Rhizosphere competence of fluorescent Pseudomonas sp. B24 genetically modified to utilise additional ferric siderophores

Abstract: The ability to utilise additional siderophores may increase the ecological fitness of biocontrol inoculants of Pseudomonas in the rhizosphere. Plasmid pCUP2 carries a copy of the gene pbu A coding for the membrane receptor of ferric pseudobactin M114. Pseudomonas sp. B24Rif containing pCUP2 can utilise ferric pseudobactin of P. fluorescens M114 in addition to its own siderophore. A larger fraction of the culturable resident fluorescent pseudomonads in the rhizosphere of sugarbeet grown in a low-iron sandy loam soil could supply siderophore-complexed iron to B24Rif(pCUP2) rather than to B24Rif. However, B24Rif and B24Rif(pCUP2) were found at similar population levels in the rhizosphere for 21 days after their inoculation on seeds. A total of 25 of 43 isolates of resident fluorescent Pseudomonas unable to cross-feed iron to B24Rif could cross-feed B24Rif(pCUP2) and they were subdivided into seven different strains by arbitrary-primed PCR fingerprinting. The siderophores produced by 11 of them were typed by HPLC and they were similar to pseudobactin M114. However, the ability to utilise ferric pseudobactin M114 did not improve the ecological fitness of B24Rif in the rhizosphere of sugarbeet although a larger fraction of the culturable resident fluorescent pseudomonads could supply pseudobactin M114-complexed iron to B24Rif(pCUP2) than to B24Rif.     

Effect of 2,4-Diacetylphloroglucinol Producing,Overproducing, and Nonproducing Pseudomonas fluorescens F113 in the Rhizosphere of Pea

Pseudomonas fluorescens F113lacZY and modified strains carrying different function modifications were assessed for their impact in the rhizosphere of pea. Strain F113lacZY naturally produces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl) useful in plant disease control. The first modified strain of F113 was repressed in production of Phl, creating the Phl negative strain F113G22. The second was a plasmid based overproducer of Phl (F113Rif (pCUGP)). Both the F113lacZY and the F113Rif (pCUGP) strains increased the rhizoplane fungal populations, whereas the same strains reduced the rhizosphere soil fungal populations with respect to the control. Similar results were found with the rhizoplane and rhizosphere soil bacterial populations. The F113G22 treatment resulted in a significantly greater indigenous fluorescent Pseudomonas population than the F113lacZY and F113Rif (pCUGP) treatments and a greater total Pseudomonas population than the control, F113lacZY, and F113Rif (pCUGP) treatments. Overproduction of Phl did not affect the establishment of the introduced Pseudomonas population. None of the inocula displaced the indigenous populations, but the F113G22 inocula had an additive effect on the total Pseudomonas population. P (phosphatase), S (sulphatase), and N (urease) cycle enzyme activities were increased while C (glucosidase, NAGase) cycle activities were decreased by the F113lacZY and F113Rif (pCUGP) treatments, suggesting C leakage from the roots. Overall, most effects of inoculation compared to the wild type were found with the non-Phl-producing strain. Overproduction of Phl had little environmental effect in relation to wild-type inocula.     

Residual Impact of the Biocontrol Inoculant Pseudomonas fluorescens F113 on the Resident Population of Rhizobia Nodulating a Red Clover Rotation Crop

A field trial was previously conducted in which sugarbeet seeds were either untreated, inoculated with the biocontrol strain Pseudomonas fluorescens F113Rif, or treated with chemical fungicides. Following harvest of sugarbeet, the field site was sown with uninoculated red clover. The aim of this study was to assess the residual impact of the microbial inoculant (and the fungicide treatment) on the diversity of resident rhizobia nodulating the red clover rotation crop. The percentage of nodules yielding rhizobial isolates after surface disinfection was 67% in the control and 70% in the P. fluorescens F113Rif treatment, but only 23% in the chemical treatment. Isolates were characterized by RAPD analysis. The main RAPD cluster (arbitrarily defined at 70% similarity) was prevalent in all three treatments. In addition, the distribution of RAPD clusters followed a log series model, regardless of the treatment applied, indicating that neither the microbial inoculant nor the fungicide treatment had caused a strong perturbation of the rhizobial population. When the P. fluorescens F113Rif and control treatments were compared using diversity indices, however, it appeared that the genetic diversity of rhizobia was significantly less in the inoculated treatment. The percentage of rhizobia sensitive to 2,4-diacetylphloroglucinol (Phl; the antimicrobial metabolite produced by P. fluorescens F113Rif) fluctuated according to field site heterogeneity, and treatments had no effect on this percentage. Yet, the proportion of Phl-sensitive isolates in the main RAPD cluster was lower in the P. fluorescens F113Rif treatment compared with the control, raising the possibility that the residual impact of the inoculant could have been partly mediated by production of Phl. This impact on the rhizobial population took place without affecting the functioning of the Rhizobium–clover symbiosis.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Assessment of genotypic diversity of antibiotic-producing pseudomonas species in the rhizosphere by denaturing gradient gel electrophoresis

