiency of combined inoculation of rhizobacteria on Vanilla plants. Based on the in vitro performance of indigenous Trichoderma spp. and Pseudomonas spp., four effective antagonists were selected and screened under greenhouse experiment for their growth enhancement potential. The maximum percentage of growth enhancement were observed in the combination of Trichoderma harzianum with Pseudomonas fluorescens treatment followed by Pseudomonas fluorescens, Trichoderma harzianum, Pseudomonas putida and Trichoderma virens, respectively in decreasing order. Combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens registered the maximum length of vine (82.88 cm), highest number of leaves (26.67/plant), recorded the highest fresh weight of shoots (61.54 g plant(-1)), fresh weight of roots (4.46 g plant(-1)) and dry weight of shoot (4.56 g plant(-1)) where as the highest dry weight of roots (2.0806 g plant(-1)) were achieved with treatments of Pseudomonas fluorescens. Among the inoculated strains, combined inoculation of Trichoderma harzianum and Pseudomonas fluorescens recorded the maximum nitrogen uptake (61.28 mg plant(-1)) followed by the combined inoculation of Trichoderma harzianum (std) and Pseudomonas fluorescens (std) (55.03 mg plant(-1)) and the highest phosphorus uptake (38.80 mg plant(-1)) was recorded in dual inoculation of Trichoderma harzianum and Pseudomonas fluorescens.

Synergistic antimicrobial effect against early biofilm formation: micropatterned surface plus antibiotic treatment.

The detrimental effects of biofilms are a cause of great concern in medical, industrial and environmental areas. In this study, we proposed a novel eradication strategy consisting of the combined use of micropatterned surfaces and antibiotics on biofilms to reduce the rate of bacterial colonisation. Pseudomonas fluorescens biofilms were used to perform a comparative evaluation of possible strategies to eradicate these biological layers. First, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration of planktonic cultures were determined. Subsequently, adhesion of bacteria on microstructured gold surfaces (MS) with patterned features that were similar to the bacterial diameter as well as on smooth nanostructured gold (NS) was assessed. As expected, lower bacterial attachment as well as inhibition of bacterial aggregation were observed on MS. The effect of streptomycin treatment (ST) in the concentration range 1-4 mg/L (0.25-1× MIC) on biofilms grown on MS and NS was also evaluated. The combined strategy involving the use of micropatterned surfaces and antibiotic treatment (MS+ST) to eradicate Pseudomonas biofilms was then investigated. Results showed a synergistic effect of MS+ST that yielded a reduction of ≥1000-fold in the number of surviving biofilm bacteria with respect to those obtained with single ST or MS. The combined strategy may be a significant contribution to the eradication of biofilms from different environments. In addition, the important role of early monolayer bacterial aggregates in increasing resistance to antimicrobial agents was demonstrated.

Multiple treatment meta-analysis of products evaluated for control of fire blight in the eastern United States.

The aim of this analysis was to estimate the effect sizes and consistency of products evaluated for fire blight control in the eastern United States over the last decade. Because only 3% of the 69 studies published from 2000 to 2008 explicitly presented a measure of within-study variability, a method for estimating the least significant difference (LSD) and, hence the sampling variance, for studies with at least two significant mean separations in the presented mean multiple comparisons was developed. Lin's concordance analysis indicated that the estimated LSD was an accurate predictor of the actual LSD based on 35 studies in a calibration evaluation (ρ(c) = 0.997). Separate multi-treatment random-effects meta-analyses were performed for three control categories: antibiotics, biological control, and plant defense-activating products and mean log response ratios relative to the nontreated controls ([Formula: see text]) were computed for each treatment and then back-transformed to obtain the mean percent disease control. None of the products evaluated performed as well as streptomycin, the standard product for fire blight control, for which the mean disease control was 68.6%. As a group, experimental antibiotics provided the best fire blight control with mean effect sizes ranging from 59.7 to 61.7%. Among the biological controls, the best control was noted for treatments combining the antibiotic streptomycin with a product based on Pantoea agglomerans (55.0% mean disease reduction) or Bacillus subtilis (53.9%). Mean disease control was 31.9, 25.7, and 22.6%, respectively, for products based on B. subtilis, Pantoea agglomerans, and Pseudomonas fluorescens without an antibiotic, suggesting that the higher efficacy of the combination treatments was due to the antibiotic. Among the plant defense-activating products, prohexadione calcium had the highest and most consistent effect size (50.7% control), while other products provided modest mean disease control of between 6.1 and 25.8%. Percent control values were significantly moderated by study location and cultivar used in the study, and were smaller, but more variable, when products were tested under high disease intensity compared with low disease intensity. Results indicate that wide-scale use of biological control and plant defense-activating products in the eastern United States is likely to remain low.

