Effect of genetically modified Pseudomonas fluorescens strains on the uptake of nitrogen by pea from 15N enriched organic residues

The effects of an antibiotic-producing Pseudomonas fluorescens strain (F113) carrying the marker gene cassette lac ZY and a marked, non-producing strain (F113G22) on the uptake of nitrogen from ¹⁵N enriched organic residues incorporated into a sandy soil were investigated in microcosm studies. Strain F113 produces the antibiotic 2,4-diacetylphloroglucinol (DAPG), while its modified derivative strain F113G22 has DAPG production deleted by Tn 5 mutagenesis. Uptake of nitrogen by pea ( Pisum sativum ) was estimated using isotope-ratio mass spectrometry. In addition, plant growth and microbial activity in soil were monitored. Both strains F113 and F113G22 enhanced the uptake of nitrogen from mineralized organic residues, even though the antibiotic producing strain F113 significantly reduced microbial activity in soil. It is suggested that the effect on nitrogen uptake was due to increased mineralization of organic residues by the introduced organisms, making greater quantities of inorganic nitrogen available for plant uptake. Unlike studies assessing impact in terms of perturbation to indigenous microbial communities, this study provides direct evidence of a change in ecosystem function as a result of the introduction of strains of a genetically marked bacterium, irrespective of whether its natural antibiotic-producing capacity has been genetically deleted.     

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.     

Effect of Introduced Pseudomonas fluorescens Strains on Soil Nematode and Protozoan Populations in the Rhizosphere of Wheat and Pea

Abstract Previous studies have shown that inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 increased mineralization of organic nitrogen in the rhizosphere. In contrast, inoculation of the same strains onto wheat seeds reduced mineralization of N from organic residues incorporated into soil. In the present study, we report on a likely explanation of this phenomenon, which appears to be governed by the effect of plant-microbe interactions on bacterial-feeding nematodes and protozoa. In soil microcosm tests, inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 resulted in an increase in the number of nematodes and protozoa in the rhizosphere as compared to noninoculated controls. This trend was repeated using a model sand system into which the bacteriophagous nematode Caenorhabditis elegans was introduced. It was subsequently found that non-inoculated germinating pea seeds exerted a nematicidal effect on C. elegans, which was remedied by inoculation with either strain F113lacZY or F113G22. This suggests that nematicidal compounds released by the germinating pea seeds were metabolized by the microbial inoculants before they affected nematode populations in the spermosphere or rhizosphere of pea. In contrast, inoculation of wheat plants resulted in significantly lower nematode populations in the rhizosphere, whereas protozoan numbers were unaffected. No nematicidal effects of inoculated or noninoculated wheat seeds could be found, suggesting that microfaunal populations were affected at a later stage during plant growth. Because of their key roles in accelerating the turnover of microbially immobilized N and organic matter, plants that support a larger microfaunal population are likely to benefit from a higher availability of inorganic nitrogen. Therefore, an understanding of plant-microbe interactions and their effects on soil microfaunal populations is essential in order to assess the effects of microbial inocula on plant mineral nutrition. Received: 27 May 1999; Accepted: 15 July 1999; Online Publication: 17 December 1999     

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.     

Impact on Arbuscular Mycorrhiza Formation of Pseudomonas Strains Used as Inoculants for Biocontrol of Soil-Borne Fungal Plant Pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 μM DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.     

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.     

Isolation of 2,4-Diacetylphloroglucinol from a Fluorescent Pseudomonad and Investigation of Physiological Parameters Influencing Its Production

Pseudomonas sp. strain F113 was isolated from the rhizosphere of sugar beets and shown to inhibit a range of plant pathogenic fungi by producing an antibioticlike compound. An antibiotic-negative mutant strain, F113G22, was generated by transposon mutagenesis. This mutant has lost the ability to inhibit both bacterial and fungal microorganisms on high-iron medium. The antibioticlike compound was subsequently identified as 2,4-diacetylphloroglucinol (DAPG), and a high-pressure liquid chromatographic assay was developed for to detect it quantitatively in growth culture media and soil. The growth temperature had a direct bearing on DAPG production by strain F113, with maximum production at 12°C. The iron concentration, pH, and oxygen had no influence on DAPG production by strain F113 under the assay conditions used. However, a low ratio of culture volume to surface area available to the microbe in the growth container was critical for optimum DAPG production. Different types of carbon sources influenced DAPG production by strain F113 to various degrees. For example, sucrose, fructose, and mannitol promoted high yields of DAPG by strain F113, whereas glucose and sorbose resulted in very poor DAPG production.     

