Isolation, selection and characterization of root-associated growth promoting bacteria in Brazil Pine (Araucaria angustifolia)

Araucaria angustifolia, a unique species of this genus that occurs naturally in Brazil, has a high socio-economic and environmental value and is critically endangered of extinction, since it has been submitted to intense predatory exploitation during the last century. Root-associated bacteria from A. angustifolia were isolated, selected and characterized for their biotechnological potential of growth promotion and biocontrol of plant pathogenic fungi. Ninety-seven strains were isolated and subjected to chemical tests. All isolates presented at least one positive feature, characterizing them as potential PGPR. Eighteen isolates produced indole-3-acetic acid (IAA), 27 were able to solubilize inorganic phosphate, 21 isolates were presumable diazotrophs, with pellicle formation in nitrogen-free culture medium, 83 were phosphatases producers, 37 were positive for siderophores and 45 endospore-forming isolates were antagonistic to Fusarium oxysporum, a pathogen of conifers. We also observed the presence of bacterial strains with multiple beneficial mechanisms of action. Analyzing the fatty acid methyl ester (FAME) and partial sequencing of the 16S rRNA gene of these isolates, it was possible to characterize the most effective isolates as belonging to Bacillaceae (9 isolates), Enterobacteriaceae (11) and Pseudomonadaceae (1). As far as we know, this is the first study to include the species Ewingella americana as a PGPR. (C) 2011 Elsevier GmbH. All rights reserved.

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Background and aims Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants. Methods A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique. Results The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA. Conclusion Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

Diversity of propanil-degrading bacteria isolated from rice rhizosphere and their potential for plant growth promotion

The herbicide propanil has long been used in rice production in southern Brazil. Bacteria isolated from contaminated soils in Massaranduba, Santa Catarina, Brazil, were found to be able to grow in the presence of propanil, using this compound as a carbon source. Thirty strains were identified as Pseudomonas (86.7%), Serratia (10.0%), and Acinetobacter (3.3%), based on phylogenetic analysis of 16S rDNA. Little genetic diversity was found within species, more than 95% homology, suggesting that there is selective pressure to metabolize propanil in the microbial community. Two strains of Pseudomonas (AF7 and AF1) were selected in bioreactor containing chemotactic growth medium, with the highest degradation activity of propanil exhibited by strain AF7, followed by AF1 (60 and 40%, respectively). These strains when encapsulated in alginate exhibited a high survival rate and were able to colonize the rice root surfaces. Inoculation with Pseudomonas strains AF7 and AF1 significantly improved the plant height of rice. Most of the Pseudomonas strains produced indoleacetic acid, soluble mineral phosphate, and fixed nitrogen. These bacterial strains could potentially be used for the bioremediation of propanil-contaminated soils and the promotion of plant growth.

GENETIC VARIABILITY OF SUGARCANE-ASSOCIATED DIAZOTROPHIC BACTERIA CAPABLE OF INORGANIC PHOSPHATE SOLUBILIZING

The sugarcane is a culture of great importance for the Brazilian agriculture. Every year this culture consumes great amounts of nitrogen and phosphate fertilizers. However, the use of plant growth-promoting bacteria can reduce the use of the chemical fertilizers, contributing to the economy and the environment conservation. So, the goal of this study was to select sugarcane-associated diazotrophic bacteria able to solubilize inorganic phosphate and to evaluate the genetic diversity of these bacteria. A total of 68 diazotrophic bacteria, leaf and root endophytic and rizoplane, of three sugarcane varieties. The selection of inorganic phosphate solubilizing diazotrophic bacteria was assayed by the solubilization index (SI) in solid medium containing insoluble phosphate. The genetic variability was analyzed by the BOX-PCR technique. The results showed that 74% of the diazotrophic strains were able to solubilize inorganic phosphate, presenting classes of different SI. The results showed that the vegetal tissue and the genotype plant influenced in the interaction between phosphate solubilizing diazotrophic bacteria and sugarcane plants. BOX-PCR revealed high genetic variability among the strains analyzed. So, sugarcane-associated diazotrophic bacteria express the capacity to solubilize inorganic phosphate and they present high genetic diversity.