Antagonistic control of microbial pathogens under iron limitations by siderophore producing bacteria in a chemostat setup.

Certain bacteria develop iron chelation mechanisms that allow them to scavenge dissolved iron from the environment and to make it unavailable to competitors. This is achieved by producing siderophores that bind the iron which is later liberated internally in the cell. Under conditions of iron limitation, siderophore producing bacteria have therefore an antagonistic growth advantage over other species. This has been observed in particular in agricultural and aquacultural systems, as well as in food microbiology. We investigate here the possibility of a probiotic biocontrol strategy to eradicate a well established, often pathogenic, non-chelating population by supplementing the system with generally regarded as safe siderophore producing bacteria. Set in a chemostat setup, our modeling and simulation studies suggest that this is indeed possible in a finite time treatment.

Mobilization of metals from uranium mine waste: the role of pyoverdines produced by Pseudomonas fluorescens.

Microorganisms produce chelating agents, such as siderophores and other ligands, which allow them to mobilize and scavenge essential elements from the environment when bioavailability is low. To better understand the effects of biologically mediated leaching of metals from mine waste, Pseudomonas fluorescens was cultivated in the presence of processed ore from the former uranium mine in Ranstad, southern Sweden. Light conditions, the concentration of the mineral source and oxygen availability were varied. The presence of ore in the culture flasks enhanced bacterial growth and raised the pH of the culture medium. Increasing the amount of ore or enhancing aeration of the medium further encouraged cell growth and pH rise. Bacteria mobilized Fe, Ni and Co from the ore. Fe-siderophore complexes were detected and estimated to be present at approximately 9 mum. In the presence of bacteria and light, dissolved Fe and U concentrations were higher compared to dark conditions. Increasing the amount of ore resulted in higher dissolved Ni concentrations but lower dissolved Fe, most likely due to precipitate formation. Data from this study support siderophore production by bacteria that allowed mobilization of essential nutrients from the processed ore. However, the availability of potentially toxic metals like Ni and U may also be enhanced. Microbial-promoted mobilization could contribute to leaching of toxic metals in current and historic mining areas. This process should be considered during design and implementation of remediation projects where trace metals are of environmental concern.

Diversity of the transcriptional regulation of the pch gene cluster in two indigenous p-cresol-degradative strains of Pseudomonas fluorescens.

p-Cresol methylhydroxylase (PCMH), a key enzyme responsible for the catabolism of p-cresol via the protocatechuate ortho pathway, was used as a tool to characterize catabolic differences between phenol- and p-cresol-degrading Pseudomonas fluore-scens strains PC18 and PC24. Although both strains catabolize p-cresol using PCMH, different whole-cell kinetic parameters for this compound were revealed. Affinity for the substrate and the specific growth rate were higher in PC18, whereas maximum p-cresol tolerance was higher in PC24. In addition, PCMH of strain PC18 was induced during growth on phenol. In both strains, the pchACXF operon, which encodes p-hydroxybenzaldehyde dehydrogenase and PCMH, was sequenced. Transcriptional regulation of these operons by PchR, a putative sigma(54)-dependent regulator, was shown. Although the promoters of these operons resembled sigma(54)-controlled promoters, they differed from the consensus sequence by having T instead of C at position -12. Complementation assays confirmed that the amino acid sequence differences of the PchR regulators between the two strains studied led to different effector-binding capabilities of these proteins: (1) phenol was a more efficient effector for PchR of PC18 than p-cresol, (2) phenol did not activate the regulator of PC24, and (3) both regulators responded similarly to p-cresol.

Evaluation of different carbon sources for growth and biosurfactant production by Pseudomonas fluorescens isolated from wastewaters.