The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD(+) reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD(+) genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD(+) genotypes directly in rhizosphere samples with a detection limit of approximately 5 x 10(3) CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD(+) genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD(+) isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD(+) genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD(+) genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.     

Construction of a rhizosphere pseudomonad with potential to degrade polychlorinated biphenyls and detection of bph gene expression in the rhizosphere.

The genetically engineered transposon TnPCB, contains genes (bph) encoding the biphenyl degradative pathway. TnPCB was stably inserted into the chromosome of two different rhizosphere pseudomonads. One genetically modified strain, Pseudomonas fluorescens F113pcb, was characterized in detail and found to be unaltered in important parameters such as growth rate and production of secondary metabolites. The expression of the heterologous bph genes in F113pcb was confirmed by the ability of the genetically modified microorganism to utilize biphenyl as a sole carbon source. The introduced trait remained stable in laboratory experiments, and no bph-negative isolates were found after extensive subculture in nonselective media. The bph trait was also stable in nonselective rhizosphere microcosms. Rhizosphere competence of the modified F113pcb was assessed in colonization experiments in nonsterile soil microcosms on sugar beet seedling roots. F113pcb was able to colonize as efficiently as a marked wild-type strain, and no decrease in competitiveness was observed. In situ expression of the bph genes in F113pcb was found when F113pcb bearing a bph'lacZ reporter fusion was inoculated onto sugar beet seeds. This indicates that the bph gene products may also be present under in situ conditions. These experiments demonstrated that rhizosphere-adapted microbes can be genetically manipulated to metabolize novel compounds without affecting their ecological competence. Expression of the introduced genes can be detected in the rhizosphere, indicating considerable potential for the manipulation of the rhizosphere as a self-sustaining biofilm for the bioremediation of pollutants in soil. Rhizosphere bacteria such as fluorescent Pseudomonas spp. are ecologically adapted to colonize and compete in the rhizosphere environment. Expanding the metabolic functions of such pseudomonads to degrade pollutants may prove to be a useful strategy for bioremediation.     

Analysis of rhizobacterial communities in perennial Graminaceae from polluted water meadow soil, and screening of metal-resistant, potentially plant growth-promoting bacteria

This study investigates the impact of long-term heavy metal contamination on the culturable, heterotrophic, functional and genetic diversity of rhizobacterial communities of perennial grasses in water meadow soil. The culturable heterotrophic diversity was investigated by colony appearance on solid LB medium. Genetic diversity was measured as bands in denaturing gradient gel electrophoresis (DGGE) obtained directly from rhizosphere soil and rhizoplane DNA extracts, and from the corresponding culturable communities. In the two rhizospheric fractions the DGGE profiles of the direct DNA extracts were similar and stable among replicates, whereas in the enriched cultures the profiles of the fractions differed, but among the replicates they were similar. One hundred isolates were collected into 33 different operational taxonomic units by use of amplified internal transcribed spacers and into 19 heavy metal-resistant phenotypes. The phylogenetic position of strains belonging to 18 operational taxonomic units, representing more than 80% of the isolates, was determined by 16S rRNA gene sequencing. Several heavy metal-resistant strains were isolated from rhizoplane. Finally, metal-resistant rhizobacteria were tested for plant growth-promoting characteristics; some were found to contain 1-aminocyclopropane-1-carboxylic acid deaminase and/or to produce indole acetic acid and siderophores. Two strains resistant to cadmium and zinc, Pseudomonas tolaasii RP23 and Pseudomonas fluorescens RS9, had all three plant growth-promoting characteristics. Our findings suggest that bacteria can respond to soil metal contamination, and the described methodological approach appears promising for targeting potential plant growth-promoting rhizobacteria.     