Role of 2,4-Diacetylphloroglucinol in the Interactions of the Biocontrol Pseudomonad Strain F113 with the Potato Cyst Nematode Globodera rostochiensis

The potato cyst nematode Globodera rostochiensis is an important pest of potato (Solanum tuberosum). Pseudomonas fluorescens F113, which produces 2,4-diacetylphloroglucinol (DAPG), was investigated as a potential biocontrol agent against G. rostochiensis. Exposure of nematode cysts to the pseudomonad, under in vitro conditions or in soil microcosms, almost doubled the ability of the eggs to hatch. The percentage of mobile juveniles was reduced threefold following their incubation in the presence of the pseudomonad, both in vitro and in soil. Results obtained with a transposon-induced DAPG-negative biosynthetic mutant of F113 and its complemented derivative with restored DAPG synthesis showed that the ability of strain F113 to produce DAPG was responsible for the increase in hatch ability and the reduction in juvenile mobility. Similar effects on egg hatch ability and juvenile mobility of G. rostochiensis were obtained in vitro by incubating nematode cysts and juveniles, respectively, in the presence of synthetic DAPG. DAPG-producing P. fluorescens F113 is proposed as a potential biocontrol inoculant for the protection of potato crops against the potato cyst nematode.     

Effects of Pseudomonas fluorescens F113 on Ecological Functions in the Pea Rhizosphere Are Dependent on pH

Abstract The aim of this microcosm study was to determine influence of the antibiotic 2,4-diacetylphloroglucinol (DAPG) on the effect of wild-type and functionally modified Pseudomonas fluorescens F113 strains in a sandy loam soil of pH 5.4 planted with pea (Pisum sativum var Montana). The functional modification of strain F113 was a repressed production of DAPG, useful in plant disease control, creating the DAPG negative strain F113 G22; both were marked with a lacZY gene cassette. Lowering the soil pH to 4.4 significantly reduced the plant shoot and root weights and the root length, whereas the bacterial inocula had no significant effect. Both inocula significantly reduced the shoot/root ratio at pH 5.4, but this effect was not evident at the lowered or elevated (6.4) pH levels. The decrease in pH significantly increased the fungal and yeast colony-forming units from the rhizosphere (root extract), but did not affect the total bacterial c.f.u.'s. Inoculatioin with strain F113 in the pH 4.4 soil resulted in a significantly greater total bacterial population. The fungal and yeast c.f.u.'s were not significantly affected by the inocula at any pH studied. Increasing the pH significantly increased the indigenous Pseudomonas population in comparison to the reduced pH treatment and significantly increased both the introduced and total Pseudomonas populations. The antibiotic producing strain significantly reduced the total bacterial population and the NAGase activity (related to fungal activity) at pH 6.4 where the inocula population was the greatest. Alkaline phosphatase, phosphodiesterase, aryl sulfatase, β-glucosidase, alkaline β-galactosidase, and NAGase activities significantly increased with increasing in pH. The F113 inocula reduced the acid phosphatase activity at pH 5.4 and increased the acid β-galactosidase activity over all the pH treatments. The results presented illustrate the variation in impact with soil pH, with implications for variability in efficacy of Pseudomonas fluorescens biocontrol agents with soil pH. Received: 26 June 1998; Accepted: 1 February 1999     

Fusaric acid-producing strains of Fusarium oxysporum alter 2,4-diacetylphloroglucinol biosynthetic gene expression in Pseudomonas fluorescens CHA0 in vitro and in the rhizosphere of wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA'-'lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA'-'lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA(+)) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA(-)) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion

During evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-β-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P. fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR.     

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA⁺) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA⁻) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

Microorganisms in the rhizosphere of wheat colonized by the fungusGaeumannomyces graminis var.tritici

The population of microorganisms in wheat rhizosphere changed in the presence of the fungus Gaeumannomyces graminis var. tritici causing the take-all of wheat. In the majority of cases when the soil was artificially contaminated by the fungus, both the number of bacteria in the rhizosphere and the bacteria/fungi ratio temporarily increased. At the beginning bacteria growing in the presence of NH4+ predominated, later bacteria utilizing organic N-substances prevailed. Pseudomonas fluorescens and the related species colonized the rhizosphere and the soil to a greater extent in the presence of G. graminis. The wheat rhizosphere with G. graminis was found to contain a higher level of the slime-producing bacterium Agrobacterium spp.; this microorganism occurred on hyphal surfaces (in hyphosphere) of both G. graminis growing in soil and Mucor spp. Changes in microbial populations in the wheat rhizosphere during the first stage of colonization by G. graminis can be partly explained by a simultaneous rhizosphere colonization by microorganisms which accompany this fungus in soil. In the period of increase in the number of bacteria in rhizosphere a temporary stimulation of wheat growth was observed.     