The indigenous strain Pseudomonas fluorescens, isolated from industrial wastewater, was able to produce glycolipid biosurfactants from a variety of carbon sources, including hydrophilic compounds, hydrocarbons, mineral oils, and vegetable oils. Hexadecane, mineral oils, vegetable oils, and glycerol were preferred carbon sources for growth and biosurfactant production by the strain. Biosurfactant production was detected by measuring the surface and interfacial tension, rhamnose concentration and emulsifying activity. The surface tension of supernatants varied from 28.4 mN m(-1) with phenanthrene to 49.6 mN m(-1) with naphthalene and heptane as carbon sources. The interfacial tension has changed in a narrow interval between 6.4 and 7.6 mN m(-1). The emulsifying activity was determined to be highest in media with vegetable oils as substrates. The biosurfactant production on insoluble carbon sources contributed to a significant increase of cell hydrophobicity and correlated with an increased growth of the strain on these substrates. Based on these results, a mechanism of biosurfactant-enhanced interfacial uptake of hydrophobic substrates could be proposed as predominant for the strain. With hexadecane as a carbon source, the pH value of 7.0-7.2 and temperature of (28 +/- 2) degrees C were optimum for growth and biosurfactant production by P. fluorescens cells. The increased specific protein and biosurfactant release during growth of the strain on hexadecane in the presence of NaCl at contents up to 2% could be due to increased cell permeability. The capability of P. fluorescens strain HW-6 to adapt its own metabolism to use different nutrients as energy sources and to keep up relatively high biosurfactant levels in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

Adhesion of food-borne bacteria to stainless steel is reduced by food conditioning films.

AIMS: Preconditioning of stainless steel with aqueous cod muscle extract significantly impedes subsequent bacterial adhesion most likely due to repelling effects of fish tropomyosin. The purpose of this study was to determine if other food conditioning films decrease or enhance bacterial adhesion to stainless steel. METHODS AND RESULTS: Attachment of Pseudomonas fluorescens AH2 to stainless steel coated with water-soluble coatings of animal origin was significantly reduced as compared with noncoated stainless steel or stainless steel coated with laboratory substrate or extracts of plant origin. Coating with animal extracts also decreases adhesion of other food-relevant bacteria. The manipulation of adhesion was not attributable to growth inhibitory effects. Chemical analysis revealed that the stainless steels were covered by homogenous layers of adsorbed proteins. The presence of tropomyocin was indicated by appearance of proteins with similar molecular weight based in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in several extracts that reduced adhesion but also extracts not containing this protein reduced bacterial adhesion, indicating that several molecular species may be involved in the phenomenon. CONCLUSIONS: It is a common perception that food materials facilitate bacterial adhesion to surfaces; however, this study demonstrates that aqueous coatings of food origin may actually reduce bacterial adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: Compounds from food extracts may potentially be used as nontoxic coatings to reduce bacterial attachment to inert surfaces.

Multiple physiological states of a Pseudomonas fluorescens DR54 biocontrol inoculant monitored by a new flow cytometry protocol.

A new fluorescence staining and flow cytometry protocol was developed to monitor several physiological states in biocontrol strain Pseudomonas fluorescens DR54 during storage survival in a stationary-phase culture, preparation of clay carrier for seed formulation, and establishment in a sugar beet spermosphere. The high load of impurities in the environmental samples was dealt with by adding a density-gradient purification step to the staining protocol. Staining by SYBR Green, combined with either propidium iodide or ethidium bromide (EB)+DiBAC((4))3, was used to quantify the total cell population and further divide this population into: (1) intact cells with an unaffected membrane and energy metabolism. (2) De-energized cells unable to maintain membrane export (EB exclusion). (3) Depolarized cells unable to maintain membrane potential. (4) Permeabilized cells with a damaged membrane. During both stationary-phase storage and steps for preparation of formulation carrier, loss of intact P. fluorescens DR54 cells was quantitatively accounted for by depolarized and permeabilized states. Surviving inoculum cells subsequently proliferated on the germinating seeds, but with a surprisingly high abundance of de-energized cells. The new protocol is the first for flow cytometry to include a recording of both intact and several subpopulations of physiologically affected bacteria in complex, environmental samples with high impurity loads.