Decreased abundance and diversity of culturable Pseudomonas spp. populations with increasing copper exposure in the sugar beet rhizosphere

Recent studies have indicated that culturable bacteria constitute highly sensitive bioindicators of metal-induced stress in soil. We report the impact of different copper exposure levels characteristic of contaminated agricultural soils on culturable Pseudomonas spp. in the rhizosphere of sugar beet. We observed that the abundance of Pseudomonas spp. was much more severely affected than that of the general population of culturable heterotrophic bacteria by copper. For diversity assessment, Pseudomonas isolates were divided into operational taxonomic units based on amplified ribosomal DNA restriction analysis and genomic PCR fingerprinting by universally primed PCR. Copper significantly decreased the diversity of Pseudomonas spp. in the rhizosphere and significantly increased the frequency of copper-resistant isolates. Concomitant chemical and biological analysis of copper in the rhizosphere and in bulk soil extracts indicated no rhizosphere effect and a relatively low copper bioavailability in the studied soil, suggesting that the observed effects of copper may occur at lower total concentrations in other soils. We conclude that culturable Pseudomonas sensu stricto constitutes a highly sensitive and relevant bioindicator group for the impact of copper in the rhizosphere habitat, and suggest that continued application of copper to agricultural soils poses a significant risk to successful rhizosphere colonization by Pseudomonas spp.     

Diversity of root-associated fluorescent pseudomonads as affected by ferritin overexpression in tobacco

A transgenic tobacco overexpressing ferritin (P6) was recently shown to accumulate more iron than the wild type (WT), leading to a reduced availability of iron in the rhizosphere and shifts in the pseudomonad community. The impact of the transgenic line on the community of fluorescent pseudomonads was assessed. The diversity of 635 isolates from rhizosphere soils, rhizoplane + root tissues, and root tissues of WT and P6, and that of 98 isolates from uncultivated soil was characterized. Their ability to grow under iron stress conditions was assessed by identifying their minimal inhibitory concentrations of 8-hydroxyquinoline for each isolate, pyoverdine diversity by isoelectrofocusing and genotypic diversity by random amplified polymorphism DNA. The antagonistic activity of representative isolates and of some purified pyoverdines against a plant pathogen (Pythium aphanidermatum Op4) was tested in vitro. In overall, isolates taken from P6 tobacco showed a greater ability to grow in iron stress conditions than WT isolates. The antagonism by some of the representative isolates was only expressed under iron stress conditions promoting siderophore synthesis and their pyoverdines appeared to have a specific structure as assessed by mass spectrometry. For other isolates, antagonism was still expressed in the presence of iron, suggesting the involvement of metabolites other than siderophores. Altogether, these data indicate that the transgenic tobacco that over-accumulates iron selected fluorescent pseudomonads, less susceptible to iron depletion and more antagonistic to the tested plant pathogen than those selected by the tobacco WT.     

Effects of soil compaction on the microbial populations of melon and maize rhizoplane

Effects of soil compaction on the microbial populations of melon and maize rhizoplane were investigated in quantity and quality. The numbers of culturable bacteria and fluorescent pseudomonads on the rhizoplane were higher when plants were grown in more compacted soil and the relative increase was larger in fluorescent pseudomonads. Total bacterial counts, however, did not appear to be affected by soil compaction, resulting in the increase in the culturable bacteria among total counts in more compacted soil. The determination of extracellular enzymatic properties (pectinase, -glucosidase, -glucosidase and -galactosidase) of each 100 isolates from bulk soil and root samples suggested that the microbial populations on the rhizoplane, especially when plants were grown in highly, compacted soil, were composed of high ratios of bacteria with abilities to utilize root exudates efficiently. The microbial community structure estimated from the colony forming curves of bulk soil and root samples suggested that the microbial populations on the rhizoplane, especially when plants were grown in compacted soil, were likely to be composed of more r-strategists which were defined as those who formed colonies within 2 days.     