Plant mediated interactions between Pseudomonas fluorescens, Rhizobium leguminosarum and arbuscular mycorrhizae on pea

Using undisturbed sandy loam soil cores heavily infested with mycorrhizae, the effects of the antibiotic-producing Pseudomonas fluorescens strain F113 and its non-antibiotic derivative Ps. fluorescens F113G22 on nodulation by introduced and indigenous Rhizobium were studied. Furthermore, the effects of the different microbial inocula on the colonization of the pea roots by mycorrhizae were studied. It was found that Ps. fluorescens F113 enhanced nodulation by Rhizobium fourfold, while the nodules produced were much larger and strongly pigmented (pink) compared with those in other treatments. The proportion of roots colonized by arbuscular mycorrhizae was not significantly affected by the different treatments.     

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.     

Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P. fluorescens strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed(-1) or soil(-1)) to establish high rhizosphere population densities (10(7) CFU g of root(-1)) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10(5) CFU g of root(-1) after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Impact of Field Release of Genetically Modified Pseudomonas fluorescens on Indigenous Microbial Populations of Wheat

In a field release experiment, an isolate of Pseudomonas fluorescens, which was chromosomally modified with two reporter gene cassettes (lacZY and Kan(supr)-xylE), was applied to spring wheat as a seed coating and subsequently as a foliar spray. The wild-type strain was isolated from the phylloplane of sugar beet but was found to be a common colonizer of both the rizosphere and phylloplane of wheat as well. The impact on the indigenous microbial populations resulting from release of this genetically modified microorganism (GMM) was compared with the impact of the unmodified, wild-type strain and a nontreated control until 1 month after harvest of the crop. The release of the P. fluorescens GMM and the unmodified, wild-type strain resulted in significant but transient perturbations of some of the culturable components of the indigenous microbial communities that inhabited the rhizosphere and phylloplane of wheat, but no significant perturbations of the indigenous culturable microbial populations in nonrhizosphere soil were found. Fast-growing organisms that did not produce resting structures (for example, fluorescent pseudomonads and yeasts) seemed to be most sensitive to perturbation. In terms of hazard and risk to the environment, the observed microbial perturbations that resulted from this GMM release may be considered minor for several reasons. First, the recombinant P. fluorescens strain caused changes that were, in general, not significantly different from those caused by the unmodified wild-type strain; second, perturbations resulting from bacterial inoculations were mainly small; and third, the release of bacteria had no obvious effects on plant growth and plant health.     

Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.     

Effects of Pseudomonas putida modified to produce phenazine-1-carboxylic acid and 2,4-diacetylphloroglucinol on the microflora of field grown wheat

Pseudomonas putida WCS358r, genetically modified to have improved activity against soil-borne pathogens, was released into the rhizosphere of wheat. Two genetically modified derivatives carried the phzor the phl biosynthetic gene loci and constitutively produced either the antifungal compound phenazine-1-carboxylic acid (PCA) or the antifungal and antibacterial compound 2,4-diacetylphloroglucinol (DAPG). In 1997 and 1998, effects of single introductions of PCA producing derivatives on the indigenous microflora were studied. A transient shift in the composition of the total fungal microflora, determined by amplified ribosomal DNA restiction analysis (ARDRA), was detected. Starting in 1999, effects of repeated introduction of genetically modified microorganisms (GMMs) were studied. Wheat seeds coated with the PCA producer, the DAPG producer, a mixture of the PCA and DAPG producers, or WCS358r, were sown and the densities, composition and activities of the rhizosphere microbial populations were measured. All introduced strains decreased from 10⁷CFU per gram of rhizosphere sample to below the detection limit after harvest of the wheat plants. The phz genes were stably maintained in the PCA producers, and PCA was detected in rhizosphere extracts of plants treated with this strain or with the mixture of the PCA and DAPG producers. The phl genes were also stably maintained in the DAPG producing derivative of WCS358r. Effects of the genetically modified bacteria on the rhizosphere fungi and bacteria were analyzed by using amplified ribosomal DNA restriction analysis. Introduction of the genetically modified bacterial strains caused a transient change in the composition of the rhizosphere microflora. However, introduction of the GMMs did not affect the several soil microbial activities that were investigated in this study. This revised version was published online in August 2006 with corrections to the Cover Date.