Growth potential of Salmonella spp. and Listeria monocytogenes in nine types of ready-to-eat vegetables stored at variable temperature conditions during shelf-life

Growth potential (delta) is defined as the difference between the population of a microorganism at the end of shelf-life of specific food and its initial population. The determination of 6 of Salmonella and Listeria monocytogenes in RTE vegetables can be very useful to determine likely threats to food safety. However, little is known on the behavior of these microorganisms in several RTE vegetables. Therefore, the aim of this study was to determine the delta of both pathogens in nine different types of RTE vegetables (escarole, collard green, spinach, watercress, arugula, grated carrot, green salad, and mix for yakisoba) stored at refrigeration (7 degrees C) and abuse temperature (15 degrees C). The population of aerobic microorganisms and lactic acid bacteria, including those showing antimicrobial activity has been also determined. Results indicated that L monocytogenes was able to grow (delta >= 0.5 log(10)) in more storage conditions and vegetables than Salmonella. Both microorganisms were inhibited in carrots, although a more pronounced effect has been observed against L monocytogenes. The highest 5 values were obtained when the RTE vegetables were stored 15 degrees C/6 days in collard greens (delta=3.3) and arugula (delta=3.2) (L monocytogenes) and arugula (delta=4.1) and escarole (delta=2.8) (Salmonella). In most vegetables and storage conditions studied, the counts of total aerobic microorganisms raised significantly independent of the temperature of storage (p<0.05). Counts of lactic acid bacteria were higher in vegetables partially or fully stored at abuse temperature with recovery of isolates showing antimicrobial activity. In conclusion, the results of this study show that Salmonella and L monocytogenes may grow and reach high populations in RTE vegetables depending on storage conditions and the definition of effective intervention strategies are needed to control their growth in these products. (C) 2012 Elsevier B.V. All rights reserved.

Growth, organic acids profile and sugar metabolism of Bifidobacterium lactis in co-culture with Streptococcus thermophilus: the inulin effect

The organic acids profile, sugar metabolism and biomass growth of Streptococcus thermophilus (St) and Bifidobacterium lactis (BI) have been studied in pure cultures or binary co-culture (St-BI) in skim milk either containing 40 mg/g of inulin or not. With inulin, the time required by St. BI and St-BI to complete fermentation (i.e., when the pH reached 4.5) was about 14, 8 and 49% shorter than without inulin, respectively. This prebiotic also enhanced the levels of lactic and acetic acids and volatile compounds, showing a positive synbiotic effect between pre- and probiotics. In particular, the St-BI co-culture showed final concentrations of both microorganisms about 15 and 38% higher than in their respective pure cultures, thus highlighting a clear synergistic effect between these microorganisms due to mutual interactions. In addition, the well-known bifidogenic effect of inulin was confirmed. (c) 2012 Elsevier Ltd. All rights reserved.

Eucalyptus growth promotion by endophytic Bacillus spp

Clonal eucalyptus plantings have increased in recent years; however, some clones with high production characteristics have vegetative propagation problems because of weak root and aerial development. Endophytic microorganisms live inside healthy plants without causing any damage to their hosts and can be beneficial, acting as plant growth promoters. We isolated endophytic bacteria from eucalyptus plants and evaluated their potential in plant growth promotion of clonal plantlets of Eucalyptus urophylla x E. grandis, known as the hybrid, E. urograndis. Eighteen isolates of E. urograndis, clone 4622, were tested for plant growth promotion using the same clone. These isolates were also evaluated for indole acetic acid production and their potential for nitrogen fixation and phosphate solubilization. The isolates were identified by partial sequencing of 16S rRNA. Bacillus subtilis was the most prevalent species. Several Bacillus species, including B. licheniformis and B. subtilis, were found for the first time as endophytes of eucalyptus. Bacillus sp strain EUCB 10 significantly increased the growth of the root and aerial parts of eucalyptus plantlets under greenhouse conditions, during the summer and winter seasons.

Effect of low-dose gaseous ozone on pathogenic bacteria

Background: Treatment of chronically infected wounds is a challenge, and bacterial environmental contamination is a growing issue in infection control. Ozone may have a role in these situations. The objective of this study was to determine whether a low dose of gaseous ozone/oxygen mixture eliminates pathogenic bacteria cultivated in Petri dishes. Methods: A pilot study with 6 bacterial strains was made using different concentrations of ozone in an ozone-oxygen mixture to determine a minimally effective dose that completely eliminated bacterial growth. The small and apparently bactericidal gaseous dose of 20 mu g/mL ozone/oxygen (1: 99) mixture, applied for 5min under atmospheric pressure was selected. In the 2nd phase, eight bacterial strains with well characterized resistance patterns were evaluated in vitro using agar-blood in adapted Petri dishes (10(5) bacteria/dish). The cultures were divided into 3 groups: 1-ozone-oxygen gaseous mixture containing 20 mu g of O-3/mL for 5 min; 2- 100% oxygen for 5 min; 3- baseline: no gas was used. Results: The selected ozone dose was applied to the following eight strains: Escherichia coli, oxacillin-resistant Staphylococcus aureus, oxacillin-susceptible Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii, Acinetobacter baumannii susceptible only to carbapenems, and Pseudomonas aeruginosa susceptible to imipenem and meropenem. All isolates were completely inhibited by the ozone-oxygen mixture while growth occurred in the other 2 groups. Conclusion: A single topical application by nebulization of a low ozone dose completely inhibited the growth of all potentially pathogenic bacterial strains with known resistance to antimicrobial agents.

Influence of Growth Media and Temperature on Bacterial Adhesion to Polystyrene Surfaces

Bacterial adhesion to inert surfaces is a complex process influenced by environmental conditions. In this work, the influence of growth medium and temperature on the adhesion of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes to polystyrene surfaces was studied. Most bacteria demonstrated the highest adhesion when cultured in TSYEA, except S. marcescens, which showed to be positively influenced by the pigment production, favored in poor nutrient media (lactose and peptone agar). P. aeruginosa adhesion to polystyrene increased at low temperatures whatever the medium used. The culture medium influenced the surface properties of the bacteria as assessed by the MATS test.

Sugarcane Growth Promotion by the Endophytic Bacterium Pantoea agglomerans 33.1

The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1: pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1:pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1:pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.

Effect of inulin on the growth and metabolism of a probiotic strain of Lactobacillus rhamnosus in co-culture with Streptococcus thermophilus

Metabolic studies are very important to improve quality of functional dairy products. For this purpose, the behaviors of pure cultures of Streptococcus thermophilus (St) and Lactobacillus rhamnosus (Lr) as well a co-culture of them (St-Lr) were investigated during skim milk fermentation, and the inulin effect as prebiotic was assessed. Lr was able to metabolize 6 g/100 g more galactose than St and St-Lr. Final lactic acid production by Lr was higher (9.8 g/L) compared to St (9.1 g/L) and St-Lr (9.1 g/L). Acetic acid concentration varied from 0.8 g/L (St-Lr) to 1.5 g/L (Lr) and that of ethanol from only 0.2 g/L (St-Lr) to 0.4 g/L (Lr), which suggests the occurrence in Lr of a NADH oxidase activity and citrate co-metabolization via pyruvate, both dissipating a part of the reducing power. Diacetyl and acetoin accumulated at the highest levels (18.4 and 0.8 mg/L, respectively) with St-Lr, which suggests possible synergistic interactions between these microorganisms as well as the Lr capability of co-metabolizing citrate in the presence of lactose. Inulin stimulated both biomass growth and levels of all end-products, as the likely result of fructose release from its partial hydrolysis and subsequent metabolization as an additional carbon and energy source. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria

Abstract Background Bacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells. Results A lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched β-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu. Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 μg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces. Conclusion AMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Influence of growth media and temperature on bacterial adhesion to polystyrene surfaces

Bacterial adhesion to inert surfaces is a complex process influenced by environmental conditions. In this work, the influence of growth medium and temperature on the adhesion of Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Micrococcus luteus and Listeria monocytogenes to polystyrene surfaces was studied. Most bacteria demonstrated the highest adhesion when cultured in TSYEA, except S. marcescens, which showed to be positively influenced by the pigment production, favored in poor nutrient media (lactose and peptone agar). P. aeruginosa adhesion to polystyrene increased at low temperatures whatever the medium used. The culture medium influenced the surface properties of the bacteria as assessed by the MATS test.