Influence of plant species on population dynamics, genotypic diversity and antibiotic production in the rhizosphere by indigenous Pseudomonas spp

The population dynamics, genotypic diversity and activity of naturally-occurring 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. was investigated for four plant species (wheat, sugar beet, potato, lily) grown in two different soils. All four plant species tested, except lily and in some cases wheat, supported relatively high rhizosphere populations (5 x 10(4) to 1 x 10(6) CFU/g root) of indigenous DAPG-producing Pseudomonas spp. during successive cultivation in both a take-all suppressive and a take-all conducive soil. Although lily supported on average the highest population densities of fluorescent Pseudomonas spp., it was the least supportive of DAPG-producing Pseudomonas spp. of all four plant species. The genotypic diversity of 492 DAPG-producing Pseudomonas isolates, assessed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the phlD gene, revealed a total of 7 genotypes. Some of the genotypes were found only in the rhizosphere of a specific plant, whereas the predominant genotypes were found at significantly higher frequencies in the rhizosphere of three plant species (wheat, sugar beet and potato). Statistical analysis of the phlD(+) genotype frequencies showed that the diversity of the phlD(+) isolates from lily was significantly lower than the diversity of phlD(+) isolates found on wheat, sugar beet or potato. Additionally, soil type had a significant effect on both the phlD(+) population density and the phlD(+) genotype frequencies, with the take-all suppressive soil being the most supportive. HPLC analysis further showed that the plant species had a significant effect on DAPG-production by the indigenous phlD(+) population: the wheat and potato rhizospheres supported significantly higher amounts of DAPG produced per cell basis than the rhizospheres of sugar beet and lily. Collectively, the results of this study showed that the host plant species has a significant influence on the dynamics, composition and activity of specific indigenous antagonistic Pseudomonas spp.     

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.     

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.     

Genotypic and phenotypic diversity of PGPR fluorescent pseudomonads isolated from the rhizosphere of sugarcane (Saccharum officinarum L.)

The genetic diversity of plant growth-promoting rhizobacterial (PGPR) fluorescent pseudomonads associated with the sugarcane (Saccharum officinarum L.) rhizosphere was analyzed. Selected isolates were screened for plant growthpromoting properties including production of indole acetic acid, phosphate solubilization, denitrification ability, and production of antifungal metabolites. Furthermore, 16S rDNA sequence analysis was performed to identify and differentiate these isolates. Based on 16S rDNA sequence similarity, the isolates were designated as Pseudomonas plecoglossicida, P. fluorescens, P. libaniensis, and P. aeruginosa. Differentiation of isolates belonging to the same group was achieved through different genomic DNA fingerprinting techniques, including randomly amplified polymorphic DNA (RAPD), amplified ribosomal DNA restriction analysis (ARDRA), repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and bacterial repetitive BOX elements (BOX) analyses. The genetic diversity observed among the isolates and rep-PCR-generated fingerprinting patterns revealed that PGPR fluorescent pseudomonads are associated with the rhizosphere of sugarcane and that P. plecoglossicida is a dominant species. The knowledge obtained herein regarding the genetic and functional diversity of fluorescent pseudomonads associated with the sugarcane rhizosphere is useful for understanding their ecological role and potential utilization in sustainable agriculture.     

Identification of conserved traits in fluorescent pseudomonads with antifungal activity

A collection of 29 fluorescent pseudomonads, some with known biological control activity against a range of phytopathogenic fungi, were characterized phenotypically and genotypically by comparing carbon source utilization patterns, suppression of Pythium ultimum both in planta and in vitro and the potential to produce known secondary metabolites. Fatty acid profiling and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA operon (ribotyping) were used to determine the diversity of isolates. A small group of genetically related Pseudomonas spp. with similar properties was identified; each isolate produced a diffusible bioactive product in vitro and was active against Pythium ultimum in planta . However, other isolates that were able to suppress damping off disease but did not inhibit hyphal extension in vitro clustered outside this group. Phenotypic analyses revealed that the accumulation of C17:0 cyclopropane fatty acid (17CFA) and the production of hydrogen cyanide correlated significantly with biological control activity and with the antagonism of fungal development. The potential of 17CFA as a marker for the selection of fluorescent pseudomonads with biocontrol agent (BCA) potential was demonstrated by the isolation of a novel active strain. This was selected after the screening of 13 clonal groups of fluorescent pseudomonads identified from 500 isolates from the phytosphere of sugar beet. Levels of 17CFA synthesis possibly reflect the efficacy of the rpoS allele in particular strains.     

Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113

Sugarbeet seeds used by farmers are often pelleted using an EB^TM-based mix. During the pelleting process, the seeds are dried immediately after application of the mix. In this work, the effects of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113Rif were investigated using a 1:1 EB^TM/vermiculite mix and sugarbeet seeds pelleted with this material. Growing F113Rif for 3 d (28 °C) within the EB^TM/vermiculite mix amended with nutrients (sucrose asparagine broth), instead of adding the cells to the unamended mix immediately before drying the mix or the pelleted sugarbeet seeds, resulted in improved survival of the strain in the mix or on the seeds, respectively, during subsequent storage. A slower drying (20 h instead of 3 h) of the F113Rif-inoculated EB^TM/vermiculite mix to 11% w/w water content enhanced strain survival in the mix during storage, but the drying conditions studied had no effect on inoculant survival on the seed during storage when pelleted seeds were dried to 10% w/w water content. Biological control of damping-off disease of sugarbeet (caused by Pythium spp.) in soil microcosms was achieved when F113Rif was inoculated in the unamended mix 3 d before pelleting the seeds, but not when nutrient-amended mix was used. Inoculum preparation and drying of the formulation are key factors to consider when optimizing the use of a commercial EB^TM/vermiculite seed formulation for delivery of a biocontrol Pseudomonas inoculant.     

Effect of sorghum seedlings, and previous crop, on soil fluorescent Pseudomonas spp.

Hypotheses in which sorghum seedlings [Sorghum bicolor (L.) Moench] of different genotypes will differentially modify soil microorganisms and will affect subsequent planting of wheat (Triticum aestivum L.) seedlings, were tested. Wheat cultivar Lewjain, and sorghum genotypes Redlan and RTx433, were planted into soils previously planted with wheat or sorghum in growth chamber experiments. Total culturable fungi and oomycetes, and fluorescent Pseudomonas spp. numbers (cfu) were determined. Pseudomonads were screened for hydrogen cyanide (HCN) production, for the presence of the phlD gene for 2,4-diacetylphloroglucinol production (Phl) and for a region of the operon involved in phenazine-1-carboxylic acid (PCA) production. Pasteurized soils were inoculated with rifampicin-marked strains of Pseudomonas fluorescens then planted with Lewjain, Redlan and RTx433 to assess rhizosphere and soil colonization. Effects of plant species, sorghum genotype and previous crop on culturable fungi and oomycetes, and pseudomonad numbers (cfu g^?1 soil) were statistically significant. Soils planted with RTx433 or Lewjain had greater numbers of fungal cfu than soils planted with Redlan. When Lewjain seedlings were grown in soil previously planted with RTx433, there were greater numbers of fungal cfu than when Lewjain was planted into Redlan soil. Wheat planted into wheat soil resulted in statistically significantly fewer numbers of pseudomonads than when planted into sorghum soil. Overall, percentages of HCN-producing pseudomonads increased, especially when wheat seedlings were planted in wheat soil. For most treatments, percent of isolates with Phl declined, except when Redlan was planted into Redlan soil, which resulted in increased Phl isolates. When rifampicin-marked P. fluorescens isolates were applied to pasteurized soil, sorghum seedlings sustained rhizosphere and soil populations similar to those on wheat. Sorghum genotypes may differ in associations with soil microorganisms, suggesting that they may differentially affect numbers of fluorescent pseudomonads in cropping systems.     

Phylogenetic diversity of fluorescent pseudomonads in agricultural soils from Korea

AIMS: To identify and compare the relative diversity and distribution of genotypes of culturable fluorescent pseudomonads from soils. METHODS AND RESULTS: Analysis of 160 isolates from seven soil samples using randomly amplified polymorphism DNA methods revealed 53 genotypes, which were subsequently identified by their 16S ribosomal DNA sequences. Phylogenetic analyses of the 53 genotypes along with 43 fluorescent pseudomonad type strains separated the genotypes into 10 distinct clusters that included two phylogenetic groups that were not represented by previously described type strains. CONCLUSIONS: The diversity of genotypes that was obtained from the soil samples was highly variable among the different soils and appeared to be associated with different soil management practices that also influence plant yields. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and phylogenetic analysis of these genotypes offers opportunities for study of phenotypic traits that may be associated within taxonomically related groups of fluorescent pseudomonad species and how these groups vary in relation to soil management practices.     

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis.