Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

Monitoring temporal and spatial variation in rhizosphere bacterial population diversity: A community approach for the improved selection of rhizosphere competent bacteria

The potential for developing a reliable strategy for selecting rhizosphere competent bacteria, based on an improved understanding of the community diversity and population dynamics of fluorescent pseudomonads, was investigated. Isolates from a collection of over 690 fluorescent pseudomonads, obtained from sugar beet and wheat plants grown in field soils in laboratory microcosms, were genotypically and phenotypically characterised. RFLP rRNA analysis (ribotyping) revealed that the sampled population was composed of 385 related but distinct ribotypes. Most ribotypes were isolated only once and represented a transient colonising population. However, representatives of 26 ribotypes were detected more often, of which five were isolated from rhizosphere soils sampled 7 months after the first sampling. Comparative phenotypic analysis of isolates (motility, antibiotic resistance and production, adherence, fatty acid composition, substrate utilisation patterns) demonstrated that the ability to utilise organic acids as carbon sources correlated with rhizosphere competence. Single inoculum and competitive colonisation studies in planted microcosms confirmed rhizosphere competence, but also demonstrated synergistic interactions. The colonisation ability and population densities of transient strains were significantly increased when co-inoculated with rhizosphere competent isolates. These data demonstrate potential cross-feeding and combined niche exploitation, rather than direct competition, confirming the multi-factorial nature of rhizosphere competence in diverse fluorescent pseudomonad communities. They also highlight the need to consider the use of mixed inocula for plant growth promotion and the systematic selection of strains for effective biotechnological exploitation.     

Plant protection by the recombinant, root-colonizing Pseudomonas fluorescens F113rifPCB strain expressing arsenic resistance: improving rhizoremediation

AIMS: The present study was designed to evaluate the stable insertion and expression of an arsenic resistance operon in the rhizosphere competent, PCB degrading strain Pseudomonas fluorescens F113rifPCB (F113rifPCB) and to investigate its ability to protect plants from arsenic. METHODS AND RESULTS: Introduction of the clone pUM3 (arsRDABC) into F113rifPCB was carried out by triparental conjugation. The resultant arsenic resistant strain was screened through a number of phenotypic tests including ability to grow on biphenyl, its rhizosphere competence and plant protection potential. CONCLUSIONS: Insertion and expression of arsenic resistant operon arsRDABC (from plasmid R773) into F113rifPCB strain has allowed this strain to grow, colonize the root and degrade biphenyl (100 mmol l(-1)) in the presence of sodium arsenate concentrations of up to 11.5 mmol l(-1). The strain retains its ability to colonize the rhizosphere of plants and appears to provide seed germination protection to arsenic which is not seen by the wild type. SIGNIFICANCE AND IMPACT OF THE STUDY: Owing to the significantly improved growth characteristics of both this rhizobacterium and plant species, the use of F113rifPCB-ars endowed with arsenic resistance capabilities may be a promising strategy to remediate mixed organic metal-contaminated sites. These types of strain could be used in the inoculation of metal accumulation plants for phytoremediation.     

Fluorescence resonance energy transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil

Genetic analysis of the location of a mini-Tn5 promoted insertion of the LB400 bph operon in the rhizosphere coloniser Pseudomonas fluorescens F113rifPCB, allowed the development of a specific PCR detection system based on the unique DNA sequence at this insertion site. Real time PCR using both SYBR green chemistry and Fluorescence Resonance Energy Transfer probes allowed the precise identification of the recombinant strain and its quantitative detection in soil microcosms over a (bacteria/g) range of five orders of magnitude. This new assay can detect the genetically modified microorganism from soil in less than 90 min and at levels below the detection limits of standard PCR or cultivable counts on selective media.     

Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.     

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD ⁺ and of hcnAB ⁺ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD , restriction fragment length polymorphism profiling of the last phlD -positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.     

Isolation and characterization of polycyclic aromatic hydrocarbon-degrading bacteria associated with the rhizosphere of salt marsh plants

Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria were isolated from contaminated estuarine sediment and salt marsh rhizosphere by enrichment using either naphthalene, phenanthrene, or biphenyl as the sole source of carbon and energy. Pasteurization of samples prior to enrichment resulted in isolation of gram-positive, spore-forming bacteria. The isolates were characterized using a variety of phenotypic, morphologic, and molecular properties. Identification of the isolates based on their fatty acid profiles and partial 16S rRNA gene sequences assigned them to three main bacterial groups: gram-negative pseudomonads; gram-positive, non-spore-forming nocardioforms; and the gram-positive, spore-forming group, Paenibacillus. Genomic digest patterns of all isolates were used to determine unique isolates, and representatives from each bacterial group were chosen for further investigation. Southern hybridization was performed using genes for PAH degradation from Pseudomonas putida NCIB 9816-4, Comamonas testosteroni GZ42, Sphingomonas yanoikuyae B1, and Mycobacterium sp. strain PY01. None of the isolates from the three groups showed homology to the B1 genes, only two nocardioform isolates showed homology to the PY01 genes, and only members of the pseudomonad group showed homology to the NCIB 9816-4 or GZ42 probes. The Paenibacillus isolates showed no homology to any of the tested gene probes, indicating the possibility of novel genes for PAH degradation. Pure culture substrate utilization experiments using several selected isolates from each of the three groups showed that the phenanthrene-enriched isolates are able to utilize a greater number of PAHs than are the naphthalene-enriched isolates. Inoculating two of the gram-positive isolates to a marine sediment slurry spiked with a mixture of PAHs (naphthalene, fluorene, phenanthrene, and pyrene) and biphenyl resulted in rapid transformation of pyrene, in addition to the two- and three-ringed PAHs and biphenyl. This study indicates that the rhizosphere of salt marsh plants contains a diverse population of PAH-degrading bacteria, and the use of plant-associated microorganisms has the potential for bioremediation of contaminated sediments.     

Identification of Traits Shared by Rhizosphere-Competent Strains of Fluorescent Pseudomonads

Rhizosphere competence of fluorescent pseudomonads is a prerequisite for the expression of their beneficial effects on plant growth and health. To date, knowledge on bacterial traits involved in rhizosphere competence is fragmented and derived mostly from studies with model strains. Here, a population approach was taken by investigating a representative collection of 23 Pseudomonas species and strains from different origins for their ability to colonize the rhizosphere of tomato plants grown in natural soil. Rhizosphere competence of these strains was related to phenotypic traits including: (1) their carbon and energetic metabolism represented by the ability to use a wide range of organic compounds, as electron donors, and iron and nitrogen oxides, as electron acceptors, and (2) their ability to produce antibiotic compounds and N-acylhomoserine lactones (N-AHSL). All these data including origin of the strains (soil/rhizosphere), taxonomic identification, phenotypic cluster based on catabolic profiles, nitrogen dissimilating ability, siderovars, susceptibility to iron starvation, antibiotic and N-AHSL production, and rhizosphere competence were submitted to multiple correspondence analyses. Colonization assays revealed a significant diversity in rhizosphere competence with survival rates ranging from approximately 0.1?% to 61?%. Multiple correspondence analyses indicated that rhizosphere competence was associated with siderophore-mediated iron acquisition, substrate utilization, and denitrification. However, the catabolic profile of one rhizosphere-competent strain differed from the others and its competence was associated with its ability to produce antibiotics phenazines and N-AHSL. Taken together, these data suggest that competitive strains have developed two types of strategies to survive in the rhizosphere.     

A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs)

Abstract In this report we describe the development and construction of a DNA module which encodes bph genes for the metabolism of PCBs and which is capable of stable integration into the chromosome of Gram negative bacteria. Introduction of the bph -module into Pseudomonas putida KT2442, Pseudomonas sp. strain B13 and its genetically engineered derivative B13FR1 expanded the biodegradative ability of these strains to include biphenyl and 4-chlorobiphenyl. The bph operon was stably inherited under laboratory conditions. Behavior of the genetically engineered strains was evaluated under simulated natural habitat conditions in lake sediment microcosms with respect to survival and removal of 4-chlorobiphenyl. The genetically engineered strains persisted under these conditions and were effective in degrading 4-chlorobiphenyl over a five day incubation period.     

Degradation of PCB congeners by bacterial strains

Biological in situ methods are options for the remediation of contaminated sites. An approach to quantify biodegradation by soil bacteria was developed, combining experiment with mathematical modelling. We performed in vitro assays to investigate the potential and kinetics of the wild-type degrader, Burkholderia sp. strain LB400 (expressing bph) and the genetically modified Pseudomonas fluorescens strains F113pcb and F113L::1180 (expressing bph under different promoters) to metabolise individual congeners of polychlorinated biphenyls (PCBs). Kinetics of metabolism was analysed using the Monod model. Results revealed similar patterns of degradable PCB congeners for LB400 and F113L::1180. The degree of PCB degradation was comparable for LB400 and F113L::1180 but was much lower for F113rifpcb. In additional mesocosm experiments with PCB-contaminated soil, the F113 derivatives demonstrated a good survival ability in willow (Salix sp.) rhizosphere. Strain F113L::1180 in combination with willow plants is expected to degrade a large spectrum of PCB congeners in soil. The data from the experiments were used to calculate the time scale of the degradation process in a PCB-contaminated soil. The uncertainty of the model predictions due to the uncertainties of experimental removal velocities and bacterial cell density in soil was quantified.     

The dual functions of biphenyl-degrading ability of Pseudomonas sp. KKS102: energy acquisition and substrate detoxification

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.     

The interconnected rhizosphere: High network complexity dominates rhizosphere assemblages

While interactions between roots and microorganisms have been intensively studied, we know little about interactions among root‐associated microbes. We used random matrix theory‐based network analysis of 16S rRNA genes to identify bacterial networks associated with wild oat (Avena fatua) over two seasons in greenhouse microcosms. Rhizosphere networks were substantially more complex than those in surrounding soils, indicating the rhizosphere has a greater potential for interactions and niche‐sharing. Network complexity increased as plants grew, even as diversity decreased, highlighting that community organisation is not captured by univariate diversity. Covariations were predominantly positive (> 80%), suggesting that extensive mutualistic interactions may occur among rhizosphere bacteria; we identified quorum‐based signalling as one potential strategy. Putative keystone taxa often had low relative abundances, suggesting low‐abundance taxa may significantly contribute to rhizosphere function. Network complexity, a previously undescribed property of the rhizosphere microbiome, appears to be a defining characteristic of this habitat.     

Role of ptsP, orfT, and sss recombinase genes in root colonization by Pseudomonas fluorescens Q8r1-96

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.     

Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild-type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild-type F113 strain carried a gene encoding the production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG-negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild-type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY-6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic-producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot-to-root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β-glucosidase, β-galactosidase and N -acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).     

Mutational Disruption of the Biosynthesis Genes Coding for the Antifungal Metabolite 2,4-Diacetylphloroglucinol Does Not Influence the Ecological Fitness of Pseudomonas fluorescens F113 in the Rhizosphere of Sugarbeets

The ability of Pseudomonas fluorescens F113 to produce the antibiotic 2,4-diacetylphloroglucinol (DAPG) is a key factor in the biocontrol of the phytopathogenic fungus Pythium ultimum by this strain. In this study, a DAPG-producing strain (rifampin-resistant mutant F113Rif) was compared with a nearly isogenic DAPG-negative biosynthesis mutant (Tn5::lacZY derivative F113G22) in terms of the ability to colonize and persist in the rhizosphere of sugarbeets in soil microcosms during 10 plant growth-harvest cycles totaling 270 days. Both strains persisted similarly in the rhizosphere for 27 days, regardless of whether they had been inoculated singly onto seeds or coinoculated in a 1:1 ratio. In order to simulate harvest and resowing, the roots were removed from the soil and the pots were resown with uninoculated sugarbeet seeds for nine successive 27-day growth-harvest cycles. Strains F113Rif and F113G22 performed similarly with respect to colonizing the rhizosphere of sugarbeet, even after nine cycles without reinoculation. The introduced strains had a transient effect on the size of the total culturable aerobic bacterial population. The results indicate that under these experimental conditions, the inability to produce DAPG did not reduce the ecological fitness of strain F113 in the rhizosphere of sugarbeets.     

Carbon fractions in the rhizosphere of pea inoculated with 2,4 diacetylphloroglucinol producing and non-producing Pseudomonas fluorescens F113

The aim of this work was to determine the effect of wild type and functionally modified Pseudomonas fluorescens strains on C fractions in the rhizosphere of pea. The lac ZY marked F113 strain produces the antibiotic 2,4 diacetylphloroglucinol (DAPG) useful in plant disease control. The modified strain of F113 was represented in production of DAPG, creating the DAPG negative strain F113 G22. The F113 treatment resulted in a significantly lower shoot/root ratio. The F113 G22 treatment had a significantly greater indigenous and total fluorescent Pseudomonas population than the control and F113 (DAPG₊) treatment. Both strains significantly increased the water soluble carbohydrates and the total water soluble carbon in the pea rhizosphere soil. Strain F113 significantly increased the soil protein content relative to the control, but not in relation to the F113 G22 treatment. The F113 treatment had a significantly greater organic acid content than the control and F113 G22 treatments, whilst the F113 G22 treatment was also significantly greater than the control. Both inocula resulted in significantly lower phosphate contents than the control. The F113 inocula significantly increased alkaline phosphatase, sulphatase and urease activities, and reduced β glucosidase activities indicating increased carbon availability. Both inocula increased C availability ; however, antibiotic production by strain F113 reduced the utilisation of organic acids released from the plant resulting in differing effects of the two strains on nutrient availability, plant growth, soil enzyme activities and Pseudomonas populations.     

In vitro antagonists of Rhizoctonia solani tested on lettuce: rhizosphere competence, biocontrol efficiency and rhizosphere microbial community response

The rhizosphere competence of 15 in vitro antagonists of Rhizoctonia solani was determined 4 weeks after sowing inoculated lettuce seeds into nonsterile soil. Based on the colonization ability determined by selective plating, eight strains were selected for growth chamber experiments to determine their efficacy in controlling bottom rot caused by R. solani on lettuce. Although in the first experiment all antagonists colonized the rhizosphere of lettuce with CFU counts above 2 × 10⁶ g⁻¹ of root fresh weight, only four isolates significantly reduced disease severity. In subsequent experiments involving these four antagonists, only Pseudomonas jessenii RU47 showed effective and consistent disease suppression. Plate counts and denaturing gradient gel electrophoresis (DGGE) fingerprints of Pseudomonas -specific gacA genes amplified from total community DNA confirmed that RU47 established as the dominant Pseudomonas population in the rhizosphere of inoculated lettuce plants. Furthermore, the DGGE fingerprint revealed that R. solani AG1-IB inoculation severely affected the bacterial and fungal community structure in the rhizosphere of lettuce and that these effects were much less pronounced in the presence of RU47. Although the exact mechanism of antagonistic activity and the ecology of RU47 remain to be further explored, our results suggest that RU47 is a promising agent to control bottom rot of lettuce.     

Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113

Aims: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. Methods and Results: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113‐based biosensor strains were encapsulated, and their ability to detect 3‐chlorobenzoate (3‐CBA) and 3‐chlorobiphenyl (3‐CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro‐organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3‐CBA or 3‐CBP degradation. In fact, detection of 3‐CBP degradation was enhanced in encapsulated biosensors. Conclusions: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. Significance and Impact of the Study